Human Embryonic Stem Cell-Derived Mesenchymal Stroma Cells (hES-MSCs) Engraft In Vivo and Support Hematopoiesis without Suppressing Immune Function: Implications for Off-The Shelf ES-MSC Therapies

PubMed Central

Li, Ou; Tormin, Ariane; Sundberg, Berit; Hyllner, Johan; Le Blanc, Katarina; Scheding, Stefan

2013-01-01

Mesenchymal stroma cells (MSCs) have a high potential for novel cell therapy approaches in clinical transplantation. Commonly used bone marrow-derived MSCs (BM-MSCs), however, have a restricted proliferative capacity and cultures are difficult to standardize. Recently developed human embryonic stem cell-derived mesenchymal stroma cells (hES-MSCs) might represent an alternative and unlimited source of hMSCs. We therefore compared human ES-cell-derived MSCs (hES-MP002.5 cells) to normal human bone marrow-derived MSCs (BM-MSCs). hES-MP002.5 cells had lower yet reasonable CFU-F capacity compared with BM-MSC (8±3 versus 29±13 CFU-F per 100 cells). Both cell types showed similar immunophenotypic properties, i.e. cells were positive for CD105, CD73, CD166, HLA-ABC, CD44, CD146, CD90, and negative for CD45, CD34, CD14, CD31, CD117, CD19, CD 271, SSEA-4 and HLA-DR. hES-MP002.5 cells, like BM-MSCs, could be differentiated into adipocytes, osteoblasts and chondrocytes in vitro. Neither hES-MP002.5 cells nor BM-MSCs homed to the bone marrow of immune-deficient NSG mice following intravenous transplantation, whereas intra-femoral transplantation into NSG mice resulted in engraftment for both cell types. In vitro long-term culture-initiating cell assays and in vivo co-transplantation experiments with cord blood CD34+ hematopoietic cells demonstrated furthermore that hES-MP002.5 cells, like BM-MSCs, possess potent stroma support function. In contrast to BM-MSCs, however, hES-MP002.5 cells showed no or only little activity in mixed lymphocyte cultures and phytohemagglutinin (PHA) lymphocyte stimulation assays. In summary, ES-cell derived MSCs might be an attractive unlimited source for stroma transplantation approaches without suppressing immune function. PMID:23383153

MicroRNA166 Modulates Cadmium Tolerance and Accumulation in Rice.

PubMed

Ding, Yanfei; Gong, Shaohua; Wang, Yi; Wang, Feijuan; Bao, Hexigeduleng; Sun, Junwei; Cai, Chong; Yi, Keke; Chen, Zhixiang; Zhu, Cheng

2018-06-20

MicroRNAs (miRNAs) are 20- to 24-nucleotide small non-coding RNAs that regulate gene expression in eukaryotic organisms. Several plant miRNAs, such as miR166, have vital roles in plant growth, development and responses to environmental stresses. One such environmental stress encountered by crop plants is exposure to cadmium (Cd), an element highly toxic to most organisms, including humans and plants. In this study, we analyzed the role of miR166 in Cd accumulation and tolerance in rice (Oryza sativa). The expression levels of miR166 in both root and leaf tissues were significantly higher in the reproductive stage than in the seedling stage in rice. The expression of miR166 in the roots of rice seedlings was reduced after Cd treatment. Overexpression of miR166 in rice improved Cd tolerance, a result associated with the reduction of Cd-induced oxidative stress in transgenic rice plants. Furthermore, overexpression of miR166 reduced both Cd translocation from roots to shoots and Cd accumulation in the grains. miR166 targets genes encoding the class-III homeodomain-leucine zipper (HD-Zip) family proteins in plants. In rice, HOMEODOMAIN CONTAINING PROTEIN 4 (OsHB4) gene (Os03g43930), which encodes an HD-Zip protein, was up-regulated by Cd treatment but down-regulated by overexpression of miR166 in transgenic rice plants. Overexpression of OsHB4 increased Cd sensitivity and Cd accumulation in the leaves and grains of transgenic rice plants. By contrast, silencing OsHB4 by RNA interference enhanced Cd tolerance in transgenic rice plants. These results indicate a critical role for miR166 in Cd accumulation and tolerance through regulation of its target gene, OsHB4, in rice. {copyright, serif} 2018 American Society of Plant Biologists. All rights reserved.

Biotechnological and biomedical applications of mesenchymal stem cells as a therapeutic system.

PubMed

Rahimzadeh, Amirbahman; Mirakabad, Fatemeh Sadat Tabatabaei; Movassaghpour, Aliakbar; Shamsasenjan, Karim; Kariminekoo, Saber; Talebi, Mehdi; Shekari, Abolfazl; Zeighamian, Vahideh; Ghalhar, Masoud Gandomkar; Akbarzadeh, Abolfazl

2016-01-01

Mesenchymal stem cells (MSCs) are non-hematopoietic, multipotent progenitor cells which reside in bone marrow (BM), support homing of hematopoietic stem cells (HSCs) and self-renewal in the BM. These cells have the potential to differentiate into tissues of mesenchymal origin, such as fibroblasts, adipocytes, cardiomyocytes, and stromal cells. MSCs can express surface molecules like CD13, CD29, CD44, CD73, CD90, CD166, CXCL12 and toll-like receptors (TLRs). Different factors, such as TGF-β, IL-10, IDO, PGE-2, sHLA-G5, HO, and Galectin-3, secreted by MSCs, induce interaction in cell to cell immunomodulatory effects on innate and adaptive cells of the immune system. Furthermore, these cells can stimulate and increase the TH2 and regulatory T-cells through inhibitory effects on the immune system. MSCs originate from the BM and other tissues including the brain, adipose tissue, peripheral blood, cornea, thymus, spleen, fallopian tube, placenta, Wharton's jelly and umbilical cord blood. Many studies have focused on two significant features of MSC therapy: (I) MSCs can modulate T-cell-mediated immunological responses, and (II) systemically administered MSCs home in to sites of ischemia or injury. In this review, we describe the known mechanisms of immunomodulation and homing of MSCs. As a result, this review emphasizes the functional role of MSCs in modulating immune responses, their capability in homing to injured tissue, and their clinical therapeutic potential.

Cell therapy: a therapeutic alternative to treat focal cartilage lesions.

PubMed

Gimeno, M J; Maneiro, E; Rendal, E; Ramallal, M; Sanjurjo, L; Blanco, F J

2005-11-01

Human mesenchymal stem cells (MSCs) are present in most of the tissue matrix, taking part in their regeneration when injury or damage occurs. The aim of this study was to investigate the presence of cells with pluripotential characteristics in synovial membranes from osteoarthritic (OA) patients and the capacity of these cells to differentiate to chondrocytes. Synovial membranes (n = 8) from OA patients were digested with collagenase. Isolated cells were cultured with DMEM, 20% FBS, and FGFb10 ng/mL. Cells from second subculture were used to carry out phenotypic characterization experiments (flow cytometry analysis with 11 monoclonal antibodies) and chondrogenic differentiation experiments(micropellet cultured in chondrogenic medium). Chondrogenic differentiation of cells was assessment by quantification of cartilage extracellular matrix components by following techniques: Safranin O, Toluidine Blue, and Alcian Blue stains to detect proteoglycans and immunohistochemistry to detect type I and II collagen. Flow cytometry analyses showed that in our population more than 90% of cells were positive for MSC markers: CD29 (95%), CD44 (90%), CD73 (95%), CD90 (98%). Cells were negative for hematopoietic markers (CD11b, CD34, and CD45). Furthermore, cells showed positive stain to multipotent markers such as CD117 (c-kit) (98%), CD166 (74%), and STRO-1 (88%) and to quiescent satellite cells like PAX-7 (35%). The micropellet analyses showed that the culture of these cells with TGFbeta-3 for 2 and 3 weeks stimulates proteoglycan and collagen type II synthesis. Both molecules are characteristic of hyaline articular cartilage. In this work, we demonstrate the presence of a cellular population with MSC characteristics in synovial tissue from OA patients. As MSC takes part in reparative processes of adult tissues, these cells could play an important role in OA pathogenesis and treatment.

Directly auto-transplanted mesenchymal stem cells induce bone formation in a ceramic bone substitute in an ectopic sheep model.

PubMed

Boos, Anja M; Loew, Johanna S; Deschler, Gloria; Arkudas, Andreas; Bleiziffer, Oliver; Gulle, Heinz; Dragu, Adrian; Kneser, Ulrich; Horch, Raymund E; Beier, Justus P

2011-06-01

Bone tissue engineering approaches increasingly focus on the use of mesenchymal stem cells (MSC). In most animal transplantation models MSC are isolated and expanded before auto cell transplantation which might be critical for clinical application in the future. Hence this study compares the potential of directly auto-transplanted versus in vitro expanded MSC with or without bone morphogenetic protein-2 (BMP-2) to induce bone formation in a large volume ceramic bone substitute in the sheep model. MSC were isolated from bone marrow aspirates and directly auto-transplanted or expanded in vitro and characterized using fluorescence activated cell sorting (FACS) and RT-PCR analysis before subcutaneous implantation in combination with BMP-2 and β-tricalcium phosphate/hydroxyapatite (β-TCP/HA) granules. Constructs were explanted after 1 to 12 weeks followed by histological and RT-PCR evaluation. Sheep MSC were CD29(+), CD44(+) and CD166(+) after selection by Ficoll gradient centrifugation, while directly auto-transplanted MSC-populations expressed CD29 and CD166 at lower levels. Both, directly auto-transplanted and expanded MSC, were constantly proliferating and had a decreasing apoptosis over time in vivo. Directly auto-transplanted MSC led to de novo bone formation in a heterotopic sheep model using a β-TCP/HA matrix comparable to the application of 60 μg/ml BMP-2 only or implantation of expanded MSC. Bone matrix proteins were up-regulated in constructs following direct auto-transplantation and in expanded MSC as well as in BMP-2 constructs. Up-regulation was detected using immunohistology methods and RT-PCR. Dense vascularization was demonstrated by CD31 immunohistology staining in all three groups. Ectopic bone could be generated using directly auto-transplanted or expanded MSC with β-TCP/HA granules alone. Hence BMP-2 stimulation might become dispensable in the future, thus providing an attractive, clinically feasible approach to bone tissue engineering. © 2011 The Authors Journal of Cellular and Molecular Medicine © 2011 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.

Manufacture of endothelial colony-forming progenitor cells from steady-state peripheral blood leukapheresis using pooled human platelet lysate.

PubMed

Siegel, Georg; Fleck, Erika; Elser, Stefanie; Hermanutz-Klein, Ursula; Waidmann, Marc; Northoff, Hinnak; Seifried, Erhard; Schäfer, Richard

2018-05-01

Endothelial colony-forming progenitor cells (ECFCs) are promising candidates for cell therapies. However, ECFC translation to the clinic requires optimized isolation and manufacture technologies according to good manufacturing practice (GMP). ECFCs were manufactured from steady-state peripheral blood (PB) leukapheresis (11 donors), using GMP-compliant technologies including pooled human platelet (PLT) lysate, and compared to human umbilical cord endothelial cells, human aortic endothelial cells, and human cerebral microvascular endothelial cells. Specific variables assessed were growth kinetics, phenotype, trophic factors production, stimulation of tube formation, and Dil-AcLDL uptake. ECFCs could be isolated from PB leukapheresis units with mean processed volume of 5411 mL and mean white blood cell (WBC) concentration factor of 8.74. The mean frequency was 1.44 × 10 -8 ECFCs per WBC, corresponding to a mean of 177.8 ECFCs per apheresis unit. Expandable for up to 12 cumulative population doublings, calculated projection showed that approximately 730 × 10 3 ECFCs could be manufactured from 1 apheresis unit. ECFCs produced epidermal growth factor, hepatocyte growth factor, vascular endothelial growth factor (VEGF)-A, PLT-derived growth factor-B, interleukin-8, and monocyte chemoattractant protein-1, featured high potential for capillary-like tubes formation, and showed no telomerase activity. They were characterized by CD29, CD31, CD44, CD105, CD117, CD133, CD144, CD146, and VEGF-R2 expression, with the most common subpopulation CD34+CD117-CD133-. Compared to controls, ECFCs featured greater Dil-AcLDL uptake and higher expression of CD29, CD31, CD34, CD44, CD144, and VEGF-R2. Here we show that isolation of ECFCs with proangiogenic profile from steady-state PB leukapheresis is feasible, marking a first step toward ECFC product manufacture according to GMP. © 2018 AABB.

Mesenchymal stem cells from the Wharton’s jelly of umbilical cord segments provide stromal support for the maintenance of cord blood hematopoietic stem cells during long-term ex vivo culture

PubMed Central

Bakhshi, Tiki; Zabriskie, Ryan C.; Bodie, Shamanique; Kidd, Shannon; Ramin, Susan; Paganessi, Laura A.; Gregory, Stephanie A.; Fung, Henry C.; Christopherson, Kent W.

2012-01-01

BACKGROUND Hematopoietic stem cells (HSCs) are routinely obtained from marrow, mobilized peripheral blood, and umbilical cord blood. Mesenchymal stem cells (MSCs) are traditionally isolated from marrow. Bone marrow–derived MSCs (BM-MSCs) have previously demonstrated their ability to act as a feeder layer in support of ex vivo cord blood expansion. However, the use of BM-MSCs to support the growth, differentiation, and engraftment of cord blood may not be ideal for transplant purposes. Therefore, the potential of MSCs from a novel source, the Wharton’s jelly of umbilical cords, to act as stromal support for the long-term culture of cord blood HSC was evaluated. STUDY DESIGN AND METHODS Umbilical cord–derived MSCs (UC-MSCs) were cultured from the Wharton’s jelly of umbilical cord segments. The UC-MSCs were then profiled for expression of 12 cell surface receptors and tested for their ability to support cord blood HSCs in a long-term culture-initiating cell (LTC-IC) assay. RESULTS Upon culture, UC-MSCs express a defined set of cell surface markers (CD29, CD44, CD73, CD90, CD105, CD166, and HLA-A) and lack other markers (CD45, CD34, CD38, CD117, and HLA-DR) similar to BM-MSCs. Like BM-MSCs, UC-MSCs effectively support the growth of CD34+ cord blood cells in LTC-IC assays. CONCLUSION These data suggest the potential therapeutic application of Wharton’s jelly–derived UC-MSCs to provide stromal support structure for the long-term culture of cord blood HSCs as well as the possibility of cotransplantation of genetically identical, HLA-matched, or unmatched cord blood HSCs and UC-MSCs in the setting of HSC transplantation. PMID:18798803

Characterization of antibodies against ferret immunoglobulins, cytokines and CD markers.

PubMed

Martel, Cyril Jean-Marie; Aasted, Bent

2009-12-15

Ferret IgG and IgM were purified from normal serum, while ferret IgA was purified from bile. The estimated molecular weights of the immunoglobulin gamma, alpha and mu heavy chains were found to be 54kDa, 69kDa and 83kDa, respectively. For immunological (ELISA) quantification of ferret immunoglobulins, we identified and characterized polyclonal antibodies towards ferret IgG, IgM and IgA. We also identified 22 monoclonal antibodies (mAbs) raised mostly against human CD markers which cross-reacted with ferret leukocytes. These antibodies were originally specific against human CD8, CD9, CD14, CD18, CD25, CD29, CD32, CD44, CD61, CD71, CD79b, CD88, CD104, CD172a and mink CD3. Finally, we identified 4 cross-reacting mAbs with specificities against ferret interferon-gamma, TNF-alpha, interleukin-4 and interleukin-8.

Adipose-derived stem cell: a better stem cell than BMSC.

PubMed

Zhu, Yanxia; Liu, Tianqing; Song, Kedong; Fan, Xiubo; Ma, Xuehu; Cui, Zhanfeng

2008-08-01

To further study the proliferation and multi-differentiation potentials of adipose-derived stem cells (ADSCs), the cells were isolated with improved methods and their growth curves were achieved with cck-8. Surface protein expression was analyzed by flow cytometry to characterize the cell phenotype. The multi-lineage potential of ADSCs was testified by differentiating cells with adipogenic, chondrogenic, osteogenic, and myogenic inducers. The results showed that about 5 x 10(5) stem cells could be obtained from 400 to 600 mg adipose tissue. The ADSCs can be continuously cultured in vitro for up to 1 month without passage and they have several logarithmic growth phases during the culture period. Also, the flow cytometry analysis showed that ADSCs expressed high levels of stem cell-related antigens (CD13, CD29, CD44, CD105, and CD166), while did not express hematopoiesis-related antigens CD34 and CD45, and human leukocyte antigen HLA-DR was also negative. Moreover, stem cell-related transcription factors, Nanog, Oct-4, Sox-2, and Rex-1 were positively expressed in ADSCs. The expression of alkaline phosphatase (ALP) was detected in the early osteogenic induction and the calcified nodules were observed by von Kossa staining. Intracellular lipid droplets could be observed by Oil Red staining. Differentiated cardiomyocytes were observed by connexin43 fluorescent staining. In order to obtain more stem cells, we can subculture ADSCs every 14 days instead of the normal 5 days. ADSCs still keep strong proliferation ability, maintain their phenotypes, and have stronger multi-differentiation potential after 25 passages. Copyright 2008 John Wiley & Sons, Ltd.

Stem/progenitor cells from inflamed human dental pulp retain tissue regeneration potential

PubMed Central

Alongi, Dominick J; Yamaza, Takayoshi; Song, Yingjie; Fouad, Ashraf F; Romberg, Elaine E; Shi, Songtao; Tuan, Rocky S; Huang, George T-J

2011-01-01

Background Potent stem/progenitor cells have been isolated from normal human dental pulps termed dental pulp stem cells (DPSCs). However, it is unknown whether these cells exist in inflamed pulps (IPs). Aims To determine whether DPSCs can be identified and isolated from IPs; and if they can be successfully cultured, whether they retain tissue regeneration potential in vivo. Materials & methods DPSCs from freshly collected normal pulps (NPs) and IPs were characterized in vitro and their tissue regeneration potential tested using an in vivo study model. Results The immunohistochemical analysis showed that IPs expressed higher levels of mesenchymal stem cell markers STRO-1, CD90, CD105 and CD146 compared with NPs (p < 0.05). Flow cytometry analysis showed that DPSCs from both NPs and IPs expressed moderate to high levels of CD146, stage-specific embryonic antigen-4, CD73 and CD166. Total population doubling of DPSCs-IPs (44.6 ± 2.9) was lower than that of DPSCs-NPs (58.9 ± 2.5) (p < 0.05), and DPSCs-IPs appeared to have a decreased osteo/dentinogenic potential compared with DPSCs-NPs based on the mineral deposition in cultures. Nonetheless, DPSCs-IPs formed pulp/dentin complexes similar to DPSCs-NPs when transplanted into immunocompromised mice. Conclusion DPSCs-IPs can be isolated and their mesenchymal stem cell marker profiles are similar to those from NPs. Although some stem cell properties of DPSCs-IPs were altered, cells from some samples remained potent in tissue regeneration in vivo. PMID:20465527

Identification of CD166 as a Surface Marker for Enriching Prostate Stem/Progenitor and Cancer Initiating Cells

PubMed Central

Wang, Shunyou; Tran, Linh M.; Goldstein, Andrew S.; Lawson, Devon; Chen, Donghui; Li, Yunfeng; Guo, Changyong; Zhang, Baohui; Fazli, Ladan; Gleave, Martin; Witte, Owen N.; Garraway, Isla P.; Wu, Hong

2012-01-01

New therapies for late stage and castration resistant prostate cancer (CRPC) depend on defining unique properties and pathways of cell sub-populations capable of sustaining the net growth of the cancer. One of the best enrichment schemes for isolating the putative stem/progenitor cell from the murine prostate gland is Lin-;Sca1+;CD49fhi (LSChi), which results in a more than 10-fold enrichment for in vitro sphere-forming activity. We have shown previously that the LSChi subpopulation is both necessary and sufficient for cancer initiation in the Pten-null prostate cancer model. To further improve this enrichment scheme, we searched for cell surface molecules upregulated upon castration of murine prostate and identified CD166 as a candidate gene. CD166 encodes a cell surface molecule that can further enrich sphere-forming activity of WT LSChi and Pten null LSChi. Importantly, CD166 could enrich sphere-forming ability of benign primary human prostate cells in vitro and induce the formation of tubule-like structures in vivo. CD166 expression is upregulated in human prostate cancers, especially CRPC samples. Although genetic deletion of murine CD166 in the Pten null prostate cancer model does not interfere with sphere formation or block prostate cancer progression and CRPC development, the presence of CD166 on prostate stem/progenitors and castration resistant sub-populations suggest that it is a cell surface molecule with the potential for targeted delivery of human prostate cancer therapeutics. PMID:22880034

Immunophenotypical characterization of canine mesenchymal stem cells from perivisceral and subcutaneous adipose tissue by a species-specific panel of antibodies.

PubMed

Ivanovska, Ana; Grolli, Stefano; Borghetti, Paolo; Ravanetti, Francesca; Conti, Virna; De Angelis, Elena; Macchi, Francesca; Ramoni, Roberto; Martelli, Paolo; Gazza, Ferdinando; Cacchioli, Antonio

2017-10-01

Immunophenotypical characterization of mesenchymal stem cells is fundamental for the design and execution of sound experimental and clinical studies. The scarce availability of species-specific antibodies for canine antigens has hampered the immunophenotypical characterization of canine mesenchymal stem cells (MSC). The aim of this study was to select a panel of species-specific direct antibodies readily useful for canine mesenchymal stem cells characterization. They were isolated from perivisceral and subcutaneous adipose tissue samples collected during regular surgeries from 8 dogs. Single color flow cytometric analysis of mesenchymal stem cells (P3) deriving from subcutaneous and perivisceral adipose tissue with a panel of 7 direct anti-canine antibodies revealed two largely homogenous cell populations with a similar pattern: CD29 + , CD44 + , CD73 + , CD90 + , CD34 - , CD45 - and MHC-II - with no statistically significant differences among them. Antibody reactivity was demonstrated on canine peripheral blood mononuclear cells. The similarities are reinforced by their in vitro cell morphology, trilineage differentiation ability and RT-PCR analysis (CD90 + , CD73 + , CD105 + , CD44 + , CD13 + , CD29 + , Oct-4 + gene and CD31 - and CD45 - expression). Our results report for the first time a comparison between the immunophenotypic profile of canine MSC deriving from perivisceral and subcutaneous adipose tissue. The substantial equivalence between the two populations has practical implication on clinical applications, giving the opportunity to choose the source depending on the patient needs. The results contribute to routine characterization of MSC populations grown in vitro, a mandatory process for the definition of solid and reproducible laboratory and therapeutic procedures. Copyright © 2017 Elsevier Ltd. All rights reserved.

Isolation, identification and multipotential differentiation of mouse adipose tissue-derived stem cells.

PubMed

Taha, Masoumeh Fakhr; Hedayati, Vahideh

2010-08-01

Bone marrow and adipose tissue have provided two suitable sources of mesenchymal stem cells. Although previous studies have confirmed close similarities between bone marrow-derived stem cells (BM-MSCs) and adipose tissue-derived stem cells (ADSCs), the molecular phenotype of ADSCs is still poorly identified. In the present study, mouse ADSCs were isolated from the inguinal fat pad of 12-14 weeks old mice. Freshly isolated and three passaged ADSCs were analyzed for the expression of OCT4, Sca-1, c-kit and CD34 by RT-PCR. Three passaged ADSCs were analyzed by flow cytometry for the presence of CD11b, CD45, CD31, CD29 and CD44. Moreover, cardiogenic, adipogenic and neurogenic differentiation of ADSCs were induced in vitro. Freshly isolated ADSCs showed the expression of OCT4, Sca-1, c-kit and CD34, and two days cultured ADSCs were positively immunostained with anti-OCT4 monoclonal antibody. After three passages, the expression of OCT4, c-kit and CD34 eliminated, while the expression of Sca-1 showed a striking enhancement. These cells were identified positive for CD29 and CD44 markers, and they showed the lack of CD45 and CD31 expression. Three passaged ADSCs were differentiated to adipocyte-, cardiomyocyte- and neuron-like cells that were identified based on the positive staining with Sudan black, anti-cardiac troponin I antibody and anti-map-2 antibody, respectively. In conclusion, adipose tissue contains a stem cell population that seems to be a good multipotential cell candidate for the future cell replacement therapy. Copyright 2010 Elsevier Ltd. All rights reserved.

sCD44 overexpression increases intraocular pressure and aqueous outflow resistance

PubMed Central

Giovingo, Michael; Nolan, Michael; McCarty, Ryan; Pang, Iok-Hou; Clark, Abbot F.; Beverley, Rachel M.; Schwartz, Steven; Stamer, W. Daniel; Walker, Loyal; Grybauskas, Algis; Skuran, Kevin; Kuprys, Paulius V.; Yue, Beatrice Y.J.T.

2013-01-01

Purpose CD44 plays major roles in multiple physiologic processes. The ectodomain concentration of the CD44 receptor, soluble CD44 (sCD44), is significantly increased in the aqueous humor of primary open-angle glaucoma (POAG). The purpose of this study was to determine if adenoviral constructs of CD44 and isolated 32-kDa sCD44 change intraocular pressure (IOP) in vivo and aqueous outflow resistance in vitro. Methods Adenoviral constructs of human standard CD44 (Ad-CD44S), soluble CD44 (Ad-sCD44), and empty viral cDNA were injected into the vitreous of BALB/cJ mice, followed by serial IOP measurements. Overexpression of CD44S and sCD44 was verified in vitro by enzyme-linked immunosorbent assay (ELISA) and western blot analysis. Anterior segments of porcine eyes were perfused with the isolated sCD44. sCD44-treated human trabecular meshwork (TM) cells and microdissected porcine TM were examined by confocal microscopy and Optiprep density gradient with western blot analysis to determine changes in lipid raft components. Results Intravitreous injection of adenoviral constructs with either Ad-CD44S or Ad-sCD44 vectors caused prolonged ocular hypertension in mice. Eight days after vector injection, Ad-CD44S significantly elevated IOP to 28.3±1.2 mmHg (mean±SEM, n=8; p<0.001); Ad-sCD44 increased IOP to 18.5±2.6 mmHg (n=8; p<0.01), whereas the IOP of uninjected eyes was 12.7±0.2 mmHg (n=16). The IOP elevation lasted more than 50 days. Topical administration of a γ-secretase inhibitor normalized Ad-sCD44-induced elevated IOP. sCD44 levels were significantly elevated in the aqueous humor of Ad-CD44S and Ad-sCD44 eyes versus contralateral uninjected eyes (p<0.01). Anterior segment perfusion of isolated 32-kDa sCD44 significantly decreased aqueous outflow rates. Co-administration of isolated sCD44 and CD44 neutralizing antibody or of γ-secretase inhibitor significantly enhanced flow rates. sCD44-treated human TM cells displayed cross-linked actin network formation. Optiprep density gradient and western blot analysis of human TM cells treated with sCD44 showed decreased annexin 2 expression and increased phosphorylated annexin 2 and caveolin 1 expression. Conclusions Our data suggest that sCD44 increases outflow resistance in vivo and in vitro. Viral overexpression of both CD44S and sCD44 is sufficient to cause ocular hypertension. Infusion of sCD44 in porcine anterior segment eyes significantly decreased flow rates. Notably, sCD44 enhanced cross-linked actin network formation. The elevated sCD44 levels seen in POAG aqueous humor may play an important causative role in POAG pathogenesis. PMID:24194636

Derivation of Stromal (Skeletal and Mesenchymal) Stem-Like Cells from Human Embryonic Stem Cells

PubMed Central

Harkness, Linda; Abdallah, Basem M.; Elsafadi, Mona; Al-Nbaheen, May S.; Aldahmash, Abdullah; Kassem, Moustapha

2012-01-01

Derivation of bone forming cells (osteoblasts) from human embryonic stem cells (hESCs) is a prerequisite for their use in clinical applications. However, there is no standard protocol for differentiating hESCs into osteoblastic cells. The aim of this study was to identify the emergence of a human stromal (mesenchymal and skeletal) stem cell (hMSC)-like population, known to be osteoblastic cell precursors and to test their osteoblastic differentiation capacity in ex vivo cultures and in vivo. We cultured hESCs in a feeder-free environment using serum replacement and as suspension aggregates (embryoid bodies; hEBs). Over a 20 day developmental period, the hEBs demonstrated increasing enrichment for cells expressing hMSC markers: CD29, CD44, CD63, CD56, CD71, CD73, CD105, CD106, and CD166 as revealed by immunohistochemical staining and flow cytometry (fluorescence-activated cell sorting) analysis. Ex vivo differentiation of hEBs using bone morphogenic protein 2 (BMP2) combined with standard osteoblast induction medium led to weak osteoblastic induction. Conversely, subcutaneous in vivo implantation of day 20 hEBs in immune deficient mice, mixed with hydroxyapatite/tricalcium phosphate (HA/TCP) as an osteoconductive scaffold, revealed bone and cartilage, and fibrous tissue elements after 8 weeks. These tissues were of human origin and there was no evidence of differentiation to nonmesodermal tissues. hEBs implanted in the absence of HA/TCP formed vacuolated tissue containing glandular, fibrous and muscle-like tissue elements. Conversely, implantation of undifferentiated hESCs resulted in the formation of a teratoma containing a mixture of endodermal, mesodermal, and ectodermal tissues. Our study demonstrates that hMSC-like cells can be obtained from hESCs and they can be induced to form skeletal tissues in vivo when combined with HA/TCP. These findings are relevant for tissue engineering and suggest that differentiated hEBs can provide an unlimited source for functional osteogenic cells. PMID:22612317

Recruited brain tumor-derived mesenchymal stem cells contribute to brain tumor progression.

PubMed

Behnan, Jinan; Isakson, Pauline; Joel, Mrinal; Cilio, Corrado; Langmoen, Iver A; Vik-Mo, Einar O; Badn, Wiaam

2014-05-01

The identity of the cells that contribute to brain tumor structure and progression remains unclear. Mesenchymal stem cells (MSCs) have recently been isolated from normal mouse brain. Here, we report the infiltration of MSC-like cells into the GL261 murine glioma model. These brain tumor-derived mesenchymal stem cells (BT-MSCs) are defined with the phenotype (Lin-Sca-1+CD9+CD44+CD166+/-) and have multipotent differentiation capacity. We show that the infiltration of BT-MSCs correlates to tumor progression; furthermore, BT-MSCs increased the proliferation rate of GL261 cells in vitro. For the first time, we report that the majority of GL261 cells expressed mesenchymal phenotype under both adherent and sphere culture conditions in vitro and that the non-MSC population is nontumorigenic in vivo. Although the GL261 cell line expressed mesenchymal phenotype markers in vitro, most BT-MSCs are recruited cells from host origin in both wild-type GL261 inoculated into green fluorescent protein (GFP)-transgenic mice and GL261-GFP cells inoculated into wild-type mice. We show the expression of chemokine receptors CXCR4 and CXCR6 on different recruited cell populations. In vivo, the GL261 cells change marker profile and acquire a phenotype that is more similar to cells growing in sphere culture conditions. Finally, we identify a BT-MSC population in human glioblastoma that is CD44+CD9+CD166+ both in freshly isolated and culture-expanded cells. Our data indicate that cells with MSC-like phenotype infiltrate into the tumor stroma and play an important role in tumor cell growth in vitro and in vivo. Thus, we suggest that targeting BT-MSCs could be a possible strategy for treating glioblastoma patients. © 2013 AlphaMed Press.

Heat shock protein-90 beta is expressed at the surface of multipotential mesenchymal precursor cells: generation of a novel monoclonal antibody, STRO-4, with specificity for mesenchymal precursor cells from human and ovine tissues.

PubMed

Gronthos, Stan; McCarty, Rosa; Mrozik, Krzysztof; Fitter, Stephen; Paton, Sharon; Menicanin, Danijela; Itescu, Silviu; Bartold, P Mark; Xian, Cory; Zannettino, Andrew C W

2009-11-01

Mesenchymal stromal cells (MSCs) and their precursor cells (MPCs) can proliferate and differentiate into multiple mesodermal and some ectodermal and endodermal tissues. Culture-expanded MSCs are currently being evaluated as a possible cell therapy to replace/repair injured or diseased tissues. While a number of mAb reagents with specificity to human MSCs, including STRO-1, STRO-3 (BLK ALP), CD71 (SH2, SH3), CD106 (VCAM-1), CD166, and CD271, have facilitated the isolation of purified populations of human MSCs from primary tissues, few if any mAb reagents have been described that can be used to isolate equivalent cells from other species. This is of particular relevance when assessing the tissue regenerative efficacy of MSCs in large immunocompetent, preclinical animal models of disease. In light of this, we sought to generate novel monoclonal antibodies (mAb) with specific reactivity against a cell surface molecule that is expressed at high levels by MSCs from different species. Using CD106 (VCAM-1)-selected ovine MSCs as an immunogen, mAb-producing hybridomas were selected for their reactivity to both human and ovine MSCs. One such hybridoma, termed STRO-4, produced an IgG mAb that reacted with <5% of human and ovine bone marrow (BM) mononuclear cells. As a single selection reagent, STRO-4 mAb was able to enrich colony-forming fibroblasts (CFU-F) in both human and ovine BM by 16- and 8-folds, respectively. Cells isolated with STRO-4 exhibited reactivity with markers commonly associated with MSCs isolated by plastic adherence including CD29, CD44, and CD166. Moreover, when placed in inductive culture conditions in vitro, STRO-4(+) MSCs exhibited multilineage differentiation potential and were capable of forming a mineralized matrix, lipid-filled adipocytes, and chondrocytes capable of forming a glycosaminoglycan-rich matrix. Biochemical analysis revealed that STRO-4 identified the beta isoform of heat shock protein-90 (Hsp90beta). In addition to identifying an antibody reagent that identifies a highly conserved epitope expressed by MSCs from different species, our study also points to a potential role for Hsp90beta in MSC biology.

Cooperativity of CD44 and CD49d in leukemia cell homing, migration, and survival offers a means for therapeutic attack.

PubMed

Singh, Vibuthi; Erb, Ulrike; Zöller, Margot

2013-11-15

A CD44 blockade drives leukemic cells into differentiation and apoptosis by dislodging from the osteogenic niche. Because anti-CD49d also supports hematopoietic stem cell mobilization, we sought to determine the therapeutic efficacy of a joint CD49d/CD44 blockade. To unravel the underlying mechanism, the CD49d(-) EL4 lymphoma was transfected with CD49d or point-mutated CD49d, prohibiting phosphorylation and FAK binding; additionally, a CD44(-) Jurkat subline was transfected with murine CD44, CD44 with a point mutation in the ezrin binding site, or with cytoplasmic tail-truncated CD44. Parental and transfected EL4 and Jurkat cells were evaluated for adhesion, migration, and apoptosis susceptibility in vitro and in vivo. Ligand-binding and Ab-blocking studies revealed CD44-CD49d cooperation in vitro and in vivo in adhesion, migration, and apoptosis resistance. The cooperation depends on ligand-induced proximity such that both CD44 and CD49d get access to src, FAK, and paxillin and via lck to the MAPK pathway, with the latter also supporting antiapoptotic molecule liberation. Accordingly, synergisms were only seen in leukemia cells expressing wild-type CD44 and CD49d. Anti-CD44 together with anti-CD49d efficiently dislodged EL4-CD49d/Jurkat-CD44 in bone marrow and spleen. Dislodging was accompanied by increased apoptosis susceptibility that strengthened low-dose chemotherapy, the combined treatment most strongly interfering with metastatic settlement and being partly curative. Ab treatment also promoted NK and Ab-dependent cellular cytotoxicity activation, which affected leukemia cells independent of CD44/CD49d tail mutations. Thus, mostly owing to a blockade of joint signaling, anti-CD44 and anti-CD49d hamper leukemic cell settlement and break apoptosis resistance, which strongly supports low-dose chemotherapy.

CD44 Splice Variants as Potential Players in Alzheimer's Disease Pathology.

PubMed

Pinner, Elhanan; Gruper, Yaron; Ben Zimra, Micha; Kristt, Don; Laudon, Moshe; Naor, David; Zisapel, Nava

2017-01-01

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by cognitive deficits, deposition of amyloid-β (Aβ) plaques, intracellular neurofibrillary tangles, and neuronal cell death. Neuroinflammation is commonly believed to participate in AD pathogenesis. CD44 is an inflammation-related gene encoding a widely-distributed family of alternatively spliced cell surface glycoproteins that have been implicated in inflammation, metastases, and inflammation-linked neuronal injuries. Here we investigated the expression patterns of CD44S (which does not contain any alternative exon) and CD44 splice variants in postmortem hippocampal samples from AD patients and matched non-AD controls. The expression of CD44S and CD44 splice variants CD44V3, CD44V6, and CD44V10 was significantly higher in AD patients compared to non-AD controls. Immunohistochemistry of human hippocampal sections revealed that CD44S differentially localized to neuritic plaques and astrocytes, whereas CD44V3, CD44V6, and CD44V10 expression was mostly neuronal. Consistent with these findings, we found that the expression of CD44V6 and CD44V10 was induced by Aβ peptide in neuroblastoma cells and primary neurons. Furthermore, in loss of function studies we found that both CD44V10-specific siRNA and CD44V10 antibody protected neuronal cells from Aβ-induced toxicity, suggesting a causal relationship between CD44V10 and neuronal cell death. These data indicate that certain CD44 splice variants contribute to AD pathology and that CD44V10 inhibition may serve as a new neuroprotective treatment strategy for this disease.

CD44 standard and CD44v10 isoform expression on leukemia cells distinctly influences niche embedding of hematopoietic stem cells.

PubMed

Erb, Ulrike; Megaptche, Amelie Pajip; Gu, Xiaoyu; Büchler, Markus W; Zöller, Margot

2014-03-31

A blockade of CD44 is considered a therapeutic option for the elimination of leukemia initiating cells. However, anti-panCD44 can interfere with hematopoiesis. Therefore we explored, whether a CD44 variant isoform (CD44v)-specific antibody can inhibit leukemia growth without attacking hematopoiesis. As a model we used CD44v10 transfected EL4 thymoma cells (EL4-v10). The therapeutic efficacy of anti-panCD44 and anti-CD44v10 was evaluated after intravenous application of EL4/EL4-v10. Ex vivo and in vitro studies evaluated the impact of anti-panCD44 and anti-CD44v10 as well as of EL4 and EL4-v10 on hematopoietic stem cells (HSC) in cocultures with bone marrow stroma cells with a focus on adhesion, migration, cell cycle progression and apoptosis resistance. Intravenously injected EL4-v10 grow in bone marrow and spleen. Anti-panCD44 and, more pronounced anti-CD44v10 prolong the survival time. The higher efficacy of anti-CD44v10 compared to anti-panCD44 does not rely on stronger antibody-dependent cellular cytotoxicity or on promoting EL4-v10 apoptosis. Instead, EL4 compete with HSC niche embedding. This has consequences on quiescence and apoptosis-protecting signals provided by the stroma. Anti-panCD44, too, more efficiently affected embedding of HSC than of EL4 in the bone marrow stroma. EL4-v10, by catching osteopontin, migrated on bone marrow stroma and did not or weakly interfere with HSC adhesion. Anti-CD44v10, too, did not affect the HSC--bone marrow stroma crosstalk. The therapeutic effect of anti-panCD44 and anti-CD44v10 is based on stimulation of antibody-dependent cellular cytotoxicity. The superiority of anti-CD44v10 is partly due to blocking CD44v10-stimulated osteopontin expression that could drive HSC out of the niche. However, the main reason for the superiority of anti-CD44v10 relies on neither EL4-v10 nor anti-CD44v10 severely interfering with HSC--stroma cell interactions that, on the other hand, are affected by EL4 and anti-panCD44. Anti-panCD44 disturbing HSC embedding in the osteogenic niche weakens its therapeutic effect towards EL4. Thus, as far as leukemic cells express CD44v isoforms, the therapeutic use of anti-panCD44 should be avoided in favor of CD44v-specific antibodies.

CD44 standard and CD44v10 isoform expression on leukemia cells distinctly influences niche embedding of hematopoietic stem cells

PubMed Central

2014-01-01

Background A blockade of CD44 is considered a therapeutic option for the elimination of leukemia initiating cells. However, anti-panCD44 can interfere with hematopoiesis. Therefore we explored, whether a CD44 variant isoform (CD44v)-specific antibody can inhibit leukemia growth without attacking hematopoiesis. As a model we used CD44v10 transfected EL4 thymoma cells (EL4-v10). Methods The therapeutic efficacy of anti-panCD44 and anti-CD44v10 was evaluated after intravenous application of EL4/EL4-v10. Ex vivo and in vitro studies evaluated the impact of anti-panCD44 and anti-CD44v10 as well as of EL4 and EL4-v10 on hematopoietic stem cells (HSC) in cocultures with bone marrow stroma cells with a focus on adhesion, migration, cell cycle progression and apoptosis resistance. Results Intravenously injected EL4-v10 grow in bone marrow and spleen. Anti-panCD44 and, more pronounced anti-CD44v10 prolong the survival time. The higher efficacy of anti-CD44v10 compared to anti-panCD44 does not rely on stronger antibody-dependent cellular cytotoxicity or on promoting EL4-v10 apoptosis. Instead, EL4 compete with HSC niche embedding. This has consequences on quiescence and apoptosis-protecting signals provided by the stroma. Anti-panCD44, too, more efficiently affected embedding of HSC than of EL4 in the bone marrow stroma. EL4-v10, by catching osteopontin, migrated on bone marrow stroma and did not or weakly interfere with HSC adhesion. Anti-CD44v10, too, did not affect the HSC – bone marrow stroma crosstalk. Conclusion The therapeutic effect of anti-panCD44 and anti-CD44v10 is based on stimulation of antibody-dependent cellular cytotoxicity. The superiority of anti-CD44v10 is partly due to blocking CD44v10-stimulated osteopontin expression that could drive HSC out of the niche. However, the main reason for the superiority of anti-CD44v10 relies on neither EL4-v10 nor anti-CD44v10 severely interfering with HSC – stroma cell interactions that, on the other hand, are affected by EL4 and anti-panCD44. Anti-panCD44 disturbing HSC embedding in the osteogenic niche weakens its therapeutic effect towards EL4. Thus, as far as leukemic cells express CD44v isoforms, the therapeutic use of anti-panCD44 should be avoided in favor of CD44v-specific antibodies. PMID:24684724

CD44s and CD44v6 Expression in Head and Neck Epithelia

PubMed Central

Mack, Brigitte; Gires, Olivier

2008-01-01

Background CD44 splice variants are long-known as being associated with cell transformation. Recently, the standard form of CD44 (CD44s) was shown to be part of the signature of cancer stem cells (CSCs) in colon, breast, and in head and neck squamous cell carcinomas (HNSCC). This is somewhat in contradiction to previous reports on the expression of CD44s in HNSCC. The aim of the present study was to clarify the actual pattern of CD44 expression in head and neck epithelia. Methods Expression of CD44s and CD44v6 was analysed by immunohistochemistry with specific antibodies in primary head and neck tissues. Scoring of all specimens followed a two-parameters system, which implemented percentages of positive cells and staining intensities from − to +++ (score = %×intensity; resulting max. score 300). In addition, cell surface expression of CD44s and CD44v6 was assessed in lymphocytes and HNSCC. Results In normal epithelia CD44s and CD44v6 were expressed in 60–95% and 50–80% of cells and yielded mean scores with a standard error of a mean (SEM) of 249.5±14.5 and 198±11.13, respectively. In oral leukoplakia and in moderately differentiated carcinomas CD44s and CD44v6 levels were slightly increased (278.9±7.16 and 242±11.7; 291.8±5.88 and 287.3±6.88). Carcinomas in situ displayed unchanged levels of both proteins whereas poorly differentiated carcinomas consistently expressed diminished CD44s and CD44v6 levels. Lymphocytes and HNSCC lines strongly expressed CD44s but not CD44v6. Conclusion CD44s and CD44v6 expression does not distinguish normal from benign or malignant epithelia of the head and neck. CD44s and CD44v6 were abundantly present in the great majority of cells in head and neck tissues, including carcinomas. Hence, the value of CD44s as a marker for the definition of a small subset of cells (i.e. less than 10%) representing head and neck cancer stem cells may need revision. PMID:18852874

CD44v10, osteopontin and lymphoma growth retardation by a CD44v10-specific antibody.

PubMed

Megaptche, Amelie Pajip; Erb, Ulrike; Büchler, Markus Wolfgang; Zöller, Margot

2014-09-01

Blockade of CD44 is considered a therapeutic option for the elimination of leukemia-initiating cells. However, the application of anti-panCD44 can be burdened by severe side effects. We determined whether these side effects could be avoided by replacing anti-panCD44 with CD44 variant isoform (CD44v)-specific antibodies in CD44v-positive hematological malignancies using the EL4 thymoma and CD44v10-transfected EL4 (EL4-v10) as models. Subcutaneous growth of EL4 and EL4-v10 was equally well inhibited by the anti-panCD44 and anti-CD44v10 antibodies, respectively. Ex vivo analysis indicated that natural killer cytotoxicity and antibody-dependent cellular cytotoxicity were the main effector mechanisms. Under local inflammation, the efficacy of anti-CD44v10 prolonged the survival time twofold compared with untreated, EL4-v10 tumor-bearing mice, and this was due to inflammation-induced expression of osteopontin (OPN). A high level of OPN in EL4-v10 tumors supported leukocyte recruitment and tumor-infiltrating T-cell activation. Taken together, in hematological malignancies expressing CD44v, anti-panCD44 can be replaced by CD44v-specific antibodies without a loss in efficacy. Furthermore, CD44v10-specific antibodies appear particularly advantageous in cutaneous leukemia therapy, as CD44v10 binding of OPN drives leukocyte recruitment and activation.

Cryopreservation of human vascular umbilical cord cells under good manufacturing practice conditions for future cell banks.

PubMed

Polchow, Bianca; Kebbel, Kati; Schmiedeknecht, Gerno; Reichardt, Anne; Henrich, Wolfgang; Hetzer, Roland; Lueders, Cora

2012-05-16

In vitro fabricated tissue engineered vascular constructs could provide an alternative to conventional substitutes. A crucial factor for tissue engineering of vascular constructs is an appropriate cell source. Vascular cells from the human umbilical cord can be directly isolated and cryopreserved until needed. Currently no cell bank for human vascular cells is available. Therefore, the establishment of a future human vascular cell bank conforming to good manufacturing practice (GMP) conditions is desirable for therapeutic applications such as tissue engineered cardiovascular constructs. A fundamental step was the adaption of conventional research and development starting materials to GMP compliant starting materials. Human umbilical cord artery derived cells (HUCAC) and human umbilical vein endothelial cells (HUVEC) were isolated, cultivated, cryopreserved (short- and long-term) directly after primary culture and recultivated subsequently. Cell viability, expression of cellular markers and proliferation potential of fresh and cryopreserved cells were studied using trypan blue staining, flow cytometry analysis, immunofluorescence staining and proliferation assays. Statistical analyses were performed using Student's t-test. Sufficient numbers of isolated cells with acceptable viabilities and homogenous expression of cellular markers confirmed that the isolation procedure was successful using GMP compliant starting materials. The influence of cryopreservation was marginal, because cryopreserved cells mostly maintain phenotypic and functional characteristics similar to those of fresh cells. Phenotypic studies revealed that fresh cultivated and cryopreserved HUCAC were positive for alpha smooth muscle actin, CD90, CD105, CD73, CD29, CD44, CD166 and negative for smoothelin. HUVEC expressed CD31, CD146, CD105 and CD144 but not alpha smooth muscle actin. Functional analysis demonstrated acceptable viability and sufficient proliferation properties of cryopreserved HUCAC and HUVEC. Adaptation of cell isolation, cultivation and cryopreservation to GMP compliant starting materials was successful. Cryopreservation did not influence cell properties with lasting impact, confirming that the application of vascular cells from the human umbilical cord is feasible for cell banking. A specific cellular marker expression profile was established for HUCAC and HUVEC using flow cytometry analysis, applicable as a GMP compliant quality control. Use of these cells for the future fabrication of advanced therapy medicinal products GMP conditions are required by the regulatory authority.

Culture of human mesenchymal stem cells using a candidate pharmaceutical grade xeno-free cell culture supplement derived from industrial human plasma pools.

PubMed

Díez, José M; Bauman, Ewa; Gajardo, Rodrigo; Jorquera, Juan I

2015-03-13

Fetal bovine serum (FBS) is an animal product used as a medium supplement. The animal origin of FBS is a concern if cultured stem cells are to be utilized for human cell therapy. Therefore, a substitute for FBS is desirable. In this study, an industrial, xeno-free, pharmaceutical-grade supplement for cell culture (SCC) under development at Grifols was tested for growth of human mesenchymal stem cells (hMSCs), cell characterization, and differentiation capacity. SCC is a freeze-dried product obtained through cold-ethanol fractionation of industrial human plasma pools from healthy donors. Bone marrow-derived hMSC cell lines were obtained from two commercial suppliers. Cell growth was evaluated by culturing hMSCs with commercial media or media supplemented with SCC or FBS. Cell viability and cell yield were assessed with an automated cell counter. Cell surface markers were studied by indirect immunofluorescence assay. Cells were cultured then differentiated into adipocytes, chondrocytes, osteoblasts, and neurons, as assessed by specific staining and microscopy observation. SCC supported the growth of commercial hMSCs. Starting from the same number of seeded cells in two consecutive passages of culture with medium supplemented with SCC, hMSC yield and cell population doubling time were equivalent to the values obtained with the commercial medium and was consistent among lots. The viability of hMSCs was higher than 90%, while maintaining the characteristic phenotype of undifferentiated hMSCs (positive for CD29, CD44, CD90, CD105, CD146, CD166 and Stro-1; negative for CD14 and CD19). Cultured hMSCs maintained the potential for differentiation into adipocytes, chondrocytes, osteoblasts, and neurons. The tested human plasma-derived SCC sustains the adequate growth of hMSCs, while preserving their differentiation capacity. SCC can be a potential candidate for cell culture supplement in advanced cell therapies.

CD44 expression is related to poor prognosis of hypopharyngeal squamous cell carcinoma.

PubMed

Uwa, Nobuhiro; Kataoka, Tatsuki R; Torii, Ikuko; Sato, Ayuko; Nishigami, Takashi; Song, Misa; Daimon, Takashi; Saeki, Nobuo; Sagawa, Kousuke; Mouri, Takeshi; Terada, Tomonori; Sakagami, Masafumi; Tsujimura, Tohru

2011-03-01

CD44 expression in hypopharyngeal squamous cell carcinomas (SCCs) is closely associated with poor prognosis for patients. CD44 may serve as a prognostic marker for hypopharyngeal SCCs. CD44, an adhesion molecule binding to extracellular matrix, is believed to participate in the progression of malignancies. To clarify the role of CD44 in the progression of hypopharyngeal SCCs, we examined CD44 expression in relation to clinical parameters in hypopharyngeal SCCs. Biopsy specimens of hypopharyngeal SCCs were collected from 40 untreated patients, and their CD44 expression was examined immunohistochemically. Hypopharyngeal SCCs were classified into two groups: CD44-low SCCs comprising < 50% CD44-positive tumor cells and CD44-high SCCs comprising ≥ 50% CD44-positive tumor cells. The relation between CD44 expression and various parameters (clinical T and N stages, distant metastasis, and pathological T and N stages) was analyzed by Fisher's exact test. The relation between CD44 expression and the 5-year disease-free survival (DFS) rate was also analyzed by log rank test. The CD44 expression in hypopharyngeal SCCs was related to pathological N stage, but not to clinical T and N stages and pathological T stage, of the patients. Distant metastasis during the follow-up occurred more frequently in patients with CD44-high SCCs than those with CD44-low SCCs. The 5-year DFS was significantly lower in the former than in the latter.

Non-Small Cell Lung Cancer Cells Expressing CD44 Are Enriched for Stem Cell-Like Properties

PubMed Central

Leung, Elaine Lai-Han; Fiscus, Ronald R.; Tung, James W.; Tin, Vicky Pui-Chi; Cheng, Lik Cheung; Sihoe, Alan Dart-Loon; Fink, Louis M.; Ma, Yupo; Wong, Maria Pik

2010-01-01

Background The cancer stem cell theory hypothesizes that cancers are perpetuated by cancer stem cells (CSC) or tumor initiating cells (TIC) possessing self-renewal and other stem cell-like properties while differentiated non-stem/initiating cells have a finite life span. To investigate whether the hypothesis is applicable to lung cancer, identification of lung CSC and demonstration of these capacities is essential. Methodology/Principal Finding The expression profiles of five stem cell markers (CD34, CD44, CD133, BMI1 and OCT4) were screened by flow cytometry in 10 lung cancer cell lines. CD44 was further investigated by testing for in vitro and in vivo tumorigenecity. Formation of spheroid bodies and in vivo tumor initiation ability were demonstrated in CD44+ cells of 4 cell lines. Serial in vivo tumor transplantability in nude mice was demonstrated using H1299 cell line. The primary xenografts initiated from CD44+ cells consisted of mixed CD44+ and CD44− cells in similar ratio as the parental H1299 cell line, supporting in vivo differentiation. Semi-quantitative Real-Time PCR (RT-PCR) showed that both freshly sorted CD44+ and CD44+ cells derived from CD44+-initiated tumors expressed the pluripotency genes OCT4/POU5F1, NANOG, SOX2. These stemness markers were not expressed by CD44− cells. Furthermore, freshly sorted CD44+ cells were more resistant to cisplatin treatment with lower apoptosis levels than CD44− cells. Immunohistochemical analysis of 141 resected non-small cell lung cancers showed tumor cell expression of CD44 in 50.4% of tumors while no CD34, and CD133 expression was observed in tumor cells. CD44 expression was associated with squamous cell carcinoma but unexpectedly, a longer survival was observed in CD44-expressing adenocarcinomas. Conclusion/Significance Overall, our results demonstrated that stem cell-like properties are enriched in CD44-expressing subpopulations of some lung cancer cell lines. Further investigation is required to clarify the role of CD44 in tumor cell renewal and cancer propagation in the in vivo environment. PMID:21124918

Nanotechniques Inactivate Cancer Stem Cells

NASA Astrophysics Data System (ADS)

Goltsev, Anatoliy N.; Babenko, Natalya N.; Gaevskaya, Yulia A.; Bondarovich, Nikolay A.; Dubrava, Tatiana G.; Ostankov, Maksim V.; Chelombitko, Olga V.; Malyukin, Yuriy V.; Klochkov, Vladimir K.; Kavok, Nataliya S.

2017-06-01

One of the tasks of current oncology is identification of cancer stem cells and search of therapeutic means capable of their specific inhibition. The paper presents the data on phenotype characteristics of Ehrlich carcinoma cells as convenient and easy-to-follow model of tumor growth. The evidence of cancer stem cells as a part of Ehrlich carcinoma and significance of CD44+ and CD44- subpopulations in maintaining the growth of this type of tumor were demonstrated. A high (tenfold) tumorigenic activity of the Ehrlich carcinoma CD44+ cells if compared to CD44- cells was proven. In this pair of comparison, the CD44+ cells had a higher potential of generating in peritoneal cavity of CD44high, CD44+CD24-, CD44+CD24+ cell subpopulations, highlighting the presence of cancer stem cells in a pool of CD44+ cells.

Phosphatidylinositol 4,5-Bisphosphate Clusters the Cell Adhesion Molecule CD44 and Assembles a Specific CD44-Ezrin Heterocomplex, as Revealed by Small Angle Neutron Scattering

DOE PAGES

Khajeh, Jahan Ali; Ju, Jeong Ho; Gupta, Yogesh K.; ...

2015-01-08

The cell adhesion molecule CD44 regulates diverse cellular functions, including cell-cell and cell-matrix interaction, cell motility, migration, differentiation, and growth. In cells, CD44 co-localizes with the membrane-cytoskeleton adapter protein Ezrin, which links the CD44 assembled receptor signaling complexes to the cytoskeletal actin and organizes the spatial and temporal localization of signaling events. Here we report that the cytoplasmic tail of CD44 (CD44ct) is largely disordered and adopts an autoinhibited conformation, which prevents CD44ct from binding directly to activated Ezrin in solution. Binding to the signaling lipid phosphatidylinositol 4,5-biphosphlate (PIP2) disrupts autoinhibition in CD44ct, and activates CD44ct to associate with Ezrin.more » Further, using contrast variation small angle neutron scattering, we show that PIP2 mediates the assembly of a specific hetero-tetramer complex of CD44ct with Ezrin. This study reveals a novel autoregulation mechanism in the cytoplasmic tail of CD44 and the role of PIP2 in mediating the assembly of multimeric CD44ct-Ezrin complexes. We hypothesize that polyvalent electrostatic interactions are responsible for the assembly of multimeric PIP2-CD44-Ezrin complexes.« less

The Selective Centrifugation Ensures a Better In Vitro Isolation of ASCs and Restores a Soft Tissue Regeneration In Vivo.

PubMed

De Francesco, Francesco; Guastafierro, Antonio; Nicoletti, Gianfranco; Razzano, Sergio; Riccio, Michele; Ferraro, Giuseppe A

2017-05-12

Autologous fat grafting procedures in plastic surgery have been extensively used to reinforce soft tissue in congenital or acquired tissue impairments. With this background, the aim of this study is firstly to examine the impact of a selective centrifugation on existing adipose stem cells (ASCs) in terms of stemness profile maintenance and, secondly, to investigate the effect of restoring volume in reconstruction on patients affected by soft tissue damage. After centrifugation, the fat graft products were separated into two layers and subsequently examined in vitro for the expression of CD34, CD90, CD117, CD105, CD29, CD31, CD44, CD73, CD133, CD14 and CD45 markers by flow cytometry and gene expression analyses were performed for Sox2, WNT3A, END, CD44, FUT4, COLL1, CTNNB1, hbEGF, KRTLG, MMP2 and VIM genes. The results showed that in the middle-high density (MHD) layer there was a peak concentration of ASCs, compared to another layer obtained after centrifugation. Research carried out on patients under treatment for soft tissue regeneration using cells obtained from MHD layer selection will be fundamental in comparative analysis. These studies will lead to an adequate standardization of outcomes, provided that treatment is performed through cell selection. Therefore, a unique procedure in tissue reconstruction and regeneration through fat grafting is presented here.

CD44v6 Regulates Growth of Brain Tumor Stem Cells Partially through the AKT-Mediated Pathway

PubMed Central

Jijiwa, Mayumi; Demir, Habibe; Gupta, Snehalata; Leung, Crystal; Joshi, Kaushal; Orozco, Nicholas; Huang, Tiffany; Yildiz, Vedat O.; Shibahara, Ichiyo; de Jesus, Jason A.; Yong, William H.; Mischel, Paul S.; Fernandez, Soledad; Kornblum, Harley I.; Nakano, Ichiro

2011-01-01

Identification of stem cell-like brain tumor cells (brain tumor stem-like cells; BTSC) has gained substantial attention by scientists and physicians. However, the mechanism of tumor initiation and proliferation is still poorly understood. CD44 is a cell surface protein linked to tumorigenesis in various cancers. In particular, one of its variant isoforms, CD44v6, is associated with several cancer types. To date its expression and function in BTSC is yet to be identified. Here, we demonstrate the presence and function of the variant form 6 of CD44 (CD44v6) in BTSC of a subset of glioblastoma multiforme (GBM). Patients with CD44high GBM exhibited significantly poorer prognoses. Among various variant forms, CD44v6 was the only isoform that was detected in BTSC and its knockdown inhibited in vitro growth of BTSC from CD44high GBM but not from CD44low GBM. In contrast, this siRNA-mediated growth inhibition was not apparent in the matched GBM sample that does not possess stem-like properties. Stimulation with a CD44v6 ligand, osteopontin (OPN), increased expression of phosphorylated AKT in CD44high GBM, but not in CD44low GBM. Lastly, in a mouse spontaneous intracranial tumor model, CD44v6 was abundantly expressed by tumor precursors, in contrast to no detectable CD44v6 expression in normal neural precursors. Furthermore, overexpression of mouse CD44v6 or OPN, but not its dominant negative form, resulted in enhanced growth of the mouse tumor stem-like cells in vitro. Collectively, these data indicate that a subset of GBM expresses high CD44 in BTSC, and its growth may depend on CD44v6/AKTpathway. PMID:21915300

PDGF Suppresses the Sulfation of CD44v and Potentiates CD44v-Mediated Binding of Colon Carcinoma Cells to Fibrin under Flow

PubMed Central

Alves, Christina S.; Konstantopoulos, Konstantinos

2012-01-01

Fibrin(ogen) mediates sustained tumor cell adhesion and survival in the pulmonary vasculature, thereby facilitating the metastatic dissemination of tumor cells. CD44 is the major functional fibrin receptor on colon carcinoma cells. Growth factors, such as platelet-derived growth factor (PDGF), induce post-translational protein modifications, which modulate ligand binding activity. In view of the roles of PDGF, fibrin(ogen) and CD44 in cancer metastasis, we aimed to delineate the effect of PDGF on CD44-fibrin recognition. By immunoprecipitating CD44 from PDGF-treated and untreated LS174T colon carcinoma cells, which express primarily CD44v, we demonstrate that PDGF enhances the adhesion of CD44v-coated beads to immobilized fibrin. Enzymatic inhibition studies coupled with flow-based adhesion assays and autoradiography reveal that PDGF augments the binding of CD44v to fibrin by significantly attenuating the extent of CD44 sulfation primarily on chondroitin and dermatan sulfate chains. Surface plasmon resonance assays confirm that PDGF enhances the affinity of CD44v-fibrin binding by markedly reducing its dissociation rate while modestly increasing the association rate. PDGF mildly reduces the affinity of CD44v-hyaluronan binding without affecting selectin-CD44v recognition. The latter is attributed to the fact that CD44v binds to selectins via sialofucosylated O-linked residues independent of heparan, dermatan and chondroitin sulfates. Interestingly, PDGF moderately reduces the sulfation of CD44s and CD44s-fibrin recognition. Collectively, these data offer a novel perspective into the mechanism by which PGDF regulates CD44-dependent binding of metastatic colon carcinoma cells to fibrin(ogen). PMID:23056168

Intracellular Domain Fragment of CD44 Alters CD44 Function in Chondrocytes*

PubMed Central

Mellor, Liliana; Knudson, Cheryl B.; Hida, Daisuke; Askew, Emily B.; Knudson, Warren

2013-01-01

The hyaluronan receptor CD44 undergoes sequential proteolytic cleavage at the cell surface. The initial cleavage of the CD44 extracellular domain is followed by a second intramembranous cleavage of the residual CD44 fragment, liberating the C-terminal cytoplasmic tail of CD44. In this study conditions that promote CD44 cleavage resulted in a diminished capacity to assemble and retain pericellular matrices even though sufficient non-degraded full-length CD44 remained. Using stable and transient overexpression of the cytoplasmic domain of CD44, we determined that the intracellular domain interfered with anchoring of the full-length CD44 to the cytoskeleton and disrupted the ability of the cells to bind hyaluronan and assemble a pericellular matrix. Co-immunoprecipitation assays were used to determine whether the mechanism of this interference was due to competition with actin adaptor proteins. CD44 of control chondrocytes was found to interact and co-immunoprecipitate with both the 65- and 130-kDa isoforms of ankyrin-3. Moreover, this interaction with ankyrin-3 proteins was diminished in cells overexpressing the CD44 intracellular domain. Mutating the putative ankyrin binding site of the transiently transfected CD44 intracellular domain diminished the inhibitory effects of this protein on matrix retention. Although CD44 in other cells types has been shown to interact with members of the ezrin/radixin/moesin (ERM) family of adaptor proteins, only modest interactions between CD44 and moesin could be demonstrated in chondrocytes. The data suggest that release of the CD44 intracellular domain into the cytoplasm of cells such as chondrocytes exerts a competitive or dominant-negative effect on the function of full-length CD44. PMID:23884413

Expression of CD44s and CD44v6 in transitional cell carcinomas of the urinary bladder: comparison with tumour grade, proliferative activity and p53 immunoreactivity of tumour cells.

PubMed

Kuncová, Jitka; Urban, Michael; Mandys, Václav

2007-11-01

Alterations of CD44 glycoproteins have been shown to play an important role in progression of various malignancies, including urothelial cancer. We investigated expression patterns of CD44s and CD44v6 in transitional cell carcinoma (TCC) of the urinary bladder in relation to tumour grade, proliferative activity, and immunoreactivity for p53. The selected markers were detected immunohistochemically in 122 samples of TCC. We found a close relationship between CD44s and CD44v6 expression and tumour grade. The extension of positive staining for CD44s and CD44v6 towards the luminal surface was a predominant feature of differentiated carcinomas (grades 1 and 2), suggesting deranged maturation of cancer cells related to their neoplastic transformation. Heterogeneous expression of CD44s and CD44v6 predominated in poorly differentiated tumours (G3-4). However, areas of squamous differentiation within the high-grade tumours displayed strong immunoreactivity for both CD44s and CD44v6. The proliferative activity and p53 overexpression increased with the dedifferentiation of the tumour. The results of this study are discussed in relation to the significance of CD44 expression in TCC and to the explanation for controversial results reported in previous studies on the relationship between CD44 expression and the biological behaviour of urothelial cells.

Isolation, in vitro culture and identification of a new type of mesenchymal stem cell derived from fetal bovine lung tissues.

PubMed

Hu, Pengfei; Pu, Yabin; Li, Xiayun; Zhu, Zhiqiang; Zhao, Yuhua; Guan, Weijun; Ma, Yuehui

2015-09-01

Lung‑derived mesenchymal stem cells (LMSCs) are considered to be important in lung tissue repair and regenerative processes. However, the biological characteristics and differentiation potential of LMSCs remain to be elucidated. In the present study, fetal lung‑derived mesenchymal stem cells (FLMSCs) were isolated from fetal bovine lung tissues by collagenase digestion. The in vitro culture conditions were optimized and stabilized and the self‑renewal ability and differentiation potential were evaluated. The results demonstrated that the FLMSCs were morphologically consistent with fibroblasts, were able to be cultured and passaged for at least 33 passages and the cell morphology and proliferative ability were stable during the first 10 passages. In addition, FLMSCs were found to express CD29, CD44, CD73 and CD166, however, they did not express hematopoietic cell specific markers, including CD34, CD45 and BOLA‑DRα. The growth kinetics of FLMSCs consisted of a lag phase, a logarithmic phase and a plateau phase, and as the passages increased, the proliferative ability of cells gradually decreased. The majority of FLMSCs were in G0/G1 phase. Following osteogenic induction, FLMSCs were positive for the expression of osteopontin and collagen type I α2. Following neurogenic differentiation, the cells were morphologically consistent with neuronal cells and positive for microtubule‑associated protein 2 and nestin expression. It was concluded that the isolated FLMSCs exhibited typical characteristics of mesenchymal stem cells and that the culture conditions were suitable for their proliferation and the maintenance of stemness. The present study illustrated the potential application of lung tissue as an adult stem cell source for regenerative therapies.

Phosphatidylinositol 4,5-Bisphosphate Clusters the Cell Adhesion Molecule CD44 and Assembles a Specific CD44-Ezrin Heterocomplex, as Revealed by Small Angle Neutron Scattering*

PubMed Central

Chen, Xiaodong; Khajeh, Jahan Ali; Ju, Jeong Ho; Gupta, Yogesh K.; Stanley, Christopher B.; Do, Changwoo; Heller, William T.; Aggarwal, Aneel K.; Callaway, David J. E.; Bu, Zimei

2015-01-01

The cell adhesion molecule CD44 regulates diverse cellular functions, including cell-cell and cell-matrix interaction, cell motility, migration, differentiation, and growth. In cells, CD44 co-localizes with the membrane-cytoskeleton adapter protein Ezrin that links the CD44 assembled receptor signaling complexes to the cytoskeletal actin network, which organizes the spatial and temporal localization of signaling events. Here we report that the cytoplasmic tail of CD44 (CD44ct) is largely disordered. Upon binding to the signaling lipid phosphatidylinositol 4,5-bisphosphate (PIP2), CD44ct clusters into aggregates. Further, contrary to the generally accepted model, CD44ct does not bind directly to the FERM domain of Ezrin or to the full-length Ezrin but only forms a complex with FERM or with the full-length Ezrin in the presence of PIP2. Using contrast variation small angle neutron scattering, we show that PIP2 mediates the assembly of a specific heterotetramer complex of CD44ct with Ezrin. This study reveals the role of PIP2 in clustering CD44 and in assembling multimeric CD44-Ezrin complexes. We hypothesize that polyvalent electrostatic interactions are responsible for the assembly of CD44 clusters and the multimeric PIP2-CD44-Ezrin complexes. PMID:25572402

Phosphatidylinositol 4,5-bisphosphate clusters the cell adhesion molecule CD44 and assembles a specific CD44-Ezrin heterocomplex, as revealed by small angle neutron scattering.

PubMed

Chen, Xiaodong; Khajeh, Jahan Ali; Ju, Jeong Ho; Gupta, Yogesh K; Stanley, Christopher B; Do, Changwoo; Heller, William T; Aggarwal, Aneel K; Callaway, David J E; Bu, Zimei

2015-03-06

The cell adhesion molecule CD44 regulates diverse cellular functions, including cell-cell and cell-matrix interaction, cell motility, migration, differentiation, and growth. In cells, CD44 co-localizes with the membrane-cytoskeleton adapter protein Ezrin that links the CD44 assembled receptor signaling complexes to the cytoskeletal actin network, which organizes the spatial and temporal localization of signaling events. Here we report that the cytoplasmic tail of CD44 (CD44ct) is largely disordered. Upon binding to the signaling lipid phosphatidylinositol 4,5-bisphosphate (PIP2), CD44ct clusters into aggregates. Further, contrary to the generally accepted model, CD44ct does not bind directly to the FERM domain of Ezrin or to the full-length Ezrin but only forms a complex with FERM or with the full-length Ezrin in the presence of PIP2. Using contrast variation small angle neutron scattering, we show that PIP2 mediates the assembly of a specific heterotetramer complex of CD44ct with Ezrin. This study reveals the role of PIP2 in clustering CD44 and in assembling multimeric CD44-Ezrin complexes. We hypothesize that polyvalent electrostatic interactions are responsible for the assembly of CD44 clusters and the multimeric PIP2-CD44-Ezrin complexes. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Triple negative breast cancer initiating cell subsets differ in functional and molecular characteristics and in γ-secretase inhibitor drug responses

PubMed Central

Azzam, Diana J; Zhao, Dekuang; Sun, Jun; Minn, Andy J; Ranganathan, Prathibha; Drews-Elger, Katherine; Han, Xiaoqing; Picon-Ruiz, Manuel; Gilbert, Candace A; Wander, Seth A; Capobianco, Anthony J; El-Ashry, Dorraya; Slingerland, Joyce M

2013-01-01

Increasing evidence suggests that stem-like cells mediate cancer therapy resistance and metastasis. Breast tumour-initiating stem cells (T-ISC) are known to be enriched in CD44+CD24neg/low cells. Here, we identify two T-ISC subsets within this population in triple negative breast cancer (TNBC) lines and dissociated primary breast cancer cultures: CD44+CD24low+ subpopulation generates CD44+CD24neg progeny with reduced sphere formation and tumourigenicity. CD44+CD24low+ populations contain subsets of ALDH1+ and ESA+ cells, yield more frequent spheres and/or T-ISC in limiting dilution assays, preferentially express metastatic gene signatures and show greater motility, invasion and, in the MDA-MB-231 model, metastatic potential. CD44+CD24low+ but not CD44+CD24neg express activated Notch1 intracellular domain (N1-ICD) and Notch target genes. We show N1-ICD transactivates SOX2 to increase sphere formation, ALDH1+ and CD44+CD24low+cells. Gamma secretase inhibitors (GSI) reduced sphere formation and xenograft growth from CD44+CD24low+ cells, but CD44+CD24neg were resistant. While GSI hold promise for targeting T-ISC, stem cell heterogeneity as observed herein, could limit GSI efficacy. These data suggest a breast T-ISC hierarchy in which distinct pathways drive developmentally related subpopulations with different anti-cancer drug responsiveness. PMID:23982961

Role of CD44 in lymphokine-activated killer cell-mediated killing of melanoma.

PubMed

Sun, Jingping; Law, Gabriela P; McKallip, Robert J

2012-03-01

In the current study, we examined the potential significance of CD44 expression on lymphokine-activated killer (LAK) cells in their interaction and killing of melanoma cells. Stimulation of splenocytes with IL-2 led to a significant increase in the expression of CD44 on T cells, NK cells, and NKT cells. Treatment of melanoma-bearing CD44 WT mice with IL-2 led to a significant reduction in the local tumor growth while treatment of melanoma-bearing CD44 KO mice with IL-2 was ineffective at controlling tumor growth. Furthermore, the ability of splenocytes from IL-2-treated CD44 KO mice to kill melanoma tumor targets was significantly reduced when compared to the anti-tumor activity of splenocytes from IL-2-treated CD44 WT mice. The importance of CD44 expression on the LAK cells was further confirmed by the observation that adoptively transferred CD44 WT LAK cells were significantly more effective than CD44 KO LAK cells at controlling tumor growth in vivo. Next, the significance of the increased expression of CD44 in tumor killing was examined and showed that following stimulation with IL-2, distinct populations of cells with low (CD44(lo)) or elevated (CD44(hi)) expression of CD44 are generated and that the CD44(hi) cells are responsible for killing of the melanoma cells. The reduced killing activity of the CD44 KO LAK cells did not result from reduced activation or expression of effector molecules but was due, at least in part, to a reduced ability to adhere to B16F10 tumor cells.

Cytoskeletal regulation of CD44 membrane organization and interactions with E-selectin.

PubMed

Wang, Ying; Yago, Tadayuki; Zhang, Nan; Abdisalaam, Salim; Alexandrakis, George; Rodgers, William; McEver, Rodger P

2014-12-19

Interactions of CD44 on neutrophils with E-selectin on activated endothelial cells mediate rolling under flow, a prerequisite for neutrophil arrest and migration into perivascular tissues. How CD44 functions as a rolling ligand despite its weak affinity for E-selectin is unknown. We examined the nanometer scale organization of CD44 on intact cells. CD44 on leukocytes and transfected K562 cells was cross-linked within a 1.14-nm spacer. Depolymerizing actin with latrunculin B reduced cross-linking. Fluorescence resonance energy transfer (FRET) revealed tight co-clustering between CD44 fused to yellow fluorescent protein (YFP) and CD44 fused to cyan fluorescent protein on K562 cells. Latrunculin B reduced FRET-reported co-clustering. Number and brightness analysis confirmed actin-dependent CD44-YFP clusters on living cells. CD44 lacking binding sites for ankyrin and for ezrin/radixin/moesin (ERM) proteins on its cytoplasmic domain (ΔANKΔERM) did not cluster. Unexpectedly, CD44 lacking only the ankyrin-binding site (ΔANK) formed larger but looser clusters. Fluorescence recovery after photobleaching demonstrated increased CD44 mobility by latrunculin B treatment or by deleting the cytoplasmic domain. ΔANKΔERM mobility increased only modestly, suggesting that the cytoplasmic domain engages the cytoskeleton by an additional mechanism. Ex vivo differentiated CD44-deficient neutrophils expressing exogenous CD44 rolled on E-selectin and activated Src kinases after binding anti-CD44 antibody. In contrast, differentiated neutrophils expressing ΔANK had impaired rolling and kinase activation. These data demonstrate that spectrin and actin networks regulate CD44 clustering and suggest that ankyrin enhances CD44-mediated neutrophil rolling and signaling. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Cytoskeletal Regulation of CD44 Membrane Organization and Interactions with E-selectin*

PubMed Central

Wang, Ying; Yago, Tadayuki; Zhang, Nan; Abdisalaam, Salim; Alexandrakis, George; Rodgers, William; McEver, Rodger P.

2014-01-01

Interactions of CD44 on neutrophils with E-selectin on activated endothelial cells mediate rolling under flow, a prerequisite for neutrophil arrest and migration into perivascular tissues. How CD44 functions as a rolling ligand despite its weak affinity for E-selectin is unknown. We examined the nanometer scale organization of CD44 on intact cells. CD44 on leukocytes and transfected K562 cells was cross-linked within a 1.14-nm spacer. Depolymerizing actin with latrunculin B reduced cross-linking. Fluorescence resonance energy transfer (FRET) revealed tight co-clustering between CD44 fused to yellow fluorescent protein (YFP) and CD44 fused to cyan fluorescent protein on K562 cells. Latrunculin B reduced FRET-reported co-clustering. Number and brightness analysis confirmed actin-dependent CD44-YFP clusters on living cells. CD44 lacking binding sites for ankyrin and for ezrin/radixin/moesin (ERM) proteins on its cytoplasmic domain (ΔANKΔERM) did not cluster. Unexpectedly, CD44 lacking only the ankyrin-binding site (ΔANK) formed larger but looser clusters. Fluorescence recovery after photobleaching demonstrated increased CD44 mobility by latrunculin B treatment or by deleting the cytoplasmic domain. ΔANKΔERM mobility increased only modestly, suggesting that the cytoplasmic domain engages the cytoskeleton by an additional mechanism. Ex vivo differentiated CD44-deficient neutrophils expressing exogenous CD44 rolled on E-selectin and activated Src kinases after binding anti-CD44 antibody. In contrast, differentiated neutrophils expressing ΔANK had impaired rolling and kinase activation. These data demonstrate that spectrin and actin networks regulate CD44 clustering and suggest that ankyrin enhances CD44-mediated neutrophil rolling and signaling. PMID:25359776

CD82 suppresses CD44 alternative splicing-dependent melanoma metastasis by mediating U2AF2 ubiquitination and degradation

PubMed Central

Fu, Ailing; Zhu, Huifeng; Ren, Qiao; Wang, Bochu; Xu, Xingran; Bai, Huiyuan; Dong, Cheng

2016-01-01

Melanoma is one of the most lethal forms of skin cancer due to its early metastatic spread. The variant form of CD44 (CD44v), a cell surface glycoprotein, is highly expressed on metastatic melanoma. The mechanisms of regulation of CD44 alternative splicing in melanoma and its pathogenic contributions are so far poorly understood. Here, we investigated the expression level of CD44 in a large set of melanocytic lesions at different stages. We found that the expression of CD44v8-10 and a splicing factor, U2AF2, is significantly increased during melanoma progression, while CD82/KAI1, a tetraspanin family of tumor suppressor, is reduced in metastatic melanoma. CD44v8-10 and U2AF2 expressions which are negatively correlated with CD82 levels are dramatically elevated in primary melanoma compared with dysplastic nevi and further increased in metastatic melanoma. We also showed that patients with higher CD44v8-10 and U2AF2 expression levels tended to have shorter survival. By using both in vivo and in vitro assays, we demonstrated that CD82 inhibits the production of CD44v8-10 on melanoma. Mechanistically, U2AF2 is a downstream target of CD82 and in malignant melanoma facilitates CD44v8-10 alternative splicing. U2AF2-mediated CD44 isoform switch is required for melanoma migration in vitro and lung and liver metastasis in vivo. Notably, overexpression of CD82 suppresses U2AF2 activity by inducing U2AF2 ubiquitination. In addition, our data suggested that enhancement of melanoma migration by U2AF2-dependent CD44v8-10 splicing is mediated by Src/FAK/RhoA activation and formation of stress fibers as well as CD44-E-selectin binding reinforcement. These findings uncovered a hitherto unappreciated function of CD82 in severing the linkage between U2AF2-mediated CD44 alternative splicing and cancer aggressiveness, with potential prognostic and therapeutic implications in melanoma. PMID:27041584

Lipid Raft Association Restricts CD44-Ezrin Interaction and Promotion of Breast Cancer Cell Migration

PubMed Central

Donatello, Simona; Babina, Irina S.; Hazelwood, Lee D.; Hill, Arnold D.K.; Nabi, Ivan R.; Hopkins, Ann M.

2012-01-01

Cancer cell migration is an early event in metastasis, the main cause of breast cancer-related deaths. Cholesterol-enriched membrane domains called lipid rafts influence the function of many molecules, including the raft-associated protein CD44. We describe a novel mechanism whereby rafts regulate interactions between CD44 and its binding partner ezrin in migrating breast cancer cells. Specifically, in nonmigrating cells, CD44 and ezrin localized to different membranous compartments: CD44 predominantly in rafts, and ezrin in nonraft compartments. After the induction of migration (either nonspecific or CD44-driven), CD44 affiliation with lipid rafts was decreased. This was accompanied by increased coprecipitation of CD44 and active (threonine-phosphorylated) ezrin-radixin-moesin (ERM) proteins in nonraft compartments and increased colocalization of CD44 with the nonraft protein, transferrin receptor. Pharmacological raft disruption using methyl-β-cyclodextrin also increased CD44-ezrin coprecipitation and colocalization, further suggesting that CD44 interacts with ezrin outside rafts during migration. Conversely, promoting CD44 retention inside lipid rafts by pharmacological inhibition of depalmitoylation virtually abolished CD44-ezrin interactions. However, transient single or double knockdown of flotillin-1 or caveolin-1 was not sufficient to increase cell migration over a short time course, suggesting complex crosstalk mechanisms. We propose a new model for CD44-dependent breast cancer cell migration, where CD44 must relocalize outside lipid rafts to drive cell migration. This could have implications for rafts as pharmacological targets to down-regulate cancer cell migration. PMID:23031255

A rational approach for cancer stem-like cell isolation and characterization using CD44 and prominin-1(CD133) as selection markers

PubMed Central

Lee, Yi-Jen; Wu, Chang-Cheng; Li, Jhy-Wei; Ou, Chien-Chih; Hsu, Shih-Chung; Tseng, Hsiu-Hsueh; Kao, Ming-Ching; Liu, Jah-Yao

2016-01-01

The availability of adequate cancer stem cells or cancer stem-like cell (CSC) is important in cancer study. From ovarian cancer cell lines, SKOV3 and OVCAR3, we induced peritoneal ascites tumors in immunodeficient mice. Among the cells (SKOV3.PX1 and OVCAR3.PX1) from those tumors, we sorted both CD44 and CD133 positive cells (SKOV3.PX1_133+44+, OVCAR3.PX1_133+44+), which manifest the characteristics of self-renewal, multi-lineage differentiation, chemoresistance and tumorigenicity, those of cancer stem-like cells (CSLC). Intraperitoneal transplantation of these CD44 and CD133 positive cells resulted in poorer survival in the engrafted animals. Clinically, increased CD133 expression was found in moderately and poorly differentiated (grade II and III) ovarian serous cystadenocarcinomas. The ascites tumor cells from human ovarian cancers demonstrated more CD133 and CD44 expressions than those from primary ovarian or metastatic tumors and confer tumorigenicity in immunodeficient mice. Compared to their parental cells, the SKOV3.PX1_133+44+ and OVCAR3.PX1_133+44+ cells uniquely expressed 5 CD markers (CD97, CD104, CD107a, CD121a, and CD125). Among these markers, CD97, CD104, CD107a, and CD121a are significantly more expressed in the CD133+ and CD44+ double positive cells of human ovarian ascites tumor cells (Ascites_133+44+) than those from primary ovarian or metastatic tumors. The cancer stem-like cells were enriched from 3% to more than 70% after this manipulation. This intraperitoneal enrichment of cancer stem-like cells, from ovarian cancer cell lines or primary ovarian tumor, potentially provides an adequate amount of ovarian cancer stem-like cells for the ovarian cancer study and possibly benefits cancer therapy. PMID:27655682

Activation of bone marrow-derived mesenchymal stromal cells-a new mechanism of defocused low-energy shock wave in regenerative medicine.

PubMed

Zhao, Yong; Wang, Jinbang; Wang, Muwen; Sun, Peng; Chen, Ji; Jin, Xunbo; Zhang, Haiyang

2013-12-01

Defocused low-energy shock wave (DLSW) therapy has shown effectiveness in regenerative medicine. The mechanism of action was mainly focused on the pathophysiological improvement at the wound tissues. In this study, the activation of stem cells treated by DLSW was first examined as an important pathway during the healing process. Cultured rat bone marrow-derived mesenchymal stromal cells (BMSC) were treated by DLSW before each passage. The untreated BMSC served as a control. The secretions of vascular endothelial growth factor (VEGF) and CXC ligand 5 (CXCL5) were tested by means of enzyme-linked immunoassay. Flow cytometry was performed to analyze the BMSC (passage 4) surface antigen expressions (CD166, CD44 and CD34). The expressions of proliferating cell nuclear antigen and Ki67 were analyzed by means of Western blot. The healing abilities of conditioned media of shocked and unshocked BMSC were examined by Matrigel-based capillary-like tube formation assay and rat major pelvic ganglia culture test. The shocked BMSC secreted more VEGF and CXCL5 than did those of unshocked BMSC. The expressions of CD166, CD44 and CD34 showed no significant differences (P > 0.05) between the shocked and unshocked BMSC. The shocked BMSC demonstrated higher expressions of proliferating cell nuclear antigen (P < 0.01) and Ki67 (P < 0.01) than did those of unshocked BMSC. The shocked BMSC conditioned medium showed higher ability to enhance the growth of major pelvic ganglia neurites (P < 0.05) and Matrigel-based endothelial tube-like formation (P < 0.05). DLSW did not interfere with the expressions of cell surface markers. DLSW enhanced the secretion and proliferation of BMSC and promoted angiogenesis and nerve regeneration in vitro. Copyright © 2013 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Identification and Characterization of Cells with Cancer Stem Cell Properties in Human Primary Lung Cancer Cell Lines

PubMed Central

Suo, Zhenhe; Munthe, Else; Solberg, Steinar; Ma, Liwei; Wang, Mengyu; Westerdaal, Nomdo Anton Christiaan; Kvalheim, Gunnar; Gaudernack, Gustav

2013-01-01

Lung cancer (LC) with its different subtypes is generally known as a therapy resistant cancer with the highest morbidity rate worldwide. Therapy resistance of a tumor is thought to be related to cancer stem cells (CSCs) within the tumors. There have been indications that the lung cancer is propagated and maintained by a small population of CSCs. To study this question we established a panel of 15 primary lung cancer cell lines (PLCCLs) from 20 fresh primary tumors using a robust serum-free culture system. We subsequently focused on identification of lung CSCs by studying these cell lines derived from 4 representative lung cancer subtypes such as small cell lung cancer (SCLC), large cell carcinoma (LCC), squamous cell carcinoma (SCC) and adenocarcinoma (AC). We identified a small population of cells strongly positive for CD44 (CD44high) and a main population which was either weakly positive or negative for CD44 (CD44low/−). Co-expression of CD90 further narrowed down the putative stem cell population in PLCCLs from SCLC and LCC as spheroid-forming cells were mainly found within the CD44highCD90+ sub-population. Moreover, these CD44highCD90+ cells revealed mesenchymal morphology, increased expression of mesenchymal markers N-Cadherin and Vimentin, increased mRNA levels of the embryonic stem cell related genes Nanog and Oct4 and increased resistance to irradiation compared to other sub-populations studied, suggesting the CD44highCD90+ population a good candidate for the lung CSCs. Both CD44highCD90+ and CD44highCD90− cells in the PLCCL derived from SCC formed spheroids, whereas the CD44low/− cells were lacking this potential. These results indicate that CD44highCD90+ sub-population may represent CSCs in SCLC and LCC, whereas in SCC lung cancer subtype, CSC potentials were found within the CD44high sub-population. PMID:23469181

The Selective Centrifugation Ensures a Better In Vitro Isolation of ASCs and Restores a Soft Tissue Regeneration In Vivo

PubMed Central

De Francesco, Francesco; Guastafierro, Antonio; Nicoletti, Gianfranco; Razzano, Sergio; Riccio, Michele; Ferraro, Giuseppe A.

2017-01-01

Autologous fat grafting procedures in plastic surgery have been extensively used to reinforce soft tissue in congenital or acquired tissue impairments. With this background, the aim of this study is firstly to examine the impact of a selective centrifugation on existing adipose stem cells (ASCs) in terms of stemness profile maintenance and, secondly, to investigate the effect of restoring volume in reconstruction on patients affected by soft tissue damage. After centrifugation, the fat graft products were separated into two layers and subsequently examined in vitro for the expression of CD34, CD90, CD117, CD105, CD29, CD31, CD44, CD73, CD133, CD14 and CD45 markers by flow cytometry and gene expression analyses were performed for Sox2, WNT3A, END, CD44, FUT4, COLL1, CTNNB1, hbEGF, KRTLG, MMP2 and VIM genes. The results showed that in the middle-high density (MHD) layer there was a peak concentration of ASCs, compared to another layer obtained after centrifugation. Research carried out on patients under treatment for soft tissue regeneration using cells obtained from MHD layer selection will be fundamental in comparative analysis. These studies will lead to an adequate standardization of outcomes, provided that treatment is performed through cell selection. Therefore, a unique procedure in tissue reconstruction and regeneration through fat grafting is presented here. PMID:28498335

Inhibition of CD147 expression by RNA interference reduces proliferation, invasion and increases chemosensitivity in cancer stem cell-like HT-29 cells.

PubMed

Chen, Jie; Pan, Yuqin; He, Bangshun; Ying, Houqun; Wang, Feng; Sun, Huiling; Deng, Qiwen; Liu, Xian; Lin, Kang; Peng, Hongxin; Cho, William C; Wang, Shukui

2015-10-01

The association between CD147 and cancer stem cells (CSCs) provides a new angle for cancer treatments. The aim of this study was to investigate the biological roles of CD147 in colorectal CSCs. The Oct4-green fluorescent protein (GFP) vector was used to isolate CSCs and pYr-mir30-shRNA was used to generate short hairpin RNA (shRNA) specifically for CD147. After RNA interference (RNAi), CD147 was evaluated by reverse transcription‑quantitative PCR and western blot analysis, and its biological functions were assessed by MTT and invasion assays. The results showed that the differentiation of isolated CSC-like HT-29 cells was blocked and these cells were highly positive for CD44 and CD147. RNAi-mediated CD147 silencing reduced the expression of CD147 at both mRNA and protein levels. Moreover, the activities of proliferation and invasion were decreased obviously in CSCs. Knockdown of CD147 increased the chemosensitivity of CSC-like cells to gemcitabine, cisplatin, docetaxel at 0.1, 1 and 10 µM respectively, however, there was no significant difference among the three groups to paclitaxel at 10 µM. In conclusion, these results suggest that CD147 plays an important role in colorectal CSCs and might be regarded as a novel CSC-specific targeted strategy against colorectal cancer.

Analysis of Cd44-Containing Lipid Rafts

PubMed Central

Oliferenko, Snezhana; Paiha, Karin; Harder, Thomas; Gerke, Volker; Schwärzler, Christoph; Schwarz, Heinz; Beug, Hartmut; Günthert, Ursula; Huber, Lukas A.

1999-01-01

CD44, the major cell surface receptor for hyaluronic acid (HA), was shown to localize to detergent-resistant cholesterol-rich microdomains, called lipid rafts, in fibroblasts and blood cells. Here, we have investigated the molecular environment of CD44 within the plane of the basolateral membrane of polarized mammary epithelial cells. We show that CD44 partitions into lipid rafts that contain annexin II at their cytoplasmic face. Both CD44 and annexin II were released from these lipid rafts by sequestration of plasma membrane cholesterol. Partition of annexin II and CD44 to the same type of lipid rafts was demonstrated by cross-linking experiments in living cells. First, when CD44 was clustered at the cell surface by anti-CD44 antibodies, annexin II was recruited into the cytoplasmic leaflet of CD44 clusters. Second, the formation of intracellular, submembranous annexin II–p11 aggregates caused by expression of a trans-dominant mutant of annexin II resulted in coclustering of CD44. Moreover, a frequent redirection of actin bundles to these clusters was observed. These basolateral CD44/annexin II–lipid raft complexes were stabilized by addition of GTPγS or phalloidin in a semipermeabilized and cholesterol-depleted cell system. The low lateral mobility of CD44 in the plasma membrane, as assessed with fluorescent recovery after photobleaching (FRAP), was dependent on the presence of plasma membrane cholesterol and an intact actin cytoskeleton. Disruption of the actin cytoskeleton dramatically increased the fraction of CD44 which could be recovered from the light detergent-insoluble membrane fraction. Taken together, our data indicate that in mammary epithelial cells the vast majority of CD44 interacts with annexin II in lipid rafts in a cholesterol-dependent manner. These CD44-containing lipid microdomains interact with the underlying actin cytoskeleton. PMID:10459018

Low molecular weight (LMW) heparin inhibits injury-induced femoral artery remodeling in mouse via upregulating CD44 expression.

PubMed

Zhao, Gaofeng; Shaik, Rahamthulla S; Zhao, Hang; Beagle, John; Kuo, Shuennwen; Hales, Charles A

2011-05-01

The mechanism of postangioplasty restenosis remains poorly understood. Low molecular weight (LMW) heparin has been shown to inhibit the proliferation of vascular smooth muscle cells (VSMCs), which is the principal characteristic of restenosis. Studies have shown that LMW heparin could bind to CD44. We hypothesized that LMW heparin might modulate CD44 expression thereby decreasing vascular remodeling. Vascular remodeling was induced in CD44(+/+) and CD44(-/-) mice and treated with LMW heparin. The arteries were harvested for histologic assessment and determination of CD44 expression. Bone marrow transplantation was introduced to further explore the role and functional sites of CD44. Effects of LMW heparin on growth capacity, CD44 expression were further studied using the cultured mouse VSMCs. Transluminal injury induced remarkable remodeling in mouse femoral artery (sham wall thickness percentage [WT%]: 3.4 ± 1.2% vs injury WT%: 31.8 ± 4.7%; P < .001). LMW heparin reduced the remodeling significantly (WT%: 17.8 ± 3.5%, P < .005). CD44(-/-) mice demonstrated considerably thicker arterial wall remodeling (WT%: 46.2 ± 7.6%, P = .0035), and CD44-chimeric mice exhibited equal contributions of the local and circulating CD44 signal to the neointima formation. LMW heparin markedly upregulated CD44 expression in the injured femoral arteries. In vitro, LMW heparin decreased mouse VSMC growth capacity and upregulated its CD44 expression simultaneously in a dose-dependent and time-dependent manner, which could be partially blocked by CD44 inhibitor. LMW heparin inhibits injury-induced femoral artery remodeling, at least partially, by upregulating CD44 expression. Copyright © 2011. Published by Mosby, Inc.

In Vitro and In Vivo Prostate Cancer Metastasis and Chemoresistance Can Be Modulated by Expression of either CD44 or CD147

PubMed Central

Hao, Jingli; Madigan, Michele C.; Khatri, Aparajita; Power, Carl A.; Hung, Tzong-Tyng; Beretov, Julia; Chang, Lei; Xiao, Weiwei; Cozzi, Paul J.; Graham, Peter H.; Kearsley, John H.; Li, Yong

2012-01-01

CD44 and CD147 are associated with cancer metastasis and progression. Our purpose in the study was to investigate the effects of down-regulation of CD44 or CD147 on the metastatic ability of prostate cancer (CaP) cells, their docetaxel (DTX) responsiveness and potential mechanisms involved in vitro and in vivo. CD44 and CD147 were knocked down (KD) in PC-3M-luc CaP cells using short hairpin RNA (shRNA). Expression of CD44, CD147, MRP2 (multi-drug resistance protein-2) and MCT4 (monocarboxylate tranporter-4) was evaluated using immunofluorescence and Western blotting. The DTX dose-response and proliferation was measured by MTT and colony assays, respectively. The invasive potential was assessed using a matrigel chamber assay. Signal transduction proteins in PI3K/Akt and MAPK/Erk pathways were assessed by Western blotting. An in vivo subcutaneous (s.c.) xenograft model was established to assess CaP tumorigenecity, lymph node metastases and DTX response. Our results indicated that KD of CD44 or CD147 decreased MCT4 and MRP2 expression, reduced CaP proliferation and invasive potential and enhanced DTX sensitivity; and KD of CD44 or CD147 down-regulated p-Akt and p-Erk, the main signal modulators associated with cell growth and survival. In vivo, CD44 or CD147-KD PC-3M-luc xenografts displayed suppressed tumor growth with increased DTX responsiveness compared to control xenografts. Both CD44 and CD147 enhance metastatic capacity and chemoresistance of CaP cells, potentially mediated by activation of the PI3K and MAPK pathways. Selective targeting of CD44/CD147 alone or combined with DTX may limit CaP metastasis and increase chemosensitivity, with promise for future CaP treatment. PMID:22870202

CD44 functions in Wnt signaling by regulating LRP6 localization and activation

PubMed Central

Schmitt, M; Metzger, M; Gradl, D; Davidson, G; Orian-Rousseau, V

2015-01-01

Wnt reception at the membrane is complex and not fully understood. CD44 is a major Wnt target gene in the intestine and is essential for Wnt-induced tumor progression in colorectal cancer. Here we show that CD44 acts as a positive regulator of the Wnt receptor complex. Downregulation of CD44 expression decreases, whereas CD44 overexpression increases Wnt activity in a concentration-dependent manner. Epistasis experiments place CD44 function at the level of the Wnt receptor LRP6. Mechanistically, CD44 physically associates with LRP6 upon Wnt treatment and modulates LRP6 membrane localization. Moreover, CD44 regulates Wnt signaling in the developing brain of Xenopus laevis embryos as shown by a decreased expression of Wnt targets tcf-4 and en-2 in CD44 morphants. PMID:25301071

Cells Isolated from Human Periapical Cysts Express Mesenchymal Stem Cell-like Properties

PubMed Central

Marrelli, Massimo; Paduano, Francesco; Tatullo, Marco

2013-01-01

We provide a detailed description of mesenchymal stem cells (MSCs) isolated from human periapical cysts, which we have termed hPCy-MSCs. These cells have a fibroblast-like shape and adhere to tissue culture plastic surfaces. hPCy-MSCs possess high proliferative potential and self-renewal capacity properties. We characterised the immunophenotype of hPCy-MSCs (CD73+, CD90+, CD105+, CD13+, CD29+, CD44+, CD45-, STRO-1+, CD146+) by flow cytometry and immunofluorescence. hPCy-MSCs possess the potential to differentiate into osteoblast- and adipocyte-like cells in vitro. Multi-potentiality was evaluated with culture-specific staining and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis for osteo/odontogenic and adipogenic markers. This is the first report to indicate that human periapical cysts contain cells with MSC-like properties. Taken together, our findings indicate that human periapical cysts could be a rich source of MSCs. PMID:24250252

Cells isolated from human periapical cysts express mesenchymal stem cell-like properties.

PubMed

Marrelli, Massimo; Paduano, Francesco; Tatullo, Marco

2013-01-01

We provide a detailed description of mesenchymal stem cells (MSCs) isolated from human periapical cysts, which we have termed hPCy-MSCs. These cells have a fibroblast-like shape and adhere to tissue culture plastic surfaces. hPCy-MSCs possess high proliferative potential and self-renewal capacity properties. We characterised the immunophenotype of hPCy-MSCs (CD73(+), CD90(+), CD105(+), CD13(+), CD29(+), CD44(+), CD45(-), STRO-1(+), CD146(+)) by flow cytometry and immunofluorescence. hPCy-MSCs possess the potential to differentiate into osteoblast- and adipocyte-like cells in vitro. Multi-potentiality was evaluated with culture-specific staining and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis for osteo/odontogenic and adipogenic markers. This is the first report to indicate that human periapical cysts contain cells with MSC-like properties. Taken together, our findings indicate that human periapical cysts could be a rich source of MSCs.

Adoptive cell therapy for lymphoma with CD4 T cells depleted of CD137-expressing regulatory T cells.

PubMed

Goldstein, Matthew J; Kohrt, Holbrook E; Houot, Roch; Varghese, Bindu; Lin, Jack T; Swanson, Erica; Levy, Ronald

2012-03-01

Adoptive immunotherapy with antitumor T cells is a promising novel approach for the treatment of cancer. However, T-cell therapy may be limited by the cotransfer of regulatory T cells (T(reg)). Here, we explored this hypothesis by using 2 cell surface markers, CD44 and CD137, to isolate antitumor CD4 T cells while excluding T(regs). In a murine model of B-cell lymphoma, only CD137(neg)CD44(hi) CD4 T cells infiltrated tumor sites and provided protection. Conversely, the population of CD137(pos)CD44hi CD4 T cells consisted primarily of activated T(regs). Notably, this CD137(pos) T(reg) population persisted following adoptive transfer and maintained expression of FoxP3 as well as CD137. Moreover, in vitro these CD137(pos) cells suppressed the proliferation of effector cells in a contact-dependent manner, and in vivo adding the CD137(pos)CD44(hi) CD4 cells to CD137(neg)CD44(hi) CD4 cells suppressed the antitumor immune response. Thus, CD137 expression on CD4 T cells defined a population of activated T(regs) that greatly limited antitumor immune responses. Consistent with observations in the murine model, human lymphoma biopsies also contained a population of CD137(pos) CD4 T cells that were predominantly CD25(pos)FoxP3(pos) T(regs). In conclusion, our findings identify 2 surface markers that can be used to facilitate the enrichment of antitumor CD4 T cells while depleting an inhibitory T(reg) population.

CD44+CD24+ subset of PANC-1 cells exhibits radiation resistance via decreased levels of reactive oxygen species.

PubMed

Wang, Lei; Li, Pengping; Hu, Wei; Xia, Youyou; Hu, Chenxi; Liu, Liang; Jiang, Xiaodong

2017-08-01

Emerging evidence has suggested that pancreatic adenocarcinoma is sustained by pancreatic cancer stem cells. The present study aimed to investigate the expression patterns of the pancreatic cancer stem cell surface markers cluster of differentiation CD44 and CD24 in a pancreatic adenocarcinoma cell line, and to investigate the possible mechanisms for their radiation resistance. Flow cytometry was used to analyze the expression patterns of CD44 and CD24 in the pancreatic adenocarcinoma PANC-1 cell line. In addition, a multi-target click model was used to fit cell survival curves and determine the sensitizer enhancement ratio. The apoptosis and cycle distribution of the four cell subsets was determined using flow cytometry, and the level of reactive oxygen species (ROS) was determined using the 2',7'-dichlorofluorescin diacetate probe. The present results identified that the ratios of CD44 + and CD24 + in the sorted PANC-1 cell line were 92.0 and 4.7%, respectively. Prior to radiation, no statistically significant differences were observed among the four groups. Following treatment with 6 MV of X-rays, the rate of apoptosis was decreased in the CD44 + CD24 + group compared with other subsets. The percentage of G0/G1 cells was highest in the CD44 + CD24 + group compared with the three other groups, which exhibited increased radiosensitivity. In addition, the level of ROS in the CD44 + CD24 + group was reduced compared with the other groups. In summary, the results of the present study indicated that CD44 + CD24 + exhibited stem cell properties. The lower level of ROS and apoptosis in CD44 + CD24 + cells may contribute to their resistance to radiation in pancreatic adenocarcinoma.

Overexpression of molecular chaperons GRP78 and GRP94 in CD44(hi)/CD24(lo) breast cancer stem cells.

PubMed

Nami, Babak; Ghasemi-Dizgah, Armin; Vaseghi, Akbar

2016-01-01

Breast cancer stem cell with CD44(hi)/CD24(lo) phonotype is described having stem cell properties and represented as the main driving factor in breast cancer initiation, growth, metastasis and low response to anti-cancer agents. Glucoseregulated proteins (GRPs) are heat shock protein family chaperons that are charged with regulation of protein machinery and modulation of endoplasmic reticulum homeostasis whose important roles in stem cell development and invasion of various cancers have been demonstrated. Here, we investigated the expression levels of GRP78 and GRP94 in CD44(hi)/CD24(lo) phenotype breast cancer stem cells (BCSCs). MCF7, T-47D and MDA-MB-231 breast cancer cell lines were used. CD44(hi)/CD24(lo) phenotype cell population were analyzed and sorted by fluorescence-activated cell sorting (FACS). Transcriptional and translational expression of GRP78 and GRP94 were investigated by western blotting and quantitative real time PCR. RESULTS showed different proportion of CD44(hi)/CD24(lo) phenotype cell population in their original bulk cells. The ranking of the cell lines in terms of CD44(hi)/CD24(lo) phenotype cell population was as MCF7

Expression of CD44v6 as matrix-associated ectodomain in the bone development.

PubMed

Nakajima, Kosei; Taniguchi, Kazumi; Mutoh, Ken-ichiro

2010-08-01

This study describes the expression of CD44v6 in the bone development and is the first study of its kind to the authors' best knowledge. The CD44 family is a family of transmembrane glycoproteins that acts as cell adhesion molecules binding cells to other cells as well as cells to the extracellular matrix. It has been suggested that the CD44v6, a family member of CD44, is closely related to the osteosarcoma metastasis. In general, when cancer cells metastasize, they revert to their immature forms. In the present study, therefore, we have investigated CD44v6 and the standard form of CD44 (CD44st) in two types of immature forms of bone tissues: developmentally immature stages from fetuses to adults as well as experimentally immature stages using fracture models. CD44st expression was identified in osteoblasts, osteocytes, and in the peripheral portion of the bone matrix from the fetal to young ages of rats. Many more intense reactions for CD44v6 were observed in the bone matrix than CD44st in fetal stages. In experimental fracture models, positive immunoreactions to CD44st were clearly observed in the osteoblasts and osteocytes. CD44v6-positive immunoreactivity, however, was not detected in either osteoblasts or the bone matrix. In conclusion, CD44v6 is expressed in the embryonic stages and may be involved in the bone matrix formation as a matrix-associated ectodomain during normal ontogenetic development but not involved in the process of fracture healing.

Biostabilization of cadmium contaminated sediments using indigenous sulfate reducing bacteria: Efficiency and process.

PubMed

Peng, Weihua; Li, Xiaomin; Liu, Tong; Liu, Yingying; Ren, Jinqian; Liang, Dawei; Fan, Wenhong

2018-06-01

Sulfate reducing bacteria (SRB) was used to stabilize cadmium (Cd) in sediments spiked with Cd. The study found that the Cd in sediments (≤600 mg kg -1 ) was successfully stabilized after 166 d SRB bio-treatment. This was verified by directly and indirectly examining Cd speciation in sediments, mobilization index, and Cd content in interstitial water. After 166 d bio-treatment, compared with control groups, Cd concentrations in interstitial water of Cd-spiked sediments were reduced by 77.6-96.4%. The bioavailable fractions of Cd (e.g., exchangeable and carbonate bound phases) were reduced, while more stable fractions of Cd (e.g., Fe-Mn oxide, organic bound, and residual phases) were increased. However, Cd mobilization in sediment was observed during the first part of bio-treatment (32 d), leading to an increase of Cd concentrations in the overlying water. Bacterial community composition (e.g., richness, diversity, and typical SRB) played an important role in Cd mobilization, dissolution, and stabilization. Bacterial community richness and diversity, including the typical SRB (e.g., Desulfobacteraceae and Desulfobulbaceae), were enhanced. However, bacterial communities were also influenced by Cd content and its speciations (especially the exchangeable and carbonate bound phases) in sediments, as well as total organic carbon in overlying water. Copyright © 2018 Elsevier Ltd. All rights reserved.

CD44 variant isoform 9 emerges in response to injury and contributes to the regeneration of the gastric epithelium

PubMed Central

Bertaux-Skeirik, Nina; Wunderlich, Mark; Teal, Emma; Chakrabarti, Jayati; Biesiada, Jacek; Mahe, Maxime; Sundaram, Nambirajan; Gabre, Joel; Hawkins, Jennifer; Jian, Gao; Engevik, Amy C.; Yang, Li; Wang, Jiang; Goldenring, James R.; Qualls, Joseph E.; Medvedovic, Mario; Helmrath, Michael A.; Diwan, Tayyab; Mulloy, James C.; Zavros, Yana

2017-01-01

The CD44 gene encodes several protein isoforms due to alternative splicing and post translational modifications. Given that CD44 variant isoform 9 (CD44v9) is expressed within Spasmolytic Polypeptide/TFF2-Expressing Metaplasia (SPEM) glands during repair, CD44v9 may be play a functional role during the process of regeneration of the gastric epithelium. Here we hypothesize that CD44v9 marks a regenerative cell lineage responsive to infiltrating macrophages during regeneration of the gastric epithelium. Ulcers were induced in CD44-decient (CD44KO) and C57BL/6 (BL6) mice by a localized application of acetic acid to the serosal surface of the stomach. Gastric organoids expressing CD44v9 were derived from mouse stomachs and transplanted at the ulcer site of CD44KO mice. Ulcers, CD44v9 expression, proliferation and histology were measured 1, 3, 5 and 7-days post-injury. Human-derived gastric organoids were generated from stomach tissue collected from elderly (>55 years) or young (14–20 years) patients. Organoids were transplanted into the stomachs of NOD scid gamma (NSG) mice at the site of injury. Gastric injury was induced in NRG-SGM3 (NRGS) mice harboring human-derived immune cells (hnNRGS) and the immune profile analyzed by CyTOF. CD44v9 expression emerged within regenerating glands the ulcer margin in response to injury. While ulcers in BL6 mice healed within 7-days post-injury, CD44KO mice exhibited loss of repair and epithelial regeneration. Ulcer healing was promoted in CD44KO mice by transplanted CD55v9-expressing gastric organoids. NSG mice exhibited loss of CD44v9 expression and gastric repair. Transplantation of human-derived gastric organoids from young, but not aged stomachs promoted repair in NSG mouse stomachs in response to injury. Finally, compared to NRGS mice, huNRGS animals exhibited reduced ulcer sizes, an infiltration of human CD162+ macrophages and an emergence of CD44v9 expression in SPEM. Thus, during repair of the gastric epithelium CD44v9 emerges within a regenerative cell lineage t hat coincides with macrophage infiltration within the injured mucosa. PMID:28497484

STAT3 as a potential therapeutic target in ALDH+ and CD44+/CD24+ stem cell-like pancreatic cancer cells.

PubMed

Lin, Li; Jou, David; Wang, Yina; Ma, Haiyan; Liu, Tianshu; Fuchs, James; Li, Pui-Kai; Lü, Jiagao; Li, Chenglong; Lin, Jiayuh

2016-12-01

Persistent activation of signal transducers and activators of transcription 3 (STAT3) is commonly detected in many types of cancer including pancreatic cancer. Whether STAT3 is activated in stem cell-like pancreatic cancer cells and the effect of STAT3 inhibition, is still unknown. Flow cytometry was used to isolate pancreatic cancer stem-like cells which are identified by both aldehyde dehydrogenase (ALDH)-positive (ALDH+) as well as cluster of differentiation (CD) 44-positive/CD24-positive subpopulations (CD44+/CD24+). STAT3 activation and the effects of STAT3 inhibition by STAT3 inhibitors, LLL12, FLLL32, and Stattic in ALDH+ and CD44+/CD24+ cells were examined. Our results showed that ALDH+ and CD44+/CD24+ pancreatic cancer stem-like cells expressed higher levels of phosphorylated STAT3, an active form of STAT3, compared to ALDH-negative (ALDH-) and CD44-negative/CD24-negative (CD44-/CD24-) pancreatic cancer cells, suggesting that STAT3 is activated in pancreatic cancer stem-like cells. Small molecular STAT3 inhibitors inhibited STAT3 phosphorylation, STAT3 downstream target gene expression, cell viability, and tumorsphere formation in ALDH+ and CD44+/CD24+ cells. Our results indicate that STAT3 is a novel therapeutic target in pancreatic cancer stem-like cells and inhibition of activated STAT3 in these cells by STAT3 inhibitors may offer an effective treatment for pancreatic cancer.

Cryopreservation of human vascular umbilical cord cells under good manufacturing practice conditions for future cell banks

PubMed Central

2012-01-01

Background In vitro fabricated tissue engineered vascular constructs could provide an alternative to conventional substitutes. A crucial factor for tissue engineering of vascular constructs is an appropriate cell source. Vascular cells from the human umbilical cord can be directly isolated and cryopreserved until needed. Currently no cell bank for human vascular cells is available. Therefore, the establishment of a future human vascular cell bank conforming to good manufacturing practice (GMP) conditions is desirable for therapeutic applications such as tissue engineered cardiovascular constructs. Materials and methods A fundamental step was the adaption of conventional research and development starting materials to GMP compliant starting materials. Human umbilical cord artery derived cells (HUCAC) and human umbilical vein endothelial cells (HUVEC) were isolated, cultivated, cryopreserved (short- and long-term) directly after primary culture and recultivated subsequently. Cell viability, expression of cellular markers and proliferation potential of fresh and cryopreserved cells were studied using trypan blue staining, flow cytometry analysis, immunofluorescence staining and proliferation assays. Statistical analyses were performed using Student’s t-test. Results Sufficient numbers of isolated cells with acceptable viabilities and homogenous expression of cellular markers confirmed that the isolation procedure was successful using GMP compliant starting materials. The influence of cryopreservation was marginal, because cryopreserved cells mostly maintain phenotypic and functional characteristics similar to those of fresh cells. Phenotypic studies revealed that fresh cultivated and cryopreserved HUCAC were positive for alpha smooth muscle actin, CD90, CD105, CD73, CD29, CD44, CD166 and negative for smoothelin. HUVEC expressed CD31, CD146, CD105 and CD144 but not alpha smooth muscle actin. Functional analysis demonstrated acceptable viability and sufficient proliferation properties of cryopreserved HUCAC and HUVEC. Conclusion Adaptation of cell isolation, cultivation and cryopreservation to GMP compliant starting materials was successful. Cryopreservation did not influence cell properties with lasting impact, confirming that the application of vascular cells from the human umbilical cord is feasible for cell banking. A specific cellular marker expression profile was established for HUCAC and HUVEC using flow cytometry analysis, applicable as a GMP compliant quality control. Use of these cells for the future fabrication of advanced therapy medicinal products GMP conditions are required by the regulatory authority. PMID:22591741

Activated ERK1/2 increases CD44 in glomerular parietal epithelial cells leading to matrix expansion

PubMed Central

Roeder, Sebastian S.; Barnes, Taylor J.; Lee, Jonathan S.; Kato, India; Eng, Diana G.; Kaverina, Natalya V.; Sunseri, Maria W.; Daniel, Christoph; Amann, Kerstin; Pippin, Jeffrey W.; Shankland, Stuart J.

2017-01-01

The glycoprotein CD44 is barely detected in normal mouse and human glomeruli, but is increased in glomerular parietal epithelial cells following podocyte injury in focal segmental glomerulosclerosis (FSGS). To determine the biological role and regulation of CD44 in these cells, we employed an in vivo and in vitro approach. Experimental FSGS was induced in CD44 knockout and wildtype mice with a cytotoxic podocyte antibody. Albuminuria, focal and global glomerulosclerosis (periodic acid-Schiff stain) and collagen IV staining were lower in CD44 knockout compared with wild type mice with FSGS. Parietal epithelial cells had lower migration from Bowman’s capsule to the glomerular tuft in CD44 knockout mice with disease compared with wild type mice. In cultured murine parietal epithelial cells, overexpressing CD44 with a retroviral vector encoding CD44 was accompanied by significantly increased collagen IV expression and parietal epithelial cells migration. Because our results showed de novo co-staining for activated ERK1/2 (pERK) in parietal epithelial cells in experimental FSGS, and also in biopsies from patients with FSGS, two in vitro strategies were employed to prove that pERK regulated CD44 levels. First, mouse parietal epithelial cells were infected with a retroviral vector for the upstream kinase MEK-DD to increase pERK, which was accompanied by increased CD44 levels. Second, in CD44 overexpressing parietal epithelial cells, decreasing pERK with U0126 was accompanied by reduced CD44. Finally, parietal epithelial cell migration was higher in cells with increased and reduced in cells with decreased pERK. Thus, pERK is a regulator of CD44 expression and increased CD44 expression leads to a pro-sclerotic and migratory parietal epithelial cells phenotype. PMID:27998643

Lung cancer tumorigenicity and drug resistance are maintained through ALDH(hi)CD44(hi) tumor initiating cells.

PubMed

Liu, Jing; Xiao, Zhijie; Wong, Sunny Kit-Man; Tin, Vicky Pui-Chi; Ho, Ka-Yan; Wang, Junwen; Sham, Mai-Har; Wong, Maria Pik

2013-10-01

Limited improvement in long term survival of lung cancer patients has been achieved by conventional chemotherapy or targeted therapy. To explore the potentials of tumor initiating cells (TIC)-directed therapy, it is essential to identify the cell targets and understand their maintenance mechanisms. We have analyzed the performance of ALDH/CD44 co-expression as TIC markers and treatment targets of lung cancer using well-validated in vitro and in vivo analyses in multiple established and patient-derived lung cancer cells. The ALDH(hi)CD44(hi) subset showed the highest enhancement of stem cell phenotypic properties compared to ALDH(hi)CD44(lo), ALDH(lo)CD44(hi), ALDH(lo)CD44(lo) cells and unsorted controls. They showed higher invasion capacities, pluripotency genes and epithelial-mesenchymal transition transcription factors expression, lower intercellular adhesion protein expression and higher G2/M phase cell cycle fraction. In immunosuppressed mice, the ALDH(hi)CD44(hi)xenografts showed the highest tumor induction frequency, serial transplantability, shortest latency, largest volume and highest growth rates. Inhibition of sonic Hedgehog and Notch developmental pathways reduced ALDH+CD44+ compartment. Chemotherapy and targeted therapy resulted in higher AALDH(hi)CD44(hi) subset viability and ALDH(lo)CD44(lo) subset apoptosis fraction. ALDH inhibition and CD44 knockdown led to reduced stemness gene expression and sensitization to drug treatment. In accordance, clinical lung cancers containing a higher abundance of ALDH and CD44-coexpressing cells was associated with lower recurrence-free survival. Together, results suggested theALDH(hi)CD44(hi)compartment was the cellular mediator of tumorigenicity and drug resistance. Further investigation of the regulatory mechanisms underlying ALDH(hi)CD44(hi)TIC maintenance would be beneficial for the development of long term lung cancer control.

Visualization of CD44 and CD133 in Normal Pancreas and Pancreatic Ductal Adenocarcinomas

PubMed Central

Immervoll, Heike; Hoem, Dag; Steffensen, Ole Johnny; Miletic, Hrvoje; Molven, Anders

2011-01-01

Tumor-initiating cells of pancreatic ductal adenocarcinoma (PDAC) have been isolated based on expression of either CD133 or CD44. The authors aimed to visualize pancreatic cells simultaneously expressing both these cell surface markers by employing the same antibodies commonly used in cell-sorting studies. Normal and diseased pancreatic tissue, including 51 PDAC cases, were analyzed. CD44 and CD133 expression was determined by immunohistochemical double staining on formalin-fixed material and subcellular protein distribution evaluated by immunofluorescence/confocal microscopy. In the normal pancreas, CD44 and CD133 were coexpressed in the centroacinar regions but in non-overlapping subcellular compartments. As expected, CD44 was found mainly basolaterally, whereas CD133 was present on the apical/endoluminal membrane. This was also the case in chronically inflamed/atrophic pancreatic tissue and in PDAC. In some malignant ducts, CD44 was found at the apical cell membrane adjacent to but never overlapping with CD133 expression. CD44 level was significantly associated with the patient’s lymph node status. In conclusion, a CD44+/CD133+ cell population does exist in the normal and neoplastic pancreas. The preferentially centroacinar localization of the doubly positive cells in the normal parenchyma suggests that this population could be of particular interest in attempts to identify tumor-initiating cells in PDAC. This article contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials. PMID:21411814

Immunohistochemical CD271 expression correlates with melanoma progress in a case-control study.

PubMed

Nielsen, Patricia Switten; Riber-Hansen, Rikke; Steiniche, Torben

2018-06-01

Putative cancer stem cell (CSC) markers have arisen from melanoma mouse and in vitro models, but their expression in paraffin embedded patient samples relative to clinical outcome remains largely unexplored. Rather than cells of the tumour bulk, conceivably, CSC drive tumour progression. Accordingly, complete eradication may prevent melanoma relapse. Because elevated tumour-cell proliferation is an established indicator of aggressive disease, this study aimed to investigate the correlation between melanoma recurrence and proliferation of putative CSC that express CD271, CD166, or CD20. Additionally, the expression of these markers was studied in naevi, melanomas, and their recurrence. In melanoma patients, 30 with relapse (cases) and 30 without (controls) were matched for tumour thickness, ulceration, Clark level, subtype, site, gender, and age. One paraffin-embedded section of the patients' primary melanoma (n = 60), relapse (n = 21), and naevus (n = 17) were immunohistochemically double-stained for Ki-67/MART1 and single-stained for CD271, CD166, and CD20. Their whole slide images were aligned as virtual quadruple stains. Image analysis established proliferation indices of each putative stem cell marker and the tumour bulk in addition to the markers' percentage level in tumour areas and the epidermis. In cases vs controls, median dermal proliferation indices (no./mm 2 ) were 211 vs 103 (p = 0.04) for CD271, 512 vs 227 (p = 0.3) for CD166, 184 vs 97 (p = 0.3) for CD20, and 95 vs 103 (p = 0.6) for the tumour bulk. Of additional interest, epidermal CD271 + keratinocytes totalled 8.8% in naevi and 0.98% in melanomas (p = 0.0007). Even though differences between naevi and melanomas also were observed for CD166 in both the epidermis (p = 0.002) and dermis (p = 0.006), they were visually less apparent. CD20 + MART1 + cells were absent in half of the melanomas, and all naevi and relapses. In conclusion, high levels of CD271 + Ki-67 + MART1 + cells were linked to melanoma relapse as opposed to common Ki-67 indices in this particular case-control study. With further investigation, such cells could be potential targets of therapy. Especially, loss of epidermal CD271 + keratinocytes seemed necessary for melanoma development; hence, identification may serve as a diagnostic tool with additional research. Copyright © 2018 Royal College of Pathologists of Australasia. Published by Elsevier B.V. All rights reserved.

Up-regulation of CD44 in the development of metastasis, recurrence and drug resistance of ovarian cancer

PubMed Central

Gao, Yan; Foster, Rosemary; Yang, Xiaoqian; Feng, Yong; Shen, Jacson K.; Mankin, Henry J.; Hornicek, Francis J.; Amiji, Mansoor M.; Duan, Zhenfeng

2015-01-01

The clinical significance of Cluster of Differentiation 44 (CD44) remains controversial in human ovarian cancer. The aim of this study is to evaluate the clinical significance of CD44 expression by using a unique tissue microarray, and then to determine the biological functions of CD44 in ovarian cancer. In this study, a unique ovarian cancer tissue microarray (TMA) was constructed with paired primary, metastatic, and recurrent tumor tissues from 26 individual patients. CD44 expression in TMA was assessed by immunohistochemistry. Both the metastatic and recurrent ovarian cancer tissues expressed higher level of CD44 than the patient-matched primary tumor. A significant association has been shown between CD44 expression and both the disease free survival and overall survival. A strong increase of CD44 was found in the tumor recurrence of mouse model. Finally, when CD44 was knocked down, proliferation, migration/invasion activity, and spheroid formation were significantly suppressed, while drug sensitivity was enhanced. Thus, up-regulation of CD44 represents a crucial event in the development of metastasis, recurrence, and drug resistance to current treatments in ovarian cancer. Developing strategies to target CD44 may prevent metastasis, recurrence, and drug resistance in ovarian cancer. PMID:25823654

Up-regulation of CD44 in the development of metastasis, recurrence and drug resistance of ovarian cancer.

PubMed

Gao, Yan; Foster, Rosemary; Yang, Xiaoqian; Feng, Yong; Shen, Jacson K; Mankin, Henry J; Hornicek, Francis J; Amiji, Mansoor M; Duan, Zhenfeng

2015-04-20

The clinical significance of Cluster of Differentiation 44 (CD44) remains controversial in human ovarian cancer. The aim of this study is to evaluate the clinical significance of CD44 expression by using a unique tissue microarray, and then to determine the biological functions of CD44 in ovarian cancer. In this study, a unique ovarian cancer tissue microarray (TMA) was constructed with paired primary, metastatic, and recurrent tumor tissues from 26 individual patients. CD44 expression in TMA was assessed by immunohistochemistry. Both the metastatic and recurrent ovarian cancer tissues expressed higher level of CD44 than the patient-matched primary tumor. A significant association has been shown between CD44 expression and both the disease free survival and overall survival. A strong increase of CD44 was found in the tumor recurrence of mouse model. Finally, when CD44 was knocked down, proliferation, migration/invasion activity, and spheroid formation were significantly suppressed, while drug sensitivity was enhanced. Thus, up-regulation of CD44 represents a crucial event in the development of metastasis, recurrence, and drug resistance to current treatments in ovarian cancer. Developing strategies to target CD44 may prevent metastasis, recurrence, and drug resistance in ovarian cancer.

Immunophenotypic characterization and tenogenic differentiation of mesenchymal stromal cells isolated from equine umbilical cord blood.

PubMed

Mohanty, Niharika; Gulati, Baldev R; Kumar, Rajesh; Gera, Sandeep; Kumar, Pawan; Somasundaram, Rajesh K; Kumar, Sandeep

2014-06-01

Mesenchymal stem cells (MSCs) isolated from umbilical cord blood (UCB) in equines have not been well characterized with respect to the expression of pluripotency and mesenchymal markers and for tenogenic differentiation potential in vitro. The plastic adherent fibroblast-like cells isolated from 13 out of 20 UCB samples could proliferate till passage 20. The cells expressed pluripotency markers (OCT4, NANOG, and SOX2) and MSC surface markers (CD90, CD73, and CD105) by RT-PCR, but did not express CD34, CD45, and CD14. On immunocytochemistry, the isolated cells showed expression of CD90 and CD73 proteins, but tested negative for CD34 and CD45. In flow cytometry, CD29, CD44, CD73, and CD90 were expressed by 96.36 ± 1.28%, 93.40 ± 0.70%, 73.23 ± 1.29% and 46.75 ± 3.95% cells, respectively. The UCB-MSCs could be differentiated to tenocytes by culturing in growth medium supplemented with 50 ng/ml of BMP-12 by day 10. The differentiated cells showed the expression of mohawk homeobox (Mkx), collagen type I alpha 1 (Col1α1), scleraxis (Scx), tenomodulin (Tnmd) and decorin (Dcn) by RT-PCR. In addition, flow cytometry detected tenomodulin and decorin protein in 95.65 ± 2.15% and 96.30 ± 1.00% of differentiated cells in comparison to 11.30 ± 0.10% and 19.45 ± 0.55% cells, respect vely in undifferentiated control cells. The findings support the observation that these cells may be suitable for therapeutic applications, including ruptured tendons in racehorses.

TGF-β reduces DNA ds-break repair mechanisms to heighten genetic diversity and adaptability of CD44+/CD24- cancer cells.

PubMed

Pal, Debjani; Pertot, Anja; Shirole, Nitin H; Yao, Zhan; Anaparthy, Naishitha; Garvin, Tyler; Cox, Hilary; Chang, Kenneth; Rollins, Fred; Kendall, Jude; Edwards, Leyla; Singh, Vijay A; Stone, Gary C; Schatz, Michael C; Hicks, James; Hannon, Gregory J; Sordella, Raffaella

2017-01-16

Many lines of evidence have indicated that both genetic and non-genetic determinants can contribute to intra-tumor heterogeneity and influence cancer outcomes. Among the best described sub-population of cancer cells generated by non-genetic mechanisms are cells characterized by a CD44+/CD24- cell surface marker profile. Here, we report that human CD44+/CD24- cancer cells are genetically highly unstable because of intrinsic defects in their DNA-repair capabilities. In fact, in CD44+/CD24- cells, constitutive activation of the TGF-beta axis was both necessary and sufficient to reduce the expression of genes that are crucial in coordinating DNA damage repair mechanisms. Consequently, we observed that cancer cells that reside in a CD44+/CD24- state are characterized by increased accumulation of DNA copy number alterations, greater genetic diversity and improved adaptability to drug treatment. Together, these data suggest that the transition into a CD44+/CD24- cell state can promote intra-tumor genetic heterogeneity, spur tumor evolution and increase tumor fitness.

CD44-mediated hyaluronan binding marks proliferating hematopoietic progenitor cells and promotes bone marrow engraftment

PubMed Central

Lee-Sayer, Sally S. M.; Dougan, Meghan N.; Cooper, Jesse; Sanderson, Leslie; Dosanjh, Manisha; Maxwell, Christopher A.

2018-01-01

CD44 is a widely expressed cell adhesion molecule that binds to the extracellular matrix component, hyaluronan. However, this interaction is not constitutive in most immune cells at steady state, as the ability of CD44 to engage hyaluronan is highly regulated. While activated T cells and macrophages gain the ability to bind hyaluronan by CD44, the status in other immune cells is less studied. Here we found a percentage of murine eosinophils, natural killer and natural killer T cells were capable of interacting with hyaluronan at steady state. To further investigate the consequences of hyaluronan binding by CD44 in the hematopoietic system, point mutations of CD44 that either cannot bind hyaluronan (LOF-CD44) or have an increased affinity for hyaluronan (GOF-CD44) were expressed in CD44-deficient bone marrow. Competitive bone marrow reconstitution of irradiated mice revealed an early preference for GOF-CD44 over WT-CD44 expressing cells, and for WT-CD44 over LOF-CD44 expressing cells, in the hematopoietic progenitor cell compartment. The advantage of the hyaluronan-binding cells was observed in the hematopoietic stem and progenitor populations, and was maintained throughout the immune system. Hematopoietic stem cells bound minimal hyaluronan at steady state, and this was increased when the cells were induced to proliferate whereas multipotent progenitors had an increased ability to bind hyaluronan at steady state. In vitro, the addition of hyaluronan promoted their proliferation. Thus, proliferating hematopoietic progenitors bind hyaluronan, and hyaluronan binding cells have a striking competitive advantage in bone marrow engraftment. PMID:29684048

Aberrant expression of cancer stem cell markers (CD44, CD90, and CD133) contributes to disease progression and reduced survival in hepatoblastoma patients: 4-year survival data.

PubMed

Bahnassy, Abeer A; Fawzy, Mohamed; El-Wakil, Mohamed; Zekri, Abdel-Rahman N; Abdel-Sayed, Ahmed; Sheta, Marwa

2015-03-01

Hepatoblastoma (HB) is an embryonal tumor of the liver in children. Prognosis and response to treatment in HB are highly variable. Cancer stem cells (CSCs) constitute a population of cells, which contribute to the development and progression of many tumors. However, their role in HB is not well defined yet. We assessed the prognostic and predictive values of some CSC markers in HB patients. Protein and messenger RNA expressions of the CSC markers CD133, CD90, and CD44 were assessed in 43 HB patients and 20 normal hepatic tissues using immunohistochemistry and quantitative real-time polymerase chain reaction. The expression levels of these markers were correlated to standard prognostic factors, patients' response to treatment, overall survival (OS), and disease-free survival (DFS). CD44, CD90, and CD133 proteins were detected in 48.8%, 32.6%, and 48.8% compared with 46.5%, 41.7%, and 58.1% RNA, respectively (concordance, 77.8%-96%). None of the normal tissue samples was positive for any of the markers. Significant correlations were reported between α-fetoprotein and both CD44 and CD133 (P = 0.02) as well as between tumor types CD90 and CD133 (P = 0.009). Reduced OS correlated with CD44, CD90, and CD133 expressions (P < 0.001), advanced stage (P < 0.001), response to treatment (P < 0.001), and total excision of the tumor. Reduced DFS correlated with CD44 and CD133 expressions (P < 0.001) only. In conclusion, CD133, CD44, and CD90 could be used as prognostic and predictive markers in HB. High expression of these markers is significantly associated with poor response to treatment and reduced survival. Moreover, complete surgical resection and systemic chemotherapy are essential to achieve good response and prolonged survival, especially in early stage patients. Copyright © 2015 Elsevier Inc. All rights reserved.

Analysis of human articular chondrocyte CD44 isoform expression and function in health and disease.

PubMed

Salter, D M; Godolphin, J L; Gourlay, M S; Lawson, M F; Hughes, D E; Dunne, E

1996-08-01

Interactions between articular chondrocytes and components of the extracellular matrix are of potential importance in the normal function of cartilage and in the pathophysiology of arthritis. Little is known of the basis of these interactions, but cell adhesive molecules such as CD44 are likely to be involved. Immunohistology using six well-characterized anti-CD44 monoclonal antibodies demonstrated standard CD44 isoform (CD44H) expression by all chondrocytes in normal and osteoarthrotic (OA) cartilage but absence of the CD44E variant. Polymerase chain reaction (PCR) of reverse transcribed mRNA from monolayer cultures of normal and OA chondrocytes using primer sequences which span the region containing variably spliced exons produced a predominant band representing the standard form of CD44, which lacks the variable exons 6-15 (v1-v10). No product was seen at the expected size of the epithelial variant of CD44 (CD44v8-10). Use of exon-specific primers, however, showed expression of variant exons resulting in multiple minor isoforms. Standard CD44 was also shown to be the predominantly expressed isoform identified by immunoprecipitation, but human articular chondrocytes did not adhere to hyaluronan in vitro. Chondrocyte CD44 may function as an adhesion receptor for other matrix molecules such as fibronectin or collagen.

Generation and evaluation of antibody agents for molecular imaging of CD44v6-expressing cancers

PubMed Central

Haylock, Anna-Karin; Nilvebrant, Johan; Mortensen, Anja; Velikyan, Irina; Nestor, Marika; Falk, Ronny

2017-01-01

Aim The aim of this study was to generate and characterize scFv antibodies directed to human CD44v6, as well as to radiolabel and evaluate top candidates in vitro and in vivo for their potential use in CD44v6-targeted molecular imaging in cancer patients. Materials and methods Phage display selections were used to isolate CD44v6-specific scFvs. A chain shuffling strategy was employed for affinity maturation based on a set of CD44v6-specific first-generation clones. Two second-generation scFv clones were then chosen for labeling with 111In or 125I and assessed for CD44v6-specific binding on cultured tumor cells. In vivo uptake and distribution was evaluated in tumor-bearing mice using a dual tumor model. Finally, a proof-of-concept small animal PET-CT study was performed on one of the candidates labeled with 124I. Results Two affinity-matured clones, CD44v6-scFv-A11 and CD44v6-scFv-H12, displayed promising binding kinetics. Seven out of eight radiolabeled conjugates demonstrated CD44v6-specific binding. In vivo studies on selected candidates demonstrated very advantageous tumor-to-organ ratios, in particular for iodinated conjugates, where 125I-labeled scFvs exhibited favorable kinetics and tumor-to-blood ratios above five already at 24 hours p.i.. The small animal PET-CT study using 124I-labeled CD44v6-scFv-H12 was in line with the biodistribution data, clearly visualizing the high CD44v6-expressing tumor. Conclusion The single chain fragments, CD44v6-scFv-A11 and CD44v6-scFv-H12 specifically bind to CD44v6, and the radiolabeled counterparts provide high tumor-to-blood ratios and fast clearance from organs and blood. We conclude that radioiodinated CD44v6-scFv-A11 and CD44v6-scFv-H12 possess features highly suitable for stringent molecular imaging. PMID:29029420

CD44 deficiency leads to decreased proinflammatory cytokine production in lung induced by PCV2 in mice.

PubMed

Fu, Qiang; Hou, Linbing; Xiao, Pingping; Guo, Chunhe; Chen, Yaosheng; Liu, Xiaohong

2014-12-01

Porcine circovirus type 2 (PCV2) is the primary etiological agent of postweaning multisystemic wasting syndrome (PMWS). CD44 is a widely expressed class I transmembrane glycoprotein implicated in immunological and inflammatory responses. In previous studies, the role of CD44 in host defense against microorganism infection remains controversial. The role of CD44 in host defense against PCV2 infection has never been studied before. In this study, we investigated the role of CD44 in the development of pneumonia induced by PCV2 in mice model. Upon infection, CD44 mRNA level in lung tissue was upregulated, and we confirmed a detrimental role of CD44 in host defense against PCV2 infection. The results demonstrated that CD44 deficiency could result in decreased proinflammatory cytokine production in lung induced by PCV2 in mice, suggesting a previously unrecognized role for CD44 in the development of pneumonia response to PCV2 infection. Copyright © 2014 Elsevier Ltd. All rights reserved.

A novel mechanism of regulating breast cancer cell migration via palmitoylation-dependent alterations in the lipid raft affiliation of CD44.

PubMed

Babina, Irina S; McSherry, Elaine A; Donatello, Simona; Hill, Arnold D K; Hopkins, Ann M

2014-02-10

Most breast cancer-related deaths result from metastasis, a process involving dynamic regulation of tumour cell adhesion and migration. The adhesion protein CD44, a key regulator of cell migration, is enriched in cholesterol-enriched membrane microdomains termed lipid rafts. We recently reported that raft affiliation of CD44 negatively regulates interactions with its migratory binding partner ezrin. Since raft affiliation is regulated by post-translational modifications including palmitoylation, we sought to establish the contribution of CD44 palmitoylation and lipid raft affiliation to cell migration. Recovery of CD44 and its binding partners from raft versus non-raft membrane microdomains was profiled in non-migrating and migrating breast cancer cell lines. Site-directed mutagenesis was used to introduce single or double point mutations into both CD44 palmitoylation sites (Cys286 and Cys295), whereupon the implications for lipid raft recovery, phenotype, ezrin co-precipitation and migratory behaviour was assessed. Finally CD44 palmitoylation status and lipid raft affiliation was assessed in primary cultures from a small panel of breast cancer patients. CD44 raft affiliation was increased during migration of non-invasive breast cell lines, but decreased during migration of highly-invasive breast cells. The latter was paralleled by increased CD44 recovery in non-raft fractions, and exclusive non-raft recovery of its binding partners. Point mutation of CD44 palmitoylation sites reduced CD44 raft affiliation in invasive MDA-MB-231 cells, increased CD44-ezrin co-precipitation and accordingly enhanced cell migration. Expression of palmitoylation-impaired (raft-excluded) CD44 mutants in non-invasive MCF-10a cells was sufficient to reversibly induce the phenotypic appearance of epithelial-to-mesenchymal transition and to increase cell motility. Interestingly, cell migration was associated with temporal reductions in CD44 palmitoylation in wild-type breast cells. Finally, the relevance of these findings is underscored by the fact that levels of palmitoylated CD44 were lower in primary cultures from invasive ductal carcinomas relative to non-tumour tissue, while CD44 co-localisation with a lipid raft marker was less in invasive ductal carcinoma relative to ductal carcinoma in situ cultures. Our results support a novel mechanism whereby CD44 palmitoylation and consequent lipid raft affiliation inversely regulate breast cancer cell migration, and may act as a new therapeutic target in breast cancer metastasis.

Multifunctionalized iron oxide nanoparticles for selective drug delivery to CD44-positive cancer cells

NASA Astrophysics Data System (ADS)

Aires, Antonio; Ocampo, Sandra M.; Simões, Bruno M.; Josefa Rodríguez, María; Cadenas, Jael F.; Couleaud, Pierre; Spence, Katherine; Latorre, Alfonso; Miranda, Rodolfo; Somoza, Álvaro; Clarke, Robert B.; Carrascosa, José L.; Cortajarena, Aitziber L.

2016-02-01

Nanomedicine nowadays offers novel solutions in cancer therapy and diagnosis by introducing multimodal treatments and imaging tools in one single formulation. Nanoparticles acting as nanocarriers change the solubility, biodistribution and efficiency of therapeutic molecules, reducing their side effects. In order to successfully apply these novel therapeutic approaches, efforts are focused on the biological functionalization of the nanoparticles to improve the selectivity towards cancer cells. In this work, we present the synthesis and characterization of novel multifunctionalized iron oxide magnetic nanoparticles (MNPs) with antiCD44 antibody and gemcitabine derivatives, and their application for the selective treatment of CD44-positive cancer cells. The lymphocyte homing receptor CD44 is overexpressed in a large variety of cancer cells, but also in cancer stem cells (CSCs) and circulating tumor cells (CTCs). Therefore, targeting CD44-overexpressing cells is a challenging and promising anticancer strategy. Firstly, we demonstrate the targeting of antiCD44 functionalized MNPs to different CD44-positive cancer cell lines using a CD44-negative non-tumorigenic cell line as a control, and verify the specificity by ultrastructural characterization and downregulation of CD44 expression. Finally, we show the selective drug delivery potential of the MNPs by the killing of CD44-positive cancer cells using a CD44-negative non-tumorigenic cell line as a control. In conclusion, the proposed multifunctionalized MNPs represent an excellent biocompatible nanoplatform for selective CD44-positive cancer therapy in vitro.

MicroRNA miR-328 Regulates Zonation Morphogenesis by Targeting CD44 Expression

PubMed Central

Wang, Chia-Hui; Lee, Daniel Y.; Deng, Zhaoqun; Jeyapalan, Zina; Lee, Shao-Chen; Kahai, Shireen; Lu, Wei-Yang; Zhang, Yaou; Yang, Burton B.

2008-01-01

Morphogenesis is crucial to initiate physiological development and tumor invasion. Here we show that a microRNA controls zonation morphogenesis by targeting hyaluronan receptor CD44. We have developed a novel system to study microRNA functions by generating constructs expressing pre-miRNAs and mature miRNAs. Using this system, we have demonstrated that expression of miR-328 reduced cell adhesion, aggregation, and migration, and regulated formation of capillary structure. Protein analysis indicated that miR-328 repressed CD44 expression. Activities of luciferase constructs harboring the target site in CD44, but not the one containing mutation, were repressed by miR-328. Zonation morphogenesis appeared in cells transfected by miR-328: miR-328-transfected cells were present on the surface of zonating structures while the control cells stayed in the middle. MiR-328-mediated CD44 actions was validated by anti-CD44 antibody, hyaluronidase, CD44 siRNA, and CD44 expression constructs. In vivo experiments showed that CD44-silencing cells appeared as layers on the surfaces of nodules or zonating structures. Immuno-histochemistry also exhibited CD44-negative cells on the surface layers of normal rat livers and the internal zones of Portal veins. Our results demonstrate that miR-328 targets CD44, which is essential in regulating zonation morphogenesis: silencing of CD44 expression is essential in sealing the zonation structures to facilitate their extension and to inhibit complex expansion. PMID:18560585

MicroRNA miR-328 regulates zonation morphogenesis by targeting CD44 expression.

PubMed

Wang, Chia-Hui; Lee, Daniel Y; Deng, Zhaoqun; Jeyapalan, Zina; Lee, Shao-Chen; Kahai, Shireen; Lu, Wei-Yang; Zhang, Yaou; Yang, Burton B

2008-06-18

Morphogenesis is crucial to initiate physiological development and tumor invasion. Here we show that a microRNA controls zonation morphogenesis by targeting hyaluronan receptor CD44. We have developed a novel system to study microRNA functions by generating constructs expressing pre-miRNAs and mature miRNAs. Using this system, we have demonstrated that expression of miR-328 reduced cell adhesion, aggregation, and migration, and regulated formation of capillary structure. Protein analysis indicated that miR-328 repressed CD44 expression. Activities of luciferase constructs harboring the target site in CD44, but not the one containing mutation, were repressed by miR-328. Zonation morphogenesis appeared in cells transfected by miR-328: miR-328-transfected cells were present on the surface of zonating structures while the control cells stayed in the middle. MiR-328-mediated CD44 actions was validated by anti-CD44 antibody, hyaluronidase, CD44 siRNA, and CD44 expression constructs. In vivo experiments showed that CD44-silencing cells appeared as layers on the surfaces of nodules or zonating structures. Immuno-histochemistry also exhibited CD44-negative cells on the surface layers of normal rat livers and the internal zones of Portal veins. Our results demonstrate that miR-328 targets CD44, which is essential in regulating zonation morphogenesis: silencing of CD44 expression is essential in sealing the zonation structures to facilitate their extension and to inhibit complex expansion.

Prognostic value of CD44 expression in penile squamous cell carcinoma: a pilot study.

PubMed

Minardi, Daniele; Lucarini, Guendalina; Filosa, Alessandra; Zizzi, Antonio; Simonetti, Oriana; Offidani, Anna Maria; d'Anzeo, Gianluca; Di Primio, Roberto; Montironi, Rodolfo; Muzzonigro, Giovanni

2012-10-01

Several studies have reported on the prognostic value of molecular markers for metastasis risk and survival in penile squamous cell carcinoma (SCC) patients. The usefulness of CD44 expression as such a marker has been studied in different tumors, but not in penile SCC. Our aim was to determine whether CD44 expression may serve as a prognostic marker for lymph node metastasis and survival in penile SCC patients. CD44 immunoistochemical expression was investigated in tissue specimens from 39 patients with penile SCC. CD44 cell positivity, staining intensity and distribution were analyzed and correlated with tumor stage, grade, lymph node status and disease-specific survival. CD44 expression was detected in epithelial cells of both intratumoral and normal tissues with different intensities and staining distributions. In normal tissues CD44 protein was mainly detected in cell membranes, whereas in the tumor compartments it was found in both the cell membranes and the cytoplasm. The intensities and percentages of CD44 expressing cells did not correlate with tumor stage and/or grade. Seventy-three percent of the patients with lymph node metastasis showed high intensities of CD44 staining, as compared to 44% of the patients without lymph node metastasis (P = 0.03). Lymph node-positive patients showed both cytoplasmic and membranous CD44 expression. High CD44 expression was found to be significantly correlated with a decreased 5 year overall survival (P = 0.01). CD44 levels and patterns of expression can be considered as markers for penile SCC aggressiveness and, in addition, may serve as predictive markers for lymph node metastasis, also in patients with clinically negative lymph nodes. CD44 expression may provide prognostic information for penile SCC patients, next to classical clinical-pathological factors.

Virtual screening-driven repositioning of etoposide as CD44 antagonist in breast cancer cells

PubMed Central

Aguirre-Alvarado, Charmina; Segura-Cabrera, Aldo; Velázquez-Quesada, Inés; Hernández-Esquivel, Miguel A.; García-Pérez, Carlos A.; Guerrero-Rodríguez, Sandra L.; Ruiz, Angel J.; Rodríguez-Moreno, Andrea; Pérez-Tapia, Sonia M.; Velasco-Velázquez, Marco A.

2016-01-01

CD44 is a receptor for hyaluronan (HA) that promotes epithelial-to-mesenchymal transition (EMT), induces cancer stem cell (CSC) expansion, and favors metastasis. Thus, CD44 is a target for the development of antineoplastic agents. In order to repurpose drugs as CD44 antagonists, we performed consensus-docking studies using the HA-binding domain of CD44 and 11,421 molecules. Drugs that performed best in docking were examined in molecular dynamics simulations, identifying etoposide as a potential CD44 antagonist. Ligand competition and cell adhesion assays in MDA-MB-231 cells demonstrated that etoposide decreased cell binding to HA as effectively as a blocking antibody. Etoposide-treated MDA-MB-231 cells developed an epithelial morphology; increased their expression of E-cadherin; and reduced their levels of EMT-associated genes and cell migration. By gene expression analysis, etoposide reverted an EMT signature similarly to CD44 knockdown, whereas other topoisomerase II (TOP2) inhibitors did not. Moreover, etoposide decreased the proportion of CD44+/CD24− cells, lowered chemoresistance, and blocked mammosphere formation. Our data indicate that etoposide blocks CD44 activation, impairing key cellular functions that drive malignancy, thus rendering it a candidate for further translational studies and a potential lead compound in the development of new CD44 antagonists. PMID:27009862

Virtual screening-driven repositioning of etoposide as CD44 antagonist in breast cancer cells.

PubMed

Aguirre-Alvarado, Charmina; Segura-Cabrera, Aldo; Velázquez-Quesada, Inés; Hernández-Esquivel, Miguel A; García-Pérez, Carlos A; Guerrero-Rodríguez, Sandra L; Ruiz-Moreno, Angel J; Rodríguez-Moreno, Andrea; Pérez-Tapia, Sonia M; Velasco-Velázquez, Marco A

2016-04-26

CD44 is a receptor for hyaluronan (HA) that promotes epithelial-to-mesenchymal transition (EMT), induces cancer stem cell (CSC) expansion, and favors metastasis. Thus, CD44 is a target for the development of antineoplastic agents. In order to repurpose drugs as CD44 antagonists, we performed consensus-docking studies using the HA-binding domain of CD44 and 11,421 molecules. Drugs that performed best in docking were examined in molecular dynamics simulations, identifying etoposide as a potential CD44 antagonist. Ligand competition and cell adhesion assays in MDA-MB-231 cells demonstrated that etoposide decreased cell binding to HA as effectively as a blocking antibody. Etoposide-treated MDA-MB-231 cells developed an epithelial morphology; increased their expression of E-cadherin; and reduced their levels of EMT-associated genes and cell migration. By gene expression analysis, etoposide reverted an EMT signature similarly to CD44 knockdown, whereas other topoisomerase II (TOP2) inhibitors did not. Moreover, etoposide decreased the proportion of CD44+/CD24- cells, lowered chemoresistance, and blocked mammosphere formation. Our data indicate that etoposide blocks CD44 activation, impairing key cellular functions that drive malignancy, thus rendering it a candidate for further translational studies and a potential lead compound in the development of new CD44 antagonists.

Differential regulation of CD44 expression by lipopolysaccharide (LPS) and TNF-alpha in human monocytic cells: distinct involvement of c-Jun N-terminal kinase in LPS-induced CD44 expression.

PubMed

Gee, Katrina; Lim, Wilfred; Ma, Wei; Nandan, Devki; Diaz-Mitoma, Francisco; Kozlowski, Maya; Kumar, Ashok

2002-11-15

Alterations in the regulation of CD44 expression play a critical role in modulating cell adhesion, migration, and inflammation. LPS, a bacterial cell wall component, regulates CD44 expression and may modulate CD44-mediated biological effects in monocytic cells during inflammation and immune responses. In this study, we show that in normal human monocytes, LPS and LPS-induced cytokines IL-10 and TNF-alpha enhance CD44 expression. To delineate the mechanism underlying LPS-induced CD44 expression, we investigated the role of the mitogen-activated protein kinases (MAPKs), p38, p42/44 extracellular signal-regulated kinase, and c-Jun N-terminal kinase (JNK) by using their specific inhibitors. We demonstrate the involvement, at least in part, of p38 MAPK in TNF-alpha-induced CD44 expression in both monocytes and promonocytic THP-1 cells. However, neither p38 nor p42/44 MAPKs were involved in IL-10-induced CD44 expression in monocytes. To further dissect the TNF-alpha and LPS-induced signaling pathways regulating CD44 expression independent of IL-10-mediated effects, we used IL-10 refractory THP-1 cells as a model system. Herein, we show that CD44 expression induced by the LPS-mediated pathway predominantly involved JNK activation. This conclusion was based on results derived by transfection of THP-1 cells with a dominant-negative mutant of stress-activated protein/extracellular signal-regulated kinase kinase 1, and by exposure of cells to JNK inhibitors dexamethasone and SP600125. All these treatments prevented CD44 induction in LPS-stimulated, but not in TNF-alpha-stimulated, THP-1 cells. Furthermore, we show that CD44 induction may involve JNK-dependent early growth response gene activation in LPS-stimulated monocytic cells. Taken together, these results suggest a predominant role of JNK in LPS-induced CD44 expression in monocytic cells.

CD147, CD44, and the epidermal growth factor receptor (EGFR) signaling pathway cooperate to regulate breast epithelial cell invasiveness.

PubMed

Grass, G Daniel; Tolliver, Lauren B; Bratoeva, Momka; Toole, Bryan P

2013-09-06

The immunoglobulin superfamily glycoprotein CD147 (emmprin; basigin) is associated with an invasive phenotype in various types of cancers, including malignant breast cancer. We showed recently that up-regulation of CD147 in non-transformed, non-invasive breast epithelial cells is sufficient to induce an invasive phenotype characterized by membrane type-1 matrix metalloproteinase (MT1-MMP)-dependent invadopodia activity (Grass, G. D., Bratoeva, M., and Toole, B. P. (2012) Regulation of invadopodia formation and activity by CD147. J. Cell Sci. 125, 777-788). Here we found that CD147 induces breast epithelial cell invasiveness by promoting epidermal growth factor receptor (EGFR)-Ras-ERK signaling in a manner dependent on hyaluronan-CD44 interaction. Furthermore, CD147 promotes assembly of signaling complexes containing CD147, CD44, and EGFR in lipid raftlike domains. We also found that oncogenic Ras regulates CD147 expression, hyaluronan synthesis, and formation of CD147-CD44-EGFR complexes, thus forming a positive feedback loop that may amplify invasiveness. Last, we showed that malignant breast cancer cells are heterogeneous in their expression of surface-associated CD147 and that high levels of membrane CD147 correlate with cell surface EGFR and CD44 levels, activated EGFR and ERK1, and activated invadopodia. Future studies should evaluate CD147 as a potential therapeutic target and disease stratification marker in breast cancer.

CD44 Interacts with HIF-2α to Modulate the Hypoxic Phenotype of Perinecrotic and Perivascular Glioma Cells.

PubMed

Johansson, Elinn; Grassi, Elisa S; Pantazopoulou, Vasiliki; Tong, Bei; Lindgren, David; Berg, Tracy J; Pietras, Elin J; Axelson, Håkan; Pietras, Alexander

2017-08-15

Hypoxia-inducible factors enhance glioma stemness, and glioma stem cells have an amplified hypoxic response despite residing within a perivascular niche. Still, little is known about differential HIF regulation in stem versus bulk glioma cells. We show that the intracellular domain of stem cell marker CD44 (CD44ICD) is released at hypoxia, binds HIF-2α (but not HIF-1α), enhances HIF target gene activation, and is required for hypoxia-induced stemness in glioma. In a glioma mouse model, CD44 was restricted to hypoxic and perivascular tumor regions, and in human glioma, a hypoxia signature correlated with CD44. The CD44ICD was sufficient to induce hypoxic signaling at perivascular oxygen tensions, and blocking CD44 cleavage decreased HIF-2α stabilization in CD44-expressing cells. Our data indicate that the stem cell marker CD44 modulates the hypoxic response of glioma cells and that the pseudo-hypoxic phenotype of stem-like glioma cells is achieved by stabilization of HIF-2α through interaction with CD44, independently of oxygen. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

TGF-β reduces DNA ds-break repair mechanisms to heighten genetic diversity and adaptability of CD44+/CD24− cancer cells

PubMed Central

Pal, Debjani; Pertot, Anja; Shirole, Nitin H; Yao, Zhan; Anaparthy, Naishitha; Garvin, Tyler; Cox, Hilary; Chang, Kenneth; Rollins, Fred; Kendall, Jude; Edwards, Leyla; Singh, Vijay A; Stone, Gary C; Schatz, Michael C; Hicks, James; Hannon, Gregory J; Sordella, Raffaella

2017-01-01

Many lines of evidence have indicated that both genetic and non-genetic determinants can contribute to intra-tumor heterogeneity and influence cancer outcomes. Among the best described sub-population of cancer cells generated by non-genetic mechanisms are cells characterized by a CD44+/CD24− cell surface marker profile. Here, we report that human CD44+/CD24− cancer cells are genetically highly unstable because of intrinsic defects in their DNA-repair capabilities. In fact, in CD44+/CD24− cells, constitutive activation of the TGF-beta axis was both necessary and sufficient to reduce the expression of genes that are crucial in coordinating DNA damage repair mechanisms. Consequently, we observed that cancer cells that reside in a CD44+/CD24− state are characterized by increased accumulation of DNA copy number alterations, greater genetic diversity and improved adaptability to drug treatment. Together, these data suggest that the transition into a CD44+/CD24− cell state can promote intra-tumor genetic heterogeneity, spur tumor evolution and increase tumor fitness. DOI: http://dx.doi.org/10.7554/eLife.21615.001 PMID:28092266

Pancreatic cancer cells express CD44 variant 9 and multidrug resistance protein 1 during mitosis.

PubMed

Kiuchi, Shizuka; Ikeshita, Shunji; Miyatake, Yukiko; Kasahara, Masanori

2015-02-01

Pancreatic cancer is one of the most lethal cancers with high metastatic potential and strong chemoresistance. Its intractable natures are attributed to high robustness in tumor cells for their survival. We demonstrate here that pancreatic cancer cells (PCCs) with an epithelial phenotype upregulate cell surface expression of CD44 variant 9 (CD44v9), an important cancer stem cell marker, during the mitotic phases of the cell cycle. Of five human CD44(+) PCC lines examined, three cell lines, PCI-24, PCI-43 and PCI-55, expressed E-cadherin and CD44 variants, suggesting that they have an epithelial phenotype. By contrast, PANC-1 and MIA PaCa-2 cells expressed vimentin and ZEB1, suggesting that they have a mesenchymal phenotype. PCCs with an epithelial phenotype upregulated cell surface expression of CD44v9 in prophase, metaphase, anaphase and telophase and downregulated CD44v9 expression in late-telophase, cytokinesis and interphase. Sorted CD44v9-negative PCI-55 cells resumed CD44v9 expression when they re-entered the mitotic stage. Interestingly, CD44v9(bright) mitotic cells expressed multidrug resistance protein 1 (MDR1) intracellularly. Upregulated expression of CD44v9 and MDR1 might contribute to the intractable nature of PCCs with high proliferative activity. Copyright © 2014 Elsevier Inc. All rights reserved.

Engineered Metal-Phenolic Capsules Show Tunable Targeted Delivery to Cancer Cells.

PubMed

Ju, Yi; Cui, Jiwei; Sun, Huanli; Müllner, Markus; Dai, Yunlu; Guo, Junling; Bertleff-Zieschang, Nadja; Suma, Tomoya; Richardson, Joseph J; Caruso, Frank

2016-06-13

We engineered metal-phenolic capsules with both high targeting and low nonspecific cell binding properties. The capsules were prepared by coating phenolic-functionalized hyaluronic acid (HA) and poly(ethylene glycol) (PEG) on calcium carbonate templates, followed by cross-linking the phenolic groups with metal ions and removing the templates. The incorporation of HA significantly enhanced binding and association with a CD44 overexpressing (CD44+) cancer cell line, while the incorporation of PEG reduced nonspecific interactions with a CD44 minimal-expressing (CD44-) cell line. Moreover, high specific targeting to CD44+ cells can be balanced with low nonspecific binding to CD44- cells simply by using an optimized feed-ratio of HA and PEG to vary the content of HA and PEG incorporated into the capsules. Loading an anticancer drug (i.e., doxorubicin) into the obtained capsules resulted in significantly higher cytotoxicity to CD44+ cells but lower cytotoxicity to CD44- cells.

CD44 staining of cancer stem-like cells is influenced by down-regulation of CD44 variant isoforms and up-regulation of the standard CD44 isoform in the population of cells that have undergone epithelial-to-mesenchymal transition.

PubMed

Biddle, Adrian; Gammon, Luke; Fazil, Bilal; Mackenzie, Ian C

2013-01-01

CD44 is commonly used as a cell surface marker of cancer stem-like cells in epithelial tumours, and we have previously demonstrated the existence of two different CD44(high) cancer stem-like cell populations in squamous cell carcinoma, one having undergone epithelial-to-mesenchymal transition and the other maintaining an epithelial phenotype. Alternative splicing of CD44 variant exons generates a great many isoforms, and it is not known which isoforms are expressed on the surface of the two different cancer stem-like cell phenotypes. Here, we demonstrate that cancer stem-like cells with an epithelial phenotype predominantly express isoforms containing the variant exons, whereas the cancer stem-like cells that have undergone an epithelial-to-mesenchymal transition down-regulate these variant isoforms and up-regulate expression of the standard CD44 isoform that contains no variant exons. In addition, we find that enzymatic treatments used to dissociate cells from tissue culture or fresh tumour specimens cause destruction of variant CD44 isoforms at the cell surface whereas expression of the standard CD44 isoform is preserved. This results in enrichment within the CD44(high) population of cancer stem-like cells that have undergone an epithelial-to-mesenchymal transition and depletion from the CD44(high) population of cancer stem-like cells that maintain an epithelial phenotype, and therefore greatly effects the characteristics of any cancer stem-like cell population isolated based on expression of CD44. As well as effecting the CD44(high) population, enzymatic treatment also reduces the percentage of the total epithelial cancer cell population staining CD44-positive, with potential implications for studies that aim to use CD44-positive staining as a prognostic indicator. Analyses of the properties of cancer stem-like cells are largely dependent on the ability to accurately identify and assay these populations. It is therefore critical that consideration be given to use of multiple cancer stem-like cell markers and suitable procedures for cell isolation in order that the correct populations are assayed.

CD44v6 expression in patients with stage II or stage III sporadic colorectal cancer is superior to CD44 expression for predicting progression

PubMed Central

Zhao, LH; Lin, QL; Wei, J; Huai, YL; Wang, KJ; Yan, HY

2015-01-01

Background: Currently, it is difficult to predict the prognosis of patients exhibiting stage II or stage III colorectal cancer (CRC) and to identify those patients most likely to benefit from aggressive treatment. The current study was performed to examine the clinicopathological significance of CD44 and CD44v6 protein expression in these patients. Study design: We retrospectively investigated 187 consecutive patients who underwent surgery with curative intent for stage II to III CRC from 2007 to 2013 in the Beijing Civil Aviation Hospital. CD44 and CD44v6 protein expression levels were determined using immunohistochemistry and compared to the clinicopathological data. Results: Using immunohistochemical detection, CD44 expression was observed in 108 (57.75%) of the CRC patients; and its detection was significantly associated with greater invasion depth, lymph node metastasis, angiolymphatic invasion, and a more advanced pathological tumor-lymph node-metastasis (TNM) stage. CD44v6 expression was observed in 135 (72.19%) of the CRC patients; and its expression was significantly associated with a poorly differentiated histology, greater invasion depth, lymph node metastasis, angiolymphatic invasion, and a more advanced pathological TNM stage. Expression of CD44v6 was higher than that of CD44 in stage II and stage III sporadic CRC. Conclusion: CD44v6 is a more useful marker for predicting a poor prognosis in stage II and stage III sporadic CRC as compared to CD44. PMID:25755763

CD147, CD44, and the Epidermal Growth Factor Receptor (EGFR) Signaling Pathway Cooperate to Regulate Breast Epithelial Cell Invasiveness*

PubMed Central

Grass, G. Daniel; Tolliver, Lauren B.; Bratoeva, Momka; Toole, Bryan P.

2013-01-01

The immunoglobulin superfamily glycoprotein CD147 (emmprin; basigin) is associated with an invasive phenotype in various types of cancers, including malignant breast cancer. We showed recently that up-regulation of CD147 in non-transformed, non-invasive breast epithelial cells is sufficient to induce an invasive phenotype characterized by membrane type-1 matrix metalloproteinase (MT1-MMP)-dependent invadopodia activity (Grass, G. D., Bratoeva, M., and Toole, B. P. (2012) Regulation of invadopodia formation and activity by CD147. J. Cell Sci. 125, 777–788). Here we found that CD147 induces breast epithelial cell invasiveness by promoting epidermal growth factor receptor (EGFR)-Ras-ERK signaling in a manner dependent on hyaluronan-CD44 interaction. Furthermore, CD147 promotes assembly of signaling complexes containing CD147, CD44, and EGFR in lipid raftlike domains. We also found that oncogenic Ras regulates CD147 expression, hyaluronan synthesis, and formation of CD147-CD44-EGFR complexes, thus forming a positive feedback loop that may amplify invasiveness. Last, we showed that malignant breast cancer cells are heterogeneous in their expression of surface-associated CD147 and that high levels of membrane CD147 correlate with cell surface EGFR and CD44 levels, activated EGFR and ERK1, and activated invadopodia. Future studies should evaluate CD147 as a potential therapeutic target and disease stratification marker in breast cancer. PMID:23888049

A novel mechanism of regulating breast cancer cell migration via palmitoylation-dependent alterations in the lipid raft affiliation of CD44

PubMed Central

2014-01-01

Introduction Most breast cancer-related deaths result from metastasis, a process involving dynamic regulation of tumour cell adhesion and migration. The adhesion protein CD44, a key regulator of cell migration, is enriched in cholesterol-enriched membrane microdomains termed lipid rafts. We recently reported that raft affiliation of CD44 negatively regulates interactions with its migratory binding partner ezrin. Since raft affiliation is regulated by post-translational modifications including palmitoylation, we sought to establish the contribution of CD44 palmitoylation and lipid raft affiliation to cell migration. Methods Recovery of CD44 and its binding partners from raft versus non-raft membrane microdomains was profiled in non-migrating and migrating breast cancer cell lines. Site-directed mutagenesis was used to introduce single or double point mutations into both CD44 palmitoylation sites (Cys286 and Cys295), whereupon the implications for lipid raft recovery, phenotype, ezrin co-precipitation and migratory behaviour was assessed. Finally CD44 palmitoylation status and lipid raft affiliation was assessed in primary cultures from a small panel of breast cancer patients. Results CD44 raft affiliation was increased during migration of non-invasive breast cell lines, but decreased during migration of highly-invasive breast cells. The latter was paralleled by increased CD44 recovery in non-raft fractions, and exclusive non-raft recovery of its binding partners. Point mutation of CD44 palmitoylation sites reduced CD44 raft affiliation in invasive MDA-MB-231 cells, increased CD44-ezrin co-precipitation and accordingly enhanced cell migration. Expression of palmitoylation-impaired (raft-excluded) CD44 mutants in non-invasive MCF-10a cells was sufficient to reversibly induce the phenotypic appearance of epithelial-to-mesenchymal transition and to increase cell motility. Interestingly, cell migration was associated with temporal reductions in CD44 palmitoylation in wild-type breast cells. Finally, the relevance of these findings is underscored by the fact that levels of palmitoylated CD44 were lower in primary cultures from invasive ductal carcinomas relative to non-tumour tissue, while CD44 co-localisation with a lipid raft marker was less in invasive ductal carcinoma relative to ductal carcinoma in situ cultures. Conclusion Our results support a novel mechanism whereby CD44 palmitoylation and consequent lipid raft affiliation inversely regulate breast cancer cell migration, and may act as a new therapeutic target in breast cancer metastasis. PMID:24512624

CD44-engineered mesoporous silica nanoparticles for overcoming multidrug resistance in breast cancer

NASA Astrophysics Data System (ADS)

Wang, Xin; Liu, Ying; Wang, Shouju; Shi, Donghong; Zhou, Xianguang; Wang, Chunyan; Wu, Jiang; Zeng, Zhiyong; Li, Yanjun; Sun, Jing; Wang, Jiandong; Zhang, Longjiang; Teng, Zhaogang; Lu, Guangming

2015-03-01

Multidrug resistance is a major impediment for the successful chemotherapy in breast cancer. CD44 is over-expressed in multidrug resistant human breast cancer cells. CD44 monoclonal antibody exhibits anticancer potential by inhibiting proliferation and regulating P-glycoprotein-mediated drug efflux activity in multidrug resistant cells. Thereby, CD44 monoclonal antibody in combination with chemotherapeutic drug might be result in enhancing chemosensitivity and overcoming multidrug resistance. The purpose of this study is to investigate the effects of the CD44 monoclonal antibody functionalized mesoporous silica nanoparticles containing doxorubicin on human breast resistant cancer MCF-7 cells. The data showed that CD44-modified mesoporous silica nanoparticles increased cytotoxicity and enhanced the downregulation of P-glycoprotein in comparison to CD44 antibody. Moreover, CD44-engineered mesoporous silica nanoparticles provided active target, which promoted more cellular uptake of DOX in the resistant cells and more retention of DOX in tumor tissues than unengineered counterpart. Animal studies of the resistant breast cancer xenografts demonstrated that CD44-engineered drug delivery system remarkably induced apoptosis and inhibited the tumor growth. Our results indicated that the CD44-engineered mesoporous silica nanoparticle-based drug delivery system offers an effective approach to overcome multidrug resistance in human breast cancer.

Equine Mesenchymal Stromal Cells Retain a Pericyte-Like Phenotype

PubMed Central

Sheldrake, Tara A.; Dawson, Lucy; Menghini, Timothy; Rink, Burgunde Elisabeth; Amilon, Karin; Khan, Nusrat; Péault, Bruno; Donadeu, Francesc Xavier

2017-01-01

Mesenchymal stem/stromal cells (MSCs) have been used in human and equine regenerative medicine, and interest in exploiting their potential has increased dramatically over the years. Despite significant effort to characterize equine MSCs, the actual origin of these cells and how much of their native phenotype is maintained in culture have not been determined. In this study, we investigated the relationship between MSCs, derived from adipose tissue (AT) and bone marrow (BM), and pericytes in the horse. Both pericyte (CD146, NG2, and αSMA) and MSC (CD29, CD90, and CD73) markers were detected in equine AT and colocalized around blood vessels. Importantly, as assessed by flow cytometry, both pericyte (CD146, NG2, and αSMA) and MSC (CD29, CD44, CD90, and CD105) markers were present in a majority (≥90%) of cells in cultures of AT-MSCs and BM-MSCs; however, levels of pericyte markers were variable within each of those populations. Moreover, the expression of pericyte markers was maintained for at least eight passages in both AT-MSCs and BM-MSCs. Hematopoietic (CD45) and endothelial (CD144) markers were also detected at low levels in MSCs by quantitative polymerase chain reaction (qPCR). Finally, in coculture experiments, AT-MSCs closely associated with networks produced by endothelial cells, resembling the natural perivascular location of pericytes in vivo. Our results indicate that equine MSCs originate from perivascular cells and moreover maintain a pericyte-like phenotype in culture. Therefore, we suggest that, in addition to classical MSC markers, pericyte markers such as CD146 could be used when assessing and characterizing equine MSCs. PMID:28376684

Equine Mesenchymal Stromal Cells Retain a Pericyte-Like Phenotype.

PubMed

Esteves, Cristina L; Sheldrake, Tara A; Dawson, Lucy; Menghini, Timothy; Rink, Burgunde Elisabeth; Amilon, Karin; Khan, Nusrat; Péault, Bruno; Donadeu, Francesc Xavier

2017-07-01

Mesenchymal stem/stromal cells (MSCs) have been used in human and equine regenerative medicine, and interest in exploiting their potential has increased dramatically over the years. Despite significant effort to characterize equine MSCs, the actual origin of these cells and how much of their native phenotype is maintained in culture have not been determined. In this study, we investigated the relationship between MSCs, derived from adipose tissue (AT) and bone marrow (BM), and pericytes in the horse. Both pericyte (CD146, NG2, and αSMA) and MSC (CD29, CD90, and CD73) markers were detected in equine AT and colocalized around blood vessels. Importantly, as assessed by flow cytometry, both pericyte (CD146, NG2, and αSMA) and MSC (CD29, CD44, CD90, and CD105) markers were present in a majority (≥90%) of cells in cultures of AT-MSCs and BM-MSCs; however, levels of pericyte markers were variable within each of those populations. Moreover, the expression of pericyte markers was maintained for at least eight passages in both AT-MSCs and BM-MSCs. Hematopoietic (CD45) and endothelial (CD144) markers were also detected at low levels in MSCs by quantitative polymerase chain reaction (qPCR). Finally, in coculture experiments, AT-MSCs closely associated with networks produced by endothelial cells, resembling the natural perivascular location of pericytes in vivo. Our results indicate that equine MSCs originate from perivascular cells and moreover maintain a pericyte-like phenotype in culture. Therefore, we suggest that, in addition to classical MSC markers, pericyte markers such as CD146 could be used when assessing and characterizing equine MSCs.

Conservation of CD44 exon v3 functional elements in mammals

PubMed Central

Vela, Elena; Hilari, Josep M; Delclaux, María; Fernández-Bellon, Hugo; Isamat, Marcos

2008-01-01

Background The human CD44 gene contains 10 variable exons (v1 to v10) that can be alternatively spliced to generate hundreds of different CD44 protein isoforms. Human CD44 variable exon v3 inclusion in the final mRNA depends on a multisite bipartite splicing enhancer located within the exon itself, which we have recently described, and provides the protein domain responsible for growth factor binding to CD44. Findings We have analyzed the sequence of CD44v3 in 95 mammalian species to report high conservation levels for both its splicing regulatory elements (the 3' splice site and the exonic splicing enhancer), and the functional glycosaminglycan binding site coded by v3. We also report the functional expression of CD44v3 isoforms in peripheral blood cells of different mammalian taxa with both consensus and variant v3 sequences. Conclusion CD44v3 mammalian sequences maintain all functional splicing regulatory elements as well as the GAG binding site with the same relative positions and sequence identity previously described during alternative splicing of human CD44. The sequence within the GAG attachment site, which in turn contains the Y motif of the exonic splicing enhancer, is more conserved relative to the rest of exon. Amplification of CD44v3 sequence from mammalian species but not from birds, fish or reptiles, may lead to classify CD44v3 as an exclusive mammalian gene trait. PMID:18710510

The High and Low Molecular Weight Forms of Hyaluronan Have Distinct Effects on CD44 Clustering*

PubMed Central

Yang, Cuixia; Cao, Manlin; Liu, Hua; He, Yiqing; Xu, Jing; Du, Yan; Liu, Yiwen; Wang, Wenjuan; Cui, Lian; Hu, Jiajie; Gao, Feng

2012-01-01

CD44 is a major cell surface receptor for the glycosaminoglycan hyaluronan (HA). Native high molecular weight hyaluronan (nHA) and oligosaccharides of hyaluronan (oHA) provoke distinct biological effects upon binding to CD44. Despite the importance of such interactions, however, the feature of binding with CD44 at the cell surface and the molecular basis for functional distinction between different sizes of HA is still unclear. In this study we investigated the effects of high and low molecular weight hyaluronan on CD44 clustering. For the first time, we provided direct evidence for a strong relationship between HA size and CD44 clustering in vivo. In CD44-transfected COS-7 cells, we showed that exogenous nHA stimulated CD44 clustering, which was disrupted by oHA. Moreover, naturally expressed CD44 was distributed into clusters due to abundantly expressed nHA in HK-2 cells (human renal proximal tubule cells) and BT549 cells (human breast cancer cell line) without exogenous stimulation. Our results suggest that native HA binding to CD44 selectively induces CD44 clustering, which could be inhibited by oHA. Finally, we demonstrated that HA regulates cell adhesion in a manner specifically dependent on its size. oHA promoted cell adhesion while nHA showed no effects. Our results might elucidate a molecular- and/or cellular-based mechanism for the diverse biological activities of nHA and oHA. PMID:23118219

CD44-mediated activation of α5β1-integrin, cortactin and paxillin signaling underpins adhesion of basal-like breast cancer cells to endothelium and Fibronectin-enriched matrices

PubMed Central

McFarlane, Suzanne; McFarlane, Cheryl; Montgomery, Nicola; Hill, Ashleigh; Waugh, David J.J.

2015-01-01

CD44 expression is elevated in basal-like breast cancer (BLBC) tissue, and correlates with increased efficiency of distant metastasis in patients and experimental models. We sought to characterize mechanisms underpinning CD44-promoted adhesion of BLBC cells to vascular endothelial monolayers and extracellular matrix (ECM) substrates. Stimulation with hyaluronan (HA), the native ligand for CD44, increased expression and activation of β1-integrin receptors, and increased α5-integrin subunit expression. Adhesion assays confirmed that CD44-signalling potentiated BLBC cell adhesion to endothelium and Fibronectin in an α5B1-integrin-dependent mechanism. Co-immunoprecipitation experiments confirmed HA-promoted association of CD44 with talin and the β1-integrin chain in BLBC cells. Knockdown of talin inhibited CD44 complexing with β1-integrin and repressed HA-induced, CD44-mediated activation of β1-integrin receptors. Immunoblotting confirmed that HA induced rapid phosphorylation of cortactin and paxillin, through a CD44-dependent and β1-integrin-dependent mechanism. Knockdown of CD44, cortactin or paxillin independently attenuated the adhesion of BL-BCa cells to endothelial monolayers and Fibronectin. Accordingly, we conclude that CD44 induced, integrin-mediated signaling not only underpins efficient adhesion of BLBC cells to BMECs to facilitate extravasation but initiates their adhesion to Fibronectin, enabling penetrant cancer cells to adhere more efficiently to underlying Fibronectin-enriched matrix present within the metastatic niche. PMID:26447611

Differential expression of CD44 and CD24 markers discriminates the epitheliod from the fibroblastoid subset in a sarcomatoid renal carcinoma cell line: evidence suggesting the existence of cancer stem cells in both subsets as studied with sorted cells.

PubMed

Hsieh, Chin-Hsuan; Hsiung, Shih-Chieh; Yeh, Chi-Tai; Yen, Chih-Feng; Chou, Yah-Huei Wu; Lei, Wei-Yi; Pang, See-Tong; Chuang, Cheng-Keng; Liao, Shuen-Kuei

2017-02-28

Epithelioid and fibroblastoid subsets coexist in the human sarcomatoid renal cell carcinoma (sRCC) cell line, RCC52, according to previous clonal studies. Herein, using monoclonal antibodies to CD44 and CD24 markers, we identified and isolated these two populations, and showed that CD44bright/CD24dim and CD44bright/CD24bright phenotypes correspond to epithelioid and fibroblastoid subsets, respectively. Both sorted subsets displayed different levels of tumorigenicity in xenotransplantation, indicating that each harbored its own cancer stem cells (CSCs). The CD44bright/CD24bright subset, associated with higher expression of MMP-7, -8 and TIMP-1 transcripts, showed greater migratory/invasive potential than the CD44bright/CD24dim subset, which was associated with higher expression of MMP-2, -9 and TIMP-2 transcripts. Both subsets differentially expressed stemness gene products c-Myc, Oct4A, Notch1, Notch2 and Notch3, and the RCC stem cell marker, CD105 in 4-5% of RCC52 cells. These results suggest the presence of CSCs in both sRCC subsets for the first time and should therefore be considered potential therapeutic targets for this aggressive malignancy.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yoshida, Go J., E-mail: medical21go@yahoo.co.jp; Saya, Hideyuki

Highlights: •CD44 variant8–10 and c-Myc are inversely expressed in gastric cancer cells. •Redox-stress enhances c-Myc expression via canonical Wnt signal. •CD44v, but not CD44 standard, suppresses redox stress-induced Wnt activation. •CD44v expression promotes both transcription and proteasome degradation of c-Myc. •Inversed expression pattern between CD44v and c-Myc is often recognized in vivo. -- Abstract: Cancer stem-like cells express high amount of CD44 variant8-10 which protects cancer cells from redox stress. We have demonstrated by immunohistochemical analysis and Western blotting, and reverse-transcription polymerase chain reaction, that CD44 variant8-10 and c-Myc tend to show the inversed expression manner in gastric cancer cells.more » That is attributable to the oxidative stress-induced canonical Wnt activation, and furthermore, the up-regulation of the downstream molecules, one of which is oncogenic c-Myc, is not easily to occur in CD44 variant-positive cancer cells. We have also found out that CD44v8-10 expression is associated with the turn-over of the c-Myc with the experiments using gastric cancer cell lines. This cannot be simply explained by the model of oxidative stress-induced Wnt activation. CD44v8-10-positive cancer cells are enriched at the invasive front. Tumor tissue at the invasive area is considered to be composed of heterogeneous cellular population; dormant cancer stem-like cells with CD44v8-10 {sup high}/ Fbw7 {sup high}/ c-Myc {sup low} and proliferative cancer stem-like cells with CD44v8-10 {sup high}/ Fbw7 {sup low}/ c-Myc {sup high}.« less

Activation of p44/42 in Human Natural Killer Cells Decreases Cell-surface Protein Expression: Relationship to Tributyltin-induced alterations of protein expression

PubMed Central

Dudimah, Fred D.; Abraha, Abraham; Wang, Xiaofei; Whalen, Margaret M.

2010-01-01

Tributyltin (TBT) activates the mitogen activated protein kinase (MAPK), p44/42 in human natural killer (NK) cells. TBT also reduces NK cytotoxic function and decreases the expression of several NK-cell proteins. To understand the role that p44/42 activation plays in TBT-induced loss of NK cell function, we have investigated how selective activation of p44/42 by phorbol 12-myristate 13-acetate (PMA) affects NK cells. Previously we showed that PMA caused losses of lytic function similar to those seen with TBT exposures. Here we examined activation of p44/42 in the regulation of NK-cell protein expression and how this regulation may explain the protein expression changes seen with TBT exposures. NK cells exposed to PMA were examined for levels of cell-surface proteins, granzyme mRNA, and perforin mRNA expression. The expression of CD11a, CD16, CD18, and CD56 were reduced, perforin mRNA levels were unchanged and granzyme mRNA levels were increased. To verify that activation of p44/42 was responsible for the alterations seen in CD11a, CD16, CD18, and CD56 with PMA, NK cells were treated with the p44/42 pathway inhibitor (PD98059) prior to PMA exposures. In the presence of PD98059, PMA caused no decreases in the expression of the cell-surface proteins. Results of these studies indicate that the activation of p44/42 may lead to the loss of NK cell cytotoxic function by decreasing the expression of CD11a, CD16, CD18, and CD56. Further, activation of p44/42 appears to be at least in part responsible for the TBT-induced decreases in expression of CD16, CD18, and CD56. PMID:20883105

Macrophage Migration Inhibitory Factor Mediates Proliferative GN via CD74

PubMed Central

Djudjaj, Sonja; Lue, Hongqi; Rong, Song; Papasotiriou, Marios; Klinkhammer, Barbara M.; Zok, Stephanie; Klaener, Ole; Braun, Gerald S.; Lindenmeyer, Maja T.; Cohen, Clemens D.; Bucala, Richard; Tittel, Andre P.; Kurts, Christian; Moeller, Marcus J.; Floege, Juergen; Ostendorf, Tammo

2016-01-01

Pathologic proliferation of mesangial and parietal epithelial cells (PECs) is a hallmark of various glomerulonephritides. Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine that mediates inflammation by engagement of a receptor complex involving the components CD74, CD44, CXCR2, and CXCR4. The proliferative effects of MIF may involve CD74 together with the coreceptor and PEC activation marker CD44. Herein, we analyzed the effects of local glomerular MIF/CD74/CD44 signaling in proliferative glomerulonephritides. MIF, CD74, and CD44 were upregulated in the glomeruli of patients and mice with proliferative glomerulonephritides. During disease, CD74 and CD44 were expressed de novo in PECs and colocalized in both PECs and mesangial cells. Stress stimuli induced MIF secretion from glomerular cells in vitro and in vivo, in particular from podocytes, and MIF stimulation induced proliferation of PECs and mesangial cells via CD74. In murine crescentic GN, Mif-deficient mice were almost completely protected from glomerular injury, the development of cellular crescents, and the activation and proliferation of PECs and mesangial cells, whereas wild-type mice were not. Bone marrow reconstitution studies showed that deficiency of both nonmyeloid and bone marrow–derived Mif reduced glomerular cell proliferation and injury. In contrast to wild-type mice, Cd74-deficient mice also were protected from glomerular injury and ensuing activation and proliferation of PECs and mesangial cells. Our data suggest a novel molecular mechanism and glomerular cell crosstalk by which local upregulation of MIF and its receptor complex CD74/CD44 mediate glomerular injury and pathologic proliferation in GN. PMID:26453615

Progenitor Cells from Cartilage: Grade Specific Differences in Stem Cell Marker Expression

PubMed Central

Mazor, Marija; Cesaro, Annabelle; Ali, Mazen; Best, Thomas M.; Lespessaille, Eric; Toumi, Hechmi

2017-01-01

Recent research has confirmed the presence of Mesenchymal stem cell (MSC)-like progenitors (MPC) in both normal and osteoarthritic cartilage. However, there is only limited information concerning how MPC markers are expressed with osteoarthritis (OA) progression. The purpose of this study was to compare the prevalence of various MPC markers in different OA grades. Human osteoarthritic tibial plateaus were obtained from ten patients undergoing total knee replacement. Each sample had been classified into a mild or severe group according to OARSI scoring. Tissue was taken from each specimen and mRNA expression levels of CD105, CD166, Notch 1, Sox9, Acan and Col II A1 were measured at day 0 and day 14 (2 weeks in vitro). Furthermore, MSC markers: Nucleostemin, CD90, CD73, CD166, CD105 and Notch 1 were studied by immunofluorescence. mRNA levels of MSC markers did not differ between mild and severe OA at day 0. At day 14, protein analysis showed that proliferated cells from both sources expressed all 6 MSC markers. Only cells from the mild OA subjects resulted in a significant increase of mRNA CD105 and CD166 after in vitro expansion. Moreover, cells from the mild OA subjects showed significantly higher levels of CD105, Sox9 and Acan compared with those from severe OA specimens. Results confirmed the presence of MSC markers in mild and severe OA tissue at both mRNA and protein levels. We found significant differences between cells obtained from mild compared to severe OA specimens suggests that mild OA derived cells may have a greater MSC potential. PMID:28805694

CD44 Staining of Cancer Stem-Like Cells Is Influenced by Down-Regulation of CD44 Variant Isoforms and Up-Regulation of the Standard CD44 Isoform in the Population of Cells That Have Undergone Epithelial-to-Mesenchymal Transition

PubMed Central

Biddle, Adrian; Gammon, Luke; Fazil, Bilal; Mackenzie, Ian C.

2013-01-01

CD44 is commonly used as a cell surface marker of cancer stem-like cells in epithelial tumours, and we have previously demonstrated the existence of two different CD44high cancer stem-like cell populations in squamous cell carcinoma, one having undergone epithelial-to-mesenchymal transition and the other maintaining an epithelial phenotype. Alternative splicing of CD44 variant exons generates a great many isoforms, and it is not known which isoforms are expressed on the surface of the two different cancer stem-like cell phenotypes. Here, we demonstrate that cancer stem-like cells with an epithelial phenotype predominantly express isoforms containing the variant exons, whereas the cancer stem-like cells that have undergone an epithelial-to-mesenchymal transition down-regulate these variant isoforms and up-regulate expression of the standard CD44 isoform that contains no variant exons. In addition, we find that enzymatic treatments used to dissociate cells from tissue culture or fresh tumour specimens cause destruction of variant CD44 isoforms at the cell surface whereas expression of the standard CD44 isoform is preserved. This results in enrichment within the CD44high population of cancer stem-like cells that have undergone an epithelial-to-mesenchymal transition and depletion from the CD44high population of cancer stem-like cells that maintain an epithelial phenotype, and therefore greatly effects the characteristics of any cancer stem-like cell population isolated based on expression of CD44. As well as effecting the CD44high population, enzymatic treatment also reduces the percentage of the total epithelial cancer cell population staining CD44-positive, with potential implications for studies that aim to use CD44-positive staining as a prognostic indicator. Analyses of the properties of cancer stem-like cells are largely dependent on the ability to accurately identify and assay these populations. It is therefore critical that consideration be given to use of multiple cancer stem-like cell markers and suitable procedures for cell isolation in order that the correct populations are assayed. PMID:23437366

Expression of CD43 in chronic lymphoproliferative leukemias.

PubMed

Sorigue, Marc; Juncà, Jordi; Sarrate, Edurne; Grau, Javier

2018-01-01

CD43 has been used on histological samples for the differential diagnosis of lymphoproliferative disorders but there is scarce data on its use by flow cytometry (FC). We set out to characterize the expression of CD43 by FC in B-cell lymphoproliferative disorders and to determine its possible role in the differential diagnosis of these malignancies. We analyzed the expression of CD43 in clonal B-cell lymphoproliferative disorders with exclusive peripheral blood and/or bone marrow involvement based on their Moreau chronic lymphocytic leukemia (CLL) score with particular emphasis on Moreau CLL score 3 (MS3) cases, which often present a diagnostic challenge. The cohort included 433 CLL (score 4-5), 34 MS3 and 166 lymphoproliferative disorders with lower scores. Generally, the higher the Moreau CLL score, the higher CD43-positivity (425/443 [96%] for CLL, 23/34 [67%] for MS3 and 18/166 [11%] for cases with lower scores). MS3 cases constituted 5.4% of all cases and were more frequently CD5, CD200, CD43-positive and had del(q13) than score 0-2 cases. Among MS3 cases, del(13q) cases were predominantly CD43-positive (12/13). The frequency of CD43-positivity increases sharply with the Moreau score. MS3 cases seem to include both CLL and non-CLL lymphoproliferative disorders and CD43 could aid in the differential diagnosis between the two. However, studies analyzing the correlation between CD43 expression and the underlying biologic changes of these cases are warranted. © 2017 International Clinical Cytometry Society. © 2017 International Clinical Cytometry Society.

Expression profiles of cancer stem cell markers: CD133, CD44, Musashi-1 and EpCAM in the cardiac mucosa-Barrett's esophagus-early esophageal adenocarcinoma-advanced esophageal adenocarcinoma sequence.

PubMed

Mokrowiecka, Anna; Veits, Lothar; Falkeis, Christina; Musial, Jacek; Kordek, Radzislaw; Lochowski, Mariusz; Kozak, Jozef; Wierzchniewska-Lawska, Agnieszka; Vieth, Michael; Malecka-Panas, Ewa

2017-03-01

Barrett's esophagus (BE), which develops as a result of gastroesophageal reflux disease, is a preneoplastic condition for esophageal adenocarcinoma (EAC). A new hypothesis suggests that cancer is a disease of stem cells, however, their expression and pathways in BE - EAC sequence are not fully elucidated yet. We used a panel of putative cancer stem cells markers to identify stem cells in consecutive steps of BE-related cancer progression. Immunohistochemistry was performed on formalin-fixed, paraffin-embedded blocks from 58 patients with normal cardiac mucosa (n=5), BE (n=14), early EAC (pT1) from mucosal resection (n=17) and advanced EAC (pT1-T4) from postoperative specimens (n=22). Expression of the CD133, CD44, Musashi-1 and EpCAM was analyzed using respective monoclonal antibodies. All markers showed a heterogeneous expression pattern, mainly at the base of the crypts of Barrett's epithelium and EAC, with positive stromal cells in metaplastic and dysplastic lesions. Immuno-expression of EpCAM, CD44 and CD133 in cardiac mucosa was significantly lower (mean immunoreactivity score (IRS)=1.2; 0.0; 0.4; respectively) compared to their expression in Barrett's metaplasia (mean IRS=4.3; 0.14; 0.7; respectively), in early adenocarcinoma (mean IRS=4.4; 0.29; 1.3; respectively) and in advanced adenocarcinoma (mean IRS=6.6; 0.7; 2.7; respectively) (p<0.05). On the contrary, Musashi-1 expression was higher in BE and early ADC compared to GM and advanced ADC (NS). Our results suggest that the stem cells could be present in premalignant lesions. EpCAM, CD44 and CD133 expression could be candidate markers for BE progression, whereas Musashi-1 may be a marker of the small intestinal features of Barrett's mucosa. Copyright © 2016 Elsevier GmbH. All rights reserved.

CD44 as a receptor for colonization of the pharynx by group A Streptococcus

PubMed Central

Cywes, Colette; Stamenkovic, Ivan; Wessels, Michael R.

2000-01-01

The pharynx is the primary reservoir for strains of group A Streptococcus (GAS) associated both with pharyngitis (streptococcal sore throat) and with invasive or “flesh-eating” soft tissue infections. We now report that CD44, a hyaluronic acid-binding protein that mediates human cell-cell– and cell-extracellular matrix–binding interactions, functions as a receptor for GAS colonization of the pharynx in vivo. We found that attachment of GAS to murine epithelial keratinocytes was mediated by binding of the GAS hyaluronic acid capsular polysaccharide to CD44. In studies of transgenic mice with a selective defect in epithelial expression of CD44, GAS adherence to CD44-deficient keratinocytes in vitro was reduced compared with adherence to keratinocytes expressing normal levels of CD44. After intranasal inoculation, GAS colonized the oropharynx of wild-type mice but failed to colonize transgenic mice deficient in CD44 expression. GAS colonization of wild-type mice could be blocked by coadministration of mAb to CD44 or by pretreatment of the animals with exogenous hyaluronic acid. These results provide evidence that CD44 serves as a receptor for GAS colonization of the pharynx and support the potential efficacy of disrupting the interaction between the GAS hyaluronic acid capsule and CD44 as a novel approach to preventing pharyngeal infection. PMID:11032859

Expression and significance of CD44s, CD44v6, and nm23 mRNA in human cancer.

PubMed

Liu, Yong-Jun; Yan, Pei-Song; Li, Jun; Jia, Jing-Fen

2005-11-14

To investigate the relationship between the expression levels of nm23 mRNA, CD44s, and CD44v6, and oncogenesis, development and metastasis of human gastric adenocarcinoma, colorectal adenocarcinoma, intraductal carcinoma of breast, and lung cancer. Using tissue microarray by immuhistochemical (IHC) staining and in situ hybridization (ISH), we examined the expression levels of nm23 mRNA, CD44s, and CD44v6 in 62 specimens of human gastric adenocarcinoma and 62 specimens of colorectal adenocarcinoma; the expression of CD44s and CD44v6 in 120 specimens of intraductal carcinoma of breast and 20 specimens of normal breast tissue; the expression of nm23 mRNA in 72 specimens of human lung cancer and 23 specimens of normal tissue adjacent to cancer. The expression of nm23 mRNA in the tissues of gastric and colorectal adenocarcinoma was not significantly different from that in the normal tissues adjacent to cancer (P>0.05), and was not associated with the invasion of tumor and the pathology grade of adenocarcinoma (P>0.05). However, the expression of nm23 mRNA was correlated negatively to the lymph node metastasis of gastric and colorectal adenocarcinoma (r = -0.49, P<0.01; r = -4.93, P<0.01). The expression of CD44s in the tissues of gastric and colorectal adenocarcinoma was significantly different from that in the normal tissues adjacent to cancer (P<0.05; P<0.01). CD44v6 was expressed in the tissues of gastric and colorectal adenocarcinoma only, the expression of CD44v6 was significantly associated with the lymph node metastasis, invasion and pathological grade of the tumor (r = 0.47, P<0.01; r = 5.04, P<0.01). CD44s and CD44v6 were expressed in intraductal carcinoma of breast, the expression of CD44s and CD44v6 was significantly associated with lymph node metastases and invasion (P<0.01). However, neither of them was expressed in the normal breast tissue. In addition, the expression of CD44v6 was closely related to the degree of cell differentiation of intraductal carcinoma of breast (c2 = 5.68, P<0.05). The expressional level of nm23 mRNA was closely related to the degree of cell differentiation (P<0.05) and lymph node metastasis (P<0.01), but the expression of nm23 gene was not related to sex, age, and type of histological classification (P>0.05). Patients with overexpression of CD44s and CD44v6 and low expression of nm23 mRNA have a higher lymph node metastatic rate and invasion. In addition, overexpression of CD44v6 is closely related to the degree of cell differentiation. Detection of the three genes is able to provide a reliable index to evaluate the invasion and metastasis of tumor cells.

Co-Expression of Putative Cancer Stem Cell Markers CD44 and CD133 in Prostate Carcinomas.

PubMed

Kalantari, Elham; Asgari, Mojgan; Nikpanah, Seyedehmoozhan; Salarieh, Naghme; Asadi Lari, Mohammad Hossein; Madjd, Zahra

2017-10-01

Cancer stem cells (CSCs) are the main players of prostate tumorigenesis thus; characterization of CSCs can pave the way for understanding the early detection, drug resistance, metastasis and relapse. The current study was conducted to evaluate the expression level and clinical significance of the potential CSC markers CD44 and CD133 in a series of prostate tissues. One hundred and forty eight prostate tissues composed of prostate cancer (PCa), high-grade prostatic intraepithelial neoplasia (HGPIN), and benign prostate hyperplasia (BPH) were immunostained for the putative CSC markers CD44 and CD133. Subsequently, the correlation between the expression of these markers and the clinicopathological variables was examined. A higher level of CD44 expression was observed in 42% of PCa, 57% of HGPIN, and 42% BPH tissues. In the case of CD133 expression PCa, HGPIN, and BPH samples demonstrated high immunoreactivity in 46%, 43%, and 42% of cells, respectively. Statistical analysis showed an inverse significant correlation between CD44 expression with Gleason score of PCa (P = 0.02), while no significant correlation was observed between CD133 expression and clinicopathological parameters. A significant reciprocal correlation was observed between the expression of two putative CSC markers CD44 and CD133 in PCa specimens while not indicating clinical significance. Further clinical investigation is required to consider these markers as targets of new therapeutic strategies for PCa.

Profiling of normal and malignant breast tissue show CD44high/CD24low phenotype as a predominant stem/progenitor marker when used in combination with Ep-CAM/CD49f markers

PubMed Central

2013-01-01

Background Accumulating evidence supports cancer to initiate and develop from a small population of stem-like cells termed as cancer stem cells (CSC). The exact phenotype of CSC and their counterparts in normal mammary gland is not well characterized. In this study our aim was to evaluate the phenotype and function of stem/progenitor cells in normal mammary epithelial cell populations and their malignant counterparts. Methods Freshly isolated cells from both normal and malignant human breasts were sorted using 13 widely used stem/progenitor cell markers individually or in combination by multi-parametric (up to 9 colors) cell sorting. The sorted populations were functionally evaluated by their ability to form colonies and mammospheres, in vitro. Results We have compared, for the first time, the stem/progenitor markers of normal and malignant breasts side-by-side. Amongst all markers tested, we found CD44high/CD24low cell surface marker combination to be the most efficient at selecting normal epithelial progenitors. Further fractionation of CD44high/CD24low positive cells showed that this phenotype selects for luminal progenitors within Ep-CAMhigh/CD49f + cells, and enriches for basal progenitors within Ep-CAM-/low/CD49f + cells. On the other hand, primary breast cancer samples, which were mainly luminal Ep-CAMhigh, had CD44high/CD24low cells among both CD49fneg and CD49f + cancer cell fractions. However, functionally, CSC were predominantly CD49f + proposing the use of CD44high/CD24low in combination with Ep-CAM/CD49f cell surface markers to further enrich for CSC. Conclusion Our study clearly demonstrates that both normal and malignant breast cells with the CD44high/CD24low phenotype have the highest stem/progenitor cell ability when used in combination with Ep-CAM/CD49f reference markers. We believe that this extensive characterization study will help in understanding breast cancer carcinogenesis, heterogeneity and drug resistance. PMID:23768049

HAb18G/CD147 Promotes pSTAT3-Mediated Pancreatic Cancer Development via CD44s †, ‡

PubMed Central

Li, Ling; Tang, Wenhua; Wu, Xiaoqing; Karnak, David; Meng, Xiaojie; Thompson, Rachel; Hao, Xinbao; Li, Yongmin; Qiao, Xiaotan T.; Lin, Jiayuh; Fuchs, James; Simeone, Diane M.; Chen, Zhi-Nan; Lawrence, Theodore S.; Xu, Liang

2013-01-01

Purpose STAT3 plays a critical role in initiation and progression of pancreatic cancer. However, therapeutically targeting STAT3 is failure in clinic. We previously identified HAb18G/CD147 as an effective target for cancer treatment. In this study, we aimed to investigate potential role of HAb18G/CD147 in STAT3-involved pancreatic tumorigenesis in vitro and in vivo. Experimental Design The expression of HAb18G/CD147, pSTAT3 and CD44s were determined in tissue microarrays. The tumorigenic function and molecular signaling mechanism of HAb18G/CD147 was assessed by in vitro cellular and clonogenic growth, reporter assay, immunoblot, immunofluorescence staining, immunoprecipitation, and in vivo tumor formationusing loss or gain-of-function strategies. Results Highly expressed HAb18G/CD147 promoted cellular and clonogenic growth in vitro and tumorigenicity in vivo. CyPA, a ligand of CD147, stimulated STAT3 phosphorylation and its downstream genes cyclin D1/survivin through HAb18G/CD147 dependent mechanisms. HAb18G/CD147 was associated and co-localized with cancer stem cell marker CD44s in lipid rafts. The inhibitors of STAT3 and survivin, as well as CD44s neutralizing antibodies suppressed the HAb18G/CD147-induced cell growth. High HAb18G/CD147 expression in pancreatic cancer was significantly correlated with the poor tumor differentiation, and the high co-expression of HAb18G/CD147-CD44s-STAT3 associated with poor survival of patients with pancreatic cancer. Conclusions We identified HAb18G/CD147 as a novel upstream activator of STAT3 via interacts with CD44s and plays a critical role in the development of pancreatic cancer. The data suggest HAb18G/CD147 could be a promising therapeutic target for highly aggressive pancreatic cancer and a surrogate marker in the STAT3-targeted molecular therapies. PMID:24132924

GFP Labeling and Hepatic Differentiation Potential of Human Placenta-Derived Mesenchymal Stem Cells.

PubMed

Yu, Jiong; Su, Xiaoru; Zhu, Chengxing; Pan, Qiaoling; Yang, Jinfeng; Ma, Jing; Shen, Leyao; Cao, Hongcui; Li, Lanjuan

2015-01-01

Stem cell-based therapy in liver diseases has received increasing interest over the past decade, but direct evidence of the homing and implantation of transplanted cells is conflicting. Reliable labeling and tracking techniques are essential but lacking. The purpose of this study was to establish human placenta-derived mesenchymal stem cells (hPMSCs) expressing green fluorescent protein (GFP) and to assay their hepatic functional differentiation in vitro. The GFP gene was transduced into hPMSCs using a lentivirus to establish GFP(+) hPMSCs. GFP(+) hPMSCs were analyzed for their phenotypic profile, viability and adipogenic, osteogenic and hepatic differentiation. The derived GFP(+) hepatocyte-like cells were evaluated for their metabolic, synthetic and secretory functions, respectively. GFP(+) hPMSCs expressed high levels of HLA I, CD13, CD105, CD73, CD90, CD44 and CD29, but were negative for HLA II, CD45, CD31, CD34, CD133, CD271 and CD79. They possessed adipogenic, osteogenic and hepatic differentiation potential. Hepatocyte-like cells derived from GFP(+) hPMSCs showed typical hepatic phenotypes. GFP gene transduction has no adverse influences on the cellular or biochemical properties of hPMSCs or markers. GFP gene transduction using lentiviral vectors is a reliable labeling and tracking method. GFP(+) hPMSCs can therefore serve as a tool to investigate the mechanisms of MSC-based therapy, including hepatic disease therapy. © 2015 S. Karger AG, Basel.

CD44S-hyaluronan interactions protect cells resulting from EMT against anoikis

PubMed Central

Cieply, Benjamin; Koontz, Colton; Frisch, Steven M.

2016-01-01

The detachment of normal epithelial cells from matrix triggers an apoptotic response known as anoikis, during homeostatic turnover. Metastatic tumor cells evade anoikis, by mechanisms that are only partly characterized. In particular, the epithelial–mesenchymal transition (EMT) in a subset of invasive tumor cells confers anoikis-resistance. In some cases, EMT up-regulates the cancer stem cell marker CD44S and the enzyme hyaluronic acid synthase-2 (HAS2). CD44S is the major receptor for hyaluronan in the extracellular matrix. Herein, we demonstrate that CD44S, unlike the CD44E isoform expressed in normal epithelial cells, contributes to the protection against anoikis. This protection requires the interaction of CD44S with hyaluronan (HA). CD44S–HA interaction is proposed to play an important role in tumor metastasis through enhanced cell survival under detached conditions. PMID:25937513

Radiation increases the cellular uptake of exosomes through CD29/CD81 complex formation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hazawa, Masaharu; Tomiyama, Kenichi; Saotome-Nakamura, Ai

Highlights: • Radiation increases cellular uptake of exosomes. • Radiation induces colocalization of CD29 and CD81. • Exosomes selectively bind the CD29/CD81 complex. • Radiation increases the cellular uptake of exosomes through CD29/CD81 complex formation. - Abstract: Exosomes mediate intercellular communication, and mesenchymal stem cells (MSC) or their secreted exosomes affect a number of pathophysiologic states. Clinical applications of MSC and exosomes are increasingly anticipated. Radiation therapy is the main therapeutic tool for a number of various conditions. The cellular uptake mechanisms of exosomes and the effects of radiation on exosome–cell interactions are crucial, but they are not well understood.more » Here we examined the basic mechanisms and effects of radiation on exosome uptake processes in MSC. Radiation increased the cellular uptake of exosomes. Radiation markedly enhanced the initial cellular attachment to exosomes and induced the colocalization of integrin CD29 and tetraspanin CD81 on the cell surface without affecting their expression levels. Exosomes dominantly bound to the CD29/CD81 complex. Knockdown of CD29 completely inhibited the radiation-induced uptake, and additional or single knockdown of CD81 inhibited basal uptake as well as the increase in radiation-induced uptake. We also examined possible exosome uptake processes affected by radiation. Radiation-induced changes did not involve dynamin2, reactive oxygen species, or their evoked p38 mitogen-activated protein kinase-dependent endocytic or pinocytic pathways. Radiation increased the cellular uptake of exosomes through CD29/CD81 complex formation. These findings provide essential basic insights for potential therapeutic applications of exosomes or MSC in combination with radiation.« less

CD44 Promotes intoxication by the clostridial iota-family toxins.

PubMed

Wigelsworth, Darran J; Ruthel, Gordon; Schnell, Leonie; Herrlich, Peter; Blonder, Josip; Veenstra, Timothy D; Carman, Robert J; Wilkins, Tracy D; Van Nhieu, Guy Tran; Pauillac, Serge; Gibert, Maryse; Sauvonnet, Nathalie; Stiles, Bradley G; Popoff, Michel R; Barth, Holger

2012-01-01

Various pathogenic clostridia produce binary protein toxins associated with enteric diseases of humans and animals. Separate binding/translocation (B) components bind to a protein receptor on the cell surface, assemble with enzymatic (A) component(s), and mediate endocytosis of the toxin complex. Ultimately there is translocation of A component(s) from acidified endosomes into the cytosol, leading to destruction of the actin cytoskeleton. Our results revealed that CD44, a multifunctional surface protein of mammalian cells, facilitates intoxication by the iota family of clostridial binary toxins. Specific antibody against CD44 inhibited cytotoxicity of the prototypical Clostridium perfringens iota toxin. Versus CD44(+) melanoma cells, those lacking CD44 bound less toxin and were dose-dependently resistant to C. perfringens iota, as well as Clostridium difficile and Clostridium spiroforme iota-like, toxins. Purified CD44 specifically interacted in vitro with iota and iota-like, but not related Clostridium botulinum C2, toxins. Furthermore, CD44 knockout mice were resistant to iota toxin lethality. Collective data reveal an important role for CD44 during intoxication by a family of clostridial binary toxins.

CD44 Promotes Intoxication by the Clostridial Iota-Family Toxins

PubMed Central

Wigelsworth, Darran J.; Ruthel, Gordon; Schnell, Leonie; Herrlich, Peter; Blonder, Josip; Veenstra, Timothy D.; Carman, Robert J.; Wilkins, Tracy D.; Van Nhieu, Guy Tran; Pauillac, Serge; Gibert, Maryse; Sauvonnet, Nathalie; Stiles, Bradley G.; Popoff, Michel R.; Barth, Holger

2012-01-01

Various pathogenic clostridia produce binary protein toxins associated with enteric diseases of humans and animals. Separate binding/translocation (B) components bind to a protein receptor on the cell surface, assemble with enzymatic (A) component(s), and mediate endocytosis of the toxin complex. Ultimately there is translocation of A component(s) from acidified endosomes into the cytosol, leading to destruction of the actin cytoskeleton. Our results revealed that CD44, a multifunctional surface protein of mammalian cells, facilitates intoxication by the iota family of clostridial binary toxins. Specific antibody against CD44 inhibited cytotoxicity of the prototypical Clostridium perfringens iota toxin. Versus CD44+ melanoma cells, those lacking CD44 bound less toxin and were dose-dependently resistant to C. perfringens iota, as well as Clostridium difficile and Clostridium spiroforme iota-like, toxins. Purified CD44 specifically interacted in vitro with iota and iota-like, but not related Clostridium botulinum C2, toxins. Furthermore, CD44 knockout mice were resistant to iota toxin lethality. Collective data reveal an important role for CD44 during intoxication by a family of clostridial binary toxins. PMID:23236484

ELK3 promotes the migration and invasion of liver cancer stem cells by targeting HIF-1α.

PubMed

Lee, Joon Ho; Hur, Wonhee; Hong, Sung Woo; Kim, Jung-Hee; Kim, Sung Min; Lee, Eun Byul; Yoon, Seung Kew

2017-02-01

Hepatocellular carcinoma (HCC) is the fifth most common solid cancer and the third most common cause of cancer-related mortality. HCC develops via a multistep process associated with genetic aberrations that facilitate HCC invasion and migration and promote metastasis. A growing body of evidence indicates that cancer stem cells (CSCs) are responsible for tumorigenesis, cancer cell invasion and metastasis. Despite the extremely small proportion of cancer cells represented by this subpopulation of HCC cells, CSCs play a key role in cancer metastasis and poor prognosis. ELK3 (Net/SAP-2/Erp) is a transcription factor that is activated by the Ras/extracellular signal-regulated kinase (ERK) signaling pathway. It plays several important roles in various physiological processes, including cell migration, invasion, wound healing, angiogenesis and tumorigenesis. In the present study, we investigated the role of ELK3 in cancer cell invasion and metastasis in CD133+/CD44+ liver cancer stem cells (LCSCs). We isolated LCSCs expressing CD133 and CD44 from Huh7 HCC cells and evaluated their metastatic potential using invasion and migration assays. We found that CD133+/CD44+ cells had increased metastatic potential compared with non-CD133+/CD44+ cells. We also demonstrated that ELK3 expression was upregulated in CD133+/CD44+ cells and that this aberration enhanced cell migration and invasion. In addition, we identified the molecular mechanism by which ELK3 promotes cancer cell migration and invasion. We found that silencing of ELK3 expression in CD133+/CD44+ LCSCs attenuated their metastatic potential by modulating the expression of heat shock-induced factor-1α (HIF-1α). Collectively, the results of the present study demonstrated that ELK3 overexpression promoted metastasis in CD133+/CD44+ cells by regulating HIF-1α expression and that silencing of ELK3 expression attenuated the metastatic potential of CD133+/CD44+ LCSCs. In conclusion, modulation of ELK3 expression may represent a novel therapeutic strategy for preventing HCC metastasis and invasion.

CD44 deficiency enhanced Streptococcus equi ssp. zooepidemicus dissemination and inflammation response in a mouse model.

PubMed

Fu, Qiang; Xiao, Pingping; Chen, Yaosheng; Wei, Zigong; Liu, Xiaohong

2017-12-01

Streptococcus equi ssp. zooepidemicus (S. zooepidemicus) is responsible for peritonitis, septicemia, meningitis, arthritis and several other serious diseases in various species. Recent studies have demonstrated that CD44 is implicated in the process of host defense against pathogenic microorganisms. In the present study, the role of CD44 in the host response to S. zooepidemicus infection was investigated in a mouse model. Upon intraperitoneal infection with S. zooepidemicus, the expression of CD44 on the peritoneal exudate cells from wild-type (WT) mice was increased. CD44 deficiency accelerated mortality, which was accompanied by increased peritoneal bacterial growth and dissemination to distant body sites. CD44 knock-out (KO) mice showed enhanced early inflammatory cell recruitment into the peritoneal fluid on S. zooepidemicus infection. In line with this, the expression of proinflammatory cytokines, chemokines in peritoneal exudate cells and peritoneal macrophages of CD44 KO mice were increased compared with those of WT mice. In addition, CD44 deficiency was associated with reduced expression of A20, a negative regulator in TLR signaling. Overall, the present study suggests that CD44 plays a protective role in antibacterial defense against S. zooepidemicus in mice. Copyright © 2017. Published by Elsevier Ltd.

СD44+/CD24- markers of cancer stem cells in patients with breast cancer of different molecular subtypes.

PubMed

Chekhun, S V; Zadvorny, T V; Tymovska, Yu O; Anikusko, M F; Novak, O E; Polishchuk, L Z

2015-03-01

To determine frequency of tumors with immunohistochemical markers of cancer stem cells (CSC) CD44+/CD24- in patients with breast cancer (BC) of different molecular subtype and to evaluate their prognostic value. Surgical material of 132 patients with BC stage I-II, age from 23 to 75 years, mean age - 50.2 ± 3.1 years was studied. Clinical, immunohistochemical (expression CD44+/CD24-), morphological, statistical. BC is characterized by heterogeneity of molecular subtypes and expression of markers (CD44+/CD24-). Immunohistochemical study of expression of CSC markers in surgical material has detected their expression in 34 (25.4%) patients with BC of different molecular subtypes. The highest frequency of cells with expression of CSC marker was observed in patients with basal molecular subtype (44.8% patients). Most of BC patients with phenotype CD44+/CD24 had stage I of tumor process (34.3%). Statistical processing of data has showen that Yule colligation coefficient equaled 0.28 (р > 0.05) that argues poor correlation between stage of tumor process and number of tumors with positive expression of CSC markers. Statistical processing of data has showen high correlation between presence of cells with expression of CSC markers and metastases of BC in regional lymph nodes (Yule colligation coefficient equals 0.943; р < 0.5). Difference in overall survival of patients with BC of basal molecular subtype depending on expression of CSC CD44+/CD24- markers was detected. Survival of patients with basal BC was reliably higher at lack in tumors of cells with CSC markers CD44+/CD24- and, correspondingly, lower at presence of such cells (р < 0.05). In patients with BC of luminal (A and B), HER-2-positive subtypes, significant change in survival of patients depending on expression of CSC markers was not determined (р > 0.05). Significance of tumor cells with markers CD44+/CD24- within the limits of molecular subtype of BC may be additional criterion for advanced biological characteristic of BC, and in patients with BC of basal molecular subtype - for predictive evaluation of individual potential of tumor to aggressive clinical course.

Phenotypic Heterogeneity and Plasticity of Isocortical and Hippocampal Astrocytes in the Human Brain

PubMed Central

Sosunov, Alexander A.; Wu, Xiaoping; Tsankova, Nadejda M.; Guilfoyle, Eileen; McKhann, Guy M.

2014-01-01

To examine the diversity of astrocytes in the human brain, we immunostained surgical specimens of temporal cortex and hippocampus and autopsy brains for CD44, a plasma membrane protein and extracellular matrix receptor. CD44 antibodies outline the details of astrocyte morphology to a degree not possible with glial fibrillary acidic protein (GFAP) antibodies. CD44+ astrocytes could be subdivided into two groups. First, CD44+ astrocytes with long processes were consistently found in the subpial area (“interlaminar” astrocytes), the deep isocortical layers, and the hippocampus. Many of these processes ended on blood vessels. Some were also found adjacent to large blood vessels, from which they extended long processes. We observed these CD44+, long-process astrocytes in every brain we examined, from fetal to adult. These astrocytes generally displayed high immunostaining for GFAP, S100β, and CD44, but low immunostaining for glutamine synthetase, excitatory amino-acid transporter 1 (EAAT1), and EAAT2. Aquaporin 4 (AQP4) appeared distributed all over the cell bodies and processes of the CD44+ astrocytes, while, in contrast, AQP4 localized to perivascular end feet in the CD44− protoplasmic astrocytes. Second, there were CD44+ astrocytes without long processes in the cortex. These were not present during gestation or at birth, and in adult brains varied substantially in number, shape, and immunohistochemical phenotype. Many of these displayed a “mixed” morphological and immunocytochemical phenotype between protoplasmic and fibrous astrocytes. We conclude that the diversity of astrocyte populations in the isocortex and archicortex in the human brain reflects both intrinsic and acquired phenotypes, the latter perhaps representing a shift from CD44− “protoplasmic” to CD44+ “fibrous”-like astrocytes. PMID:24501367

Transforming growth factor-β-mediated CD44/STAT3 signaling contributes to the development of atrial fibrosis and fibrillation.

PubMed

Chang, Shang-Hung; Yeh, Yung-Hsin; Lee, Jia-Lin; Hsu, Yu-Juei; Kuo, Chi-Tai; Chen, Wei-Jan

2017-09-04

Atrial fibrillation (AF) is associated with atrial fibrosis. Inhibition of atrial fibrosis might be a plausible approach for AF prevention and therapy. This study is designed to evaluate the potential role of CD44, a membrane receptor known to regulate fibrosis, and its related signaling in the pathogenesis of atrial fibrosis and AF. Treatment of cultured rat atrial fibroblasts with transforming growth factor-β (TGF-β, a key mediator of atrial fibrosis) led to a higher expression of hyaluronan (HA), CD44, STAT3, and collagen (a principal marker of fibrosis) than that of ventricular fibroblasts. In vivo, TGF-β transgenic mice and AF patients exhibited a greater expression of HA, CD44, STAT3, and collagen in their atria than wild-type mice and sinus rhythm subjects, respectively. Treating TGF-β transgenic mice with an anti-CD44 blocking antibody resulted in a lower expression of STAT3 and collagen in their atria than those with control IgG antibody. Programmed stimulation triggered less AF episodes in TGF-β transgenic mice treated with anti-CD44 blocking antibody than in those with control IgG. Blocking CD44 signaling with anti-CD44 antibody and mutated CD44 plasmids attenuated TGF-β-induced STAT3 activation and collagen expression in cultured atrial fibroblasts. Deletion and mutational analysis of the collagen promoter along with chromatin immunoprecipitation demonstrated that STAT3 served as a vital transcription factor in collagen expression. TGF-β-mediated HA/CD44/STAT3 pathway plays a crucial role in the development of atrial fibrosis and AF. Blocking CD44-dependent signaling may be a feasible way for AF management.

CD44 variant-dependent redox status regulation in liver fluke-associated cholangiocarcinoma: A target for cholangiocarcinoma treatment.

PubMed

Thanee, Malinee; Loilome, Watcharin; Techasen, Anchalee; Sugihara, Eiji; Okazaki, Shogo; Abe, Shinya; Ueda, Shiho; Masuko, Takashi; Namwat, Nisana; Khuntikeo, Narong; Titapun, Attapol; Pairojkul, Chawalit; Saya, Hideyuki; Yongvanit, Puangrat

2016-07-01

Expression of CD44, especially the variant isoforms (CD44v) of this major cancer stem cell marker, contributes to reactive oxygen species (ROS) defense through stabilizing xCT (a cystine-glutamate transporter) and promoting glutathione synthesis. This enhances cancer development and increases chemotherapy resistance. We investigate the role of CD44v in the regulation of the ROS defense system in cholangiocarcinoma (CCA). Immunohistochemical staining of CD44v and p38(MAPK) (a major ROS target) expression in Opisthorchis viverrini-induced hamster CCA tissues (at 60, 90, 120, and 180 days) reveals a decreased phospho-p38(MAPK) signal, whereas the CD44v signal was increased during bile duct transformation. Patients with CCA showed CD44v overexpression and negative-phospho-p38(MAPK) patients a significantly shorter survival rate than the low CD44v signal and positive-phospho-p38(MAPK) patients (P = 0.030). Knockdown of CD44 showed that xCT and glutathione levels were decreased, leading to a high level of ROS. We examined xCT-targeted CD44v cancer stem cell therapy using sulfasalazine. Glutathione decreased and ROS increased after the treatment, leading to inhibition of cell proliferation and induction of cell death. Thus, the accumulation of CD44v leads to the suppression of p38(MAPK) in transforming bile duct cells. The redox status regulation of CCA cells depends on the expression of CD44v to contribute the xCT function and is a link to the poor prognosis of patients. Thus, an xCT inhibitor could inhibit cell growth and activate cell death. This suggests that an xCT-targeting drug may improve CCA therapy by sensitization to the available drug (e.g. gemcitabine) by blocking the mechanism of the cell's ROS defensive system. © 2016 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

Effect of dacarbazine on CD44 in live melanoma cells as measured by atomic force microscopy-based nanoscopy.

PubMed

Huang, Xun; He, Jiexiang; Zhang, Huan-Tian; Sun, Kai; Yang, Jie; Wang, Huajun; Zhang, Hongxin; Guo, Zhenzhao; Zha, Zhen-Gang; Zhou, Changren

2017-01-01

CD44 ligand-receptor interactions are known to be involved in regulating cell migration and tumor cell metastasis. High expression levels of CD44 correlate with a poor prognosis of melanoma patients. In order to understand not only the mechanistic basis for dacarbazine (DTIC)-based melanoma treatment but also the reason for the poor prognosis of melanoma patients treated with DTIC, dynamic force spectroscopy was used to structurally map single native CD44-coupled receptors on the surface of melanoma cells. The effect of DTIC treatment was quantified by the dynamic binding strength and the ligand-binding free-energy landscape. The results demonstrated no obvious effect of DTIC on the unbinding force between CD44 ligand and its receptor, even when the CD44 nanodomains were reduced significantly. However, DTIC did perturb the kinetic and thermodynamic interactions of the CD44 ligand-receptor, with a resultant greater dissociation rate, lower affinity, lower binding free energy, and a narrower energy valley for the free-energy landscape. For cells treated with 25 and 75 μg/mL DTIC for 24 hours, the dissociation constant for CD44 increased 9- and 70-fold, respectively. The CD44 ligand binding free energy decreased from 9.94 for untreated cells to 8.65 and 7.39 kcal/mol for DTIC-treated cells, which indicated that the CD44 ligand-receptor complexes on DTIC-treated melanoma cells were less stable than on untreated cells. However, affinity remained in the micromolar range, rather than the millimolar range associated with nonaffinity ligands. Hence, the CD44 receptor could still be activated, resulting in intracellular signaling that could trigger a cellular response. These results demonstrate DTIC perturbs, but not completely inhibits, the binding of CD44 ligand to membrane receptors, suggesting a basis for the poor prognosis associated with DTIC treatment of melanoma. Overall, atomic force microscopy-based nanoscopic methods offer thermodynamic and kinetic insight into the effect of DTIC on the CD44 ligand-binding process.

Effect of dacarbazine on CD44 in live melanoma cells as measured by atomic force microscopy-based nanoscopy

PubMed Central

Huang, Xun; He, Jiexiang; Zhang, Huan-tian; Sun, Kai; Yang, Jie; Wang, Huajun; Zhang, Hongxin; Guo, Zhenzhao; Zha, Zhen-gang; Zhou, Changren

2017-01-01

CD44 ligand–receptor interactions are known to be involved in regulating cell migration and tumor cell metastasis. High expression levels of CD44 correlate with a poor prognosis of melanoma patients. In order to understand not only the mechanistic basis for dacarbazine (DTIC)-based melanoma treatment but also the reason for the poor prognosis of melanoma patients treated with DTIC, dynamic force spectroscopy was used to structurally map single native CD44-coupled receptors on the surface of melanoma cells. The effect of DTIC treatment was quantified by the dynamic binding strength and the ligand-binding free-energy landscape. The results demonstrated no obvious effect of DTIC on the unbinding force between CD44 ligand and its receptor, even when the CD44 nanodomains were reduced significantly. However, DTIC did perturb the kinetic and thermodynamic interactions of the CD44 ligand–receptor, with a resultant greater dissociation rate, lower affinity, lower binding free energy, and a narrower energy valley for the free-energy landscape. For cells treated with 25 and 75 μg/mL DTIC for 24 hours, the dissociation constant for CD44 increased 9- and 70-fold, respectively. The CD44 ligand binding free energy decreased from 9.94 for untreated cells to 8.65 and 7.39 kcal/mol for DTIC-treated cells, which indicated that the CD44 ligand–receptor complexes on DTIC-treated melanoma cells were less stable than on untreated cells. However, affinity remained in the micromolar range, rather than the millimolar range associated with nonaffinity ligands. Hence, the CD44 receptor could still be activated, resulting in intracellular signaling that could trigger a cellular response. These results demonstrate DTIC perturbs, but not completely inhibits, the binding of CD44 ligand to membrane receptors, suggesting a basis for the poor prognosis associated with DTIC treatment of melanoma. Overall, atomic force microscopy-based nanoscopic methods offer thermodynamic and kinetic insight into the effect of DTIC on the CD44 ligand-binding process. PMID:29296081

Characterization of a CD44/CD122int memory CD8 T cell subset generated under sterile inflammatory conditions.

PubMed

Mbitikon-Kobo, Florentin-Martial; Vocanson, Marc; Michallet, Marie-Cécile; Tomkowiak, Martine; Cottalorda, Anne; Angelov, Georgi S; Coupet, Charles-Antoine; Djebali, Sophia; Marçais, Antoine; Dubois, Bertrand; Bonnefoy-Bérard, Nathalie; Nicolas, Jean-François; Arpin, Christophe; Marvel, Jacqueline

2009-03-15

Most memory CD8 T cell subsets that have been hitherto defined are generated in response to infectious pathogens. In this study, we have characterized the CD8 T cells that survive priming conditions, devoid of pathogen-derived danger signals. In both a TCR-transgenic model and a model of contact hypersensitivity, we show that the priming of naive CD8 T cells under sterile inflammatory conditions generates memory. The corresponding memory CD8 T cells can be identified by their intermediate expression levels of CD44 and CD122. We also show that CD44/122(int) memory CD8 T cells spontaneously develop in wild type mice and that they display intermediate levels of several other memory traits including functional (IFN-gamma secretion capacity, CCL5 messenger stores), phenotypic, and molecular (T-bet and eomesodermin expression levels) features. We finally show that they correspond to an early differentiation stage and can further differentiate in CD44/122(high) memory T cells. Altogether, our results identify a new memory CD8 T cell subset that is generated under sterile inflammatory conditions and involved in the recall contact hypersensitivity reactions that are responsible for allergic contact dermatitis.

CD44 is a direct target of miR-199a-3p and contributes to aggressive progression in osteosarcoma

PubMed Central

Gao, Yan; Feng, Yong; Shen, Jacson K.; Lin, Min; Choy, Edwin; Cote, Gregory M.; Harmon, David C.; Mankin, Henry J.; Hornicek, Francis J.; Duan, Zhenfeng

2015-01-01

Osteosarcoma is the most common primary bone malignancy in children and adolescents. Herein, we investigated the role of cluster of differentiation 44 (CD44), a cell-surface glycoprotein involved in cell-cell interactions, cell adhesion, and migration in osteosarcoma. We constructed a human osteosarcoma tissue microarray with 114 patient tumor specimens, including tumor tissues from primary, metastatic, and recurrent stages, and determined the expression of CD44 by immunohistochemistry. Results showed that CD44 was overexpressed in metastatic and recurrent osteosarcoma as compared with primary tumors. Higher expression of CD44 was found in both patients with shorter survival and patients who exhibited unfavorable response to chemotherapy before surgical resection. Additionally, the 3′-untranslated region of CD44 mRNA was the direct target of microRNA-199a-3p (miR-199a-3p). Overexpression of miR-199a-3p significantly inhibited CD44 expression in osteosarcoma cells. miR-199a-3p is one of the most dramatically decreased miRs in osteosarcoma cells and tumor tissues as compared with normal osteoblast cells. Transfection of miR-199a-3p significantly increased the drug sensitivity through down-regulation of CD44 in osteosarcoma cells. Taken together, these results suggest that the CD44-miR-199a-3p axis plays an important role in the development of metastasis, recurrence, and drug resistance of osteosarcoma. Developing strategies to target CD44 may improve the clinical outcome of osteosarcoma. PMID:26079799

Enrichment of prostate cancer stem-like cells from human prostate cancer cell lines by culture in serum-free medium and chemoradiotherapy.

PubMed

Wang, Lei; Huang, Xing; Zheng, Xinmin; Wang, Xinghuan; Li, Shiwen; Zhang, Lin; Yang, Zhonghua; Xia, Zhiping

2013-01-01

The discovery of rare subpopulations of cancer stem cells (CSCs) has created a new focus in cancer research. As CSCs demonstrate resistance to chemoradiation therapy relative to other cancer cells, this allows the enrichment of CSC populations by killing apoptosis-susceptible cancer cells. In this study, three commonly used human prostate cancer (PCa) cell lines (DU145, PC-3 and LNCaP) were examined for their expression of the putative stem cell markers CD133 and CD44 via flow cytometric analysis. Under normal culture conditions, CD133(+)/CD44(+) cells were only present in the DU145 cell line, and comprised only a minor percentage (0.1% ± 0.01%) of the total population. However, the proportion of these CD133(+)/CD44(+) prostate CSCs could be increased in these cell lines via culture in serum-free medium (SFM), or through chemotherapy or radiotherapy. Indeed, after culture in SFM, the proportion of CD133(+)/CD44(+) cells in DU145 and PC-3 had increased to 10.3% and 3.0%, respectively. Moreover, the proportion had increased to 9.8% enriched by chemotherapy and 3.5% by radiotherapy in DU145. Colony-formation tests, cell invasion assays, and tumor xenografts in BALB/c nude mice were used to evaluate the stem cell properties of CD133(+)/CD44(+) PCa cells that were isolated via fluorescence-activated cell sorting (FACS). CD133(+)/CD44(+) cells had an enhanced colony-formation capability and invasive ability in vitro, and displayed greater tumorigenic properties in vivo. These results demonstrate the presence of CD133(+)/CD44(+) prostate CSCs in established PCa cell lines and that populations of these cells can be enriched by culture in SFM or chemoradiotherapy. Finding novel therapies to override chemoradiation resistance in the prostate CSCs is the key to improve long-term results in PCa management.

CD47 is an adverse prognostic factor and a therapeutic target in gastric cancer

PubMed Central

Yoshida, Kazumichi; Tsujimoto, Hironori; Matsumura, Kouji; Kinoshita, Manabu; Takahata, Risa; Matsumoto, Yusuke; Hiraki, Shuichi; Ono, Satoshi; Seki, Shuhji; Yamamoto, Junji; Hase, Kazuo

2015-01-01

CD47 is an antiphagocytic molecule that acts via ligation to signal regulatory protein alpha on phagocytes; its enhanced expression and therapeutic targeting have recently been reported for several malignancies. However, CD47 expression in gastric cancer is not well documented. Immunohistochemical expression of CD47 in surgical specimens was investigated. Expression of CD47 and CD44, a known gastric cancer stem cell marker, were investigated in gastric cancer cell lines by flow cytometry. MKN45 and MKN74 gastric cancer cells were sorted by fluorescence-activated cell sorting according to CD44 and CD47 expression levels, and their in vitro proliferation, spheroid-forming capacity, and in vivo tumorigenicity were studied. In vitro phagocytosis of cancer cells by human macrophages in the presence of a CD47 blocking monoclonal antibody (B6H12) and the survival of immunodeficient mice intraperitoneally engrafted with MKN45 cells and B6H12 were compared to experiments using control antibodies. Immunohistochemistry of the clinical specimens indicated that CD47 was positive in 57 out of 115 cases, and its positivity was an independent adverse prognostic factor. Approximately 90% of the MKN45 and MKN74 cells expressed CD47 and CD44. CD47hi gastric cancer cells showed significantly higher proliferation and spheroid colony formation than CD47lo, and CD44hiCD47hi cells showed the highest proliferation in vitro and tumorigenicity in vivo. B6H12 significantly enhanced in vitro phagocytosis of cancer cells by human macrophages and prolonged the survival of intraperitoneal cancer dissemination in mice compared to control antibodies. In conclusion, CD47 is an adverse prognostic factor and promising therapeutic target in gastric cancer. PMID:26077800

STAT3 as a promising chemoresistance biomarker associated with the CD44+/high/CD24-/low/ALDH+ BCSCs-like subset of the triple-negative breast cancer (TNBC) cell line.

PubMed

Moreira, Milene Pereira; da Conceição Braga, Letícia; Cassali, Geovanni Dantas; Silva, Luciana Maria

2018-02-15

The cancer stem cell (CSC) concept is currently employed to explain the mechanism of multidrug resistance that is implicated in the reduced efficacy of many chemotherapeutic agents, consequently leading to metastatic spread and disease relapse. We searched for potential predictive markers of doxorubicin (DOX) resistance in breast cancer stem cells (BCSCs) of the BT-549 human triple-negative breast cancer (TNBC) cell line classified as a claudin-low subtype. In this study, we show that BT-549 presents a BCSCs-like subset determined by a CD44 +/high /CD24 -/low /ALDH1 + phenotype. The CD44 +/high /CD24 -/low /ALDH + BCSCs-like subset presented the downregulation of a majority of the genes analyzed (64 genes), and only 3 genes were upregulated after DOX treatment. Among the upregulated genes, MAPK3, PRKCZ and STAT3, STAT3 presented a higher level of upregulation in the DOX-treated CD44 +/high /CD24 -/low /ALDH + BCSCs-like subset. The identification of biomarkers that predict antitumor responses is at the top of cancer research priorities. STAT3 was highlighted as a molecular signature in the CD44 +/high /CD24 -/low /ALDH1 + BCSCs-like subset obtained from the TNBC BT-549 cell line related to DOX resistance. A majority of the evaluated genes in the EGF pathway appear to be not associated with DOX resistance, as observed in the CD44 +/high /CD24 -/low /ALDH1 + BCSCs-like subset. Copyright © 2018 Elsevier Inc. All rights reserved.

CD44v6: A metastasis-associated biomarker in patients with gastric cancer?

PubMed Central

Lu, Li; Huang, Fei; Zhao, Zhicheng; Li, Chuan; Liu, Tong; Li, Weidong; Fu, Weihua

2016-01-01

Abstract Background: The diagnostic and prognostic value of CD44v6 in patients with gastric cancer remains unclear. Therefore, a quantitative meta-analysis was conducted to determine the clinical value of CD44v6 in patients with gastric cancer. Methods: Sixteen studies with 2177 patients were included. Pooled odds ratios (ORs) and hazard ratio (HR) with 95% confidence intervals (CIs) were calculated to estimate the impact of CD44v6 in patients with gastric cancer on clinicopathological features and 5-year overall survival (OS). Sensitivity analysis, subgroup analysis, and regression analysis were introduced to evaluate the heterogeneity across the studies. Publication bias was also explored among the studies. Results: The meta-analysis showed that the upregulated CD44v6 was associated with lymph node metastasis (OR 1.91, 95% CI 1.19–3.08; P = 0.007), distant metastasis (OR 3.41, 95% CI 2.01–5.78; P = 0.000), high TNM stage (OR 2.29, 95% CI 1.10–4.75; P = 0.026), lymphatic vessel invasion (OR 1.59, 95% CI 1.21–2.09; P = 0.001), and vascular invasion (OR 1.57, 95% CI 1.19–2.07; P = 0.001). When excluded 1 study based on sensitivity analysis, pooled HR indicated that CD44v6 positive expression was correlated poor 5-year OS (OR 1.76, 95% CI 1.30–2.39; P = 0.000), meanwhile, heterogeneity was eliminated. The heterogeneity of Lauren type mainly existed in the big sample size subgroup. Different region and publication year might contribute to the heterogeneity of differentiation type. While the heterogeneity of lymph node mainly existed in Asian and big sample size group. Publication bias was observed among 12 studies on lymph node metastasis (Ppublication bias = 0.041), and 5 studies on TNM stage (Ppublication bias = 0.026). Conclusion: Taken together, CD44v6 overexpression might be correlated to the characteristics of tumor metastasis in gastric cancer, consisting with many mechanism studies. Therefore, CD44v6 might present a metastasis-associated biomarker in patients with gastric cancer. PMID:27977599

Increased expression of CD44 is associated with more aggressive behavior in clear cell renal cell carcinoma.

PubMed

Zanjani, Leili Saeednejad; Madjd, Zahra; Abolhasani, Maryam; Rasti, Arezoo; Fodstad, Oystein; Andersson, Yvonne; Asgari, Mojgan

2018-01-01

Although CD44 has been suggested as a prognostic marker in renal cell carcinoma (RCC), the prognostic significance of this marker in three main subtypes of RCC is still unclear. Thus, the present study was conducted to evaluate the expression and prognostic significance of CD44 as a cancer stem cell marker in different histological subtypes of RCC. Methodology & results: CD44 expression was evaluated in 206 well-defined renal tumor samples using immunohistochemistry on tissue microarrays. Higher CD44 expression was associated with more aggressive behavior, tumor progression and worse prognosis in clear cell RCC (ccRCC) but not in papillary and chromophobe RCC subtypes. Cancer stem cell marker CD44 may be a promising target for cancer treatment only in ccRCC.

Breast Cancer Translational Research Center of Excellence FY12-14

DTIC Science & Technology

2014-09-01

DNA aptamers against CD44 that inhibit breast cancer invasion and metastasis. CD44 adhesion molecules are expressed in many breast cancer cells and...Somiari RI, Somiari S, Cutler ML, Mural RJ, Shriver CD. “DNA aptamers against exon v10 of CD44 inhibit breast cancer cell migration.” PLoS One

Generation of Mesenchymal-Like Stem Cells From Urine in Pediatric Patients.

PubMed

He, W; Zhu, W; Cao, Q; Shen, Y; Zhou, Q; Yu, P; Liu, X; Ma, J; Li, Y; Hong, K

2016-01-01

Mesenchymal stem cells (MSCs) have been widely used for regenerative medicine. Traditionally, the procedures of MSC isolation are usually invasive and time-consuming. Urine is merely a body waste, and recent studies have suggested that urine represents an alternative source of stem cells. We, therefore, determined whether the possibility of isolating mesenchymal-like stem cells was practical from human urine. A total of 16 urine samples were collected from pediatric patients. Urine-derived cells were isolated, expanded, and identified for specific cell surface markers using flow cytometry. Cell morphology was observed by microscopy. Osteogenic and adipogenic differentiation potential were determinded by culturing cells in specific induction medium, and assessed by alkaline phosphatase and oil red O stainings, respectively. Clones were established and passaged successfully from primary cultures of urine cells. Cultured urine-derived cells at passage 3 were fusiform and arranged with certain directionality. Urine-derived cells at passage 5 displayed expressions of cell surface markers (CD29, CD105, CD166, CD90, and CD13). There was no expression of the general hematopoietic cell markers (CD45, CD34, and HLA-DR). Under in vitro induction conditions, urine-derived cells at passage 5 were able to differentiate into osteoblasts, but not adipocytes. Urine may be a noninvasive source for mesenchymal-like stem cells. These cells could potentially provide a new source of autologous stem cells for regenerative medicine and cell therapy. Copyright © 2016 Elsevier Inc. All rights reserved.

Prognostic relevance of miR-137 in patients with hepatocellular carcinoma.

PubMed

Sakabe, Tomohiko; Azumi, Junya; Umekita, Yoshihisa; Toriguchi, Kan; Hatano, Etsuro; Hirooka, Yasuaki; Shiota, Goshi

2017-02-01

Cancer stem cells (CSCs) play a pivotal role in progression, metastasis and recurrence of cancer. Therefore, it is clinically useful to identify the relevant CSC marker that is associated with prognosis of hepatocellular carcinoma (HCC) and clarify its genetic and biological characteristics. Expression of four CSC markers, CD13, EpCAM, CD44 and CD44v9, was examined in 99 HCC patients. Biological and cDNA/miRNA microarray data were compared among CD44-positive/-negative HCC cells and normal hepatic cells. The significance of the representative miRNAs was examined with regard to prognosis of additional 110 HCC patients. CD44-positive HuH7 cells proliferated faster and showed a greater sphere forming ability than CD44-negative HuH7 cells. CD44-positive HuH7 cells exhibited higher expression of specific genes involved in resistance to reactive oxygen species, anticancer drugs and tumour invasion than CD44-negative HCC cells. Higher expression of six miRNAs was observed in CD44-positive HuH7 cells, CD44-negative HuH7 cells, and human normal hepatic cells in that order. Of the six miRNAs, miR-137 was closely associated with overall and cancer-specific survivals, as well as with invasion of hepatic vein, hepatic artery, portal vein and bile duct, and alpha-foetoprotein in additional 110 HCC patients. miR-137 may serve as a prognostic marker in patients with HCC and may be a potential target for the elimination of liver CSCs. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

The relationship between cadmium in kidney and cadmium in urine and blood in an environmentally exposed population

DOE Office of Scientific and Technical Information (OSTI.GOV)

Akerstrom, Magnus, E-mail: magnus.akerstrom@amm.gu.se; Barregard, Lars; Lundh, Thomas

Introduction: Cadmium (Cd) is toxic to the kidney and a major part of the body burden occurs here. Cd in urine (U-Cd) and blood (B-Cd) are widely-used biomarkers for assessing Cd exposure or body burden. However, empirical general population data on the relationship between Cd in kidney (K-Cd), urine, and blood are scarce. Our objectives were to determine the relationship between cadmium in kidney, urine, and blood, and calculate the elimination half-time of Cd from the kidney. Methods: Kidney cortex biopsies, urine, and blood samples were collected from 109 living kidney donors. Cd concentrations were determined and the relationships betweenmore » K-Cd, U-Cd, and B-Cd were investigated in regression models. The half-time of K-Cd was estimated from the elimination constant. Results: There was a strong association between K-Cd and U-Cd adjusted for creatinine (r{sub p} = 0.70, p < 0.001), while the association with B-Cd was weaker (r{sub p} = 0.44, p < 0.001). The relationship between K-Cd and U-Cd was nonlinear, with slower elimination of Cd at high K-Cd. Estimates of the K-Cd half-time varied between 18 and 44 years. A K-Cd of 25 μg/g corresponds to U-Cd of 0.42 μg/g creatinine in overnight urine (U-Cd/K-Cd ratio: about 1:60). Multivariate models showed Cd in blood and urinary albumin as determinants for U-Cd excretion. Discussion: In healthy individuals with low-level Cd exposure, there was a strong correlation between Cd in kidney and urine, especially after adjustment for creatinine. Urinary Cd was also affected by Cd in blood and urinary albumin. Previous estimates of the U-Cd/K-Cd ratio may underestimate K-Cd at low U-Cd. - Highlights: ► The first study of the relation between Cd in kidney, blood and urine at low U-Cd ► Simultaneous samples were collected from healthy kidney donors. ► There was a nonlinear relationship between cadmium in kidney and urine. ► Estimates of the kidney cadmium half-time were 18–44 years, depending on model used. ► Previous data seem to underestimate kidney cadmium at low urinary cadmium.« less

Localization of the T-cell response to RSV infection is altered in infant mice.

PubMed

Eichinger, Katherine M; Kosanovich, Jessica L; Empey, Kerry M

2018-02-01

Respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infections worldwide, causing disproportionate morbidity and mortality in infants and children. Infants with stronger Th1 responses have less severe disease, yet little is known about the infant T-cell response within the air space. Thus, we tested the hypothesis that RSV infected infant mice would have quantitative and qualitative deficiencies in CD4 + and CD8 + T-cell populations isolated from the bronchoalveolar lavage when compared to adults and that local delivery of IFN-γ would increase airway CD4 + Tbet + and CD8 + Tbet + T-cell responses. We compared the localization of T-cell responses in RSV-infected infant and adult mice and investigated the effects of local IFN-γ administration on infant cellular immunity. Adult CD8 + CD44 HI and CD4 + CD44 HI Tbet + T-cells accumulated in the alveolar space whereas CD4 + CD44 HI Tbet + T-cells were evenly distributed between the infant lung tissue and airway and infant lungs contained higher frequencies of CD8 + T-cells. Delivery of IFN-γ to the infant airway failed to increase the accumulation of T-cells in the airspace and unexpectedly reduced CD4 + CD44 HI Tbet + T-cells. However, intranasal IFN-γ increased RSV F protein-specific CD8 + T-cells in the alveolar space. Together, these data suggest that quantitative and qualitative defects exist in the infant T-cell response to RSV but early, local IFN-γ exposure can increase the CD8 + RSV-specific T-cell response. © 2017 Wiley Periodicals, Inc.

Characterization of mouse CD53: epitope mapping, cellular distribution and induction by T cell receptor engagement during repertoire selection.

PubMed

Tomlinson, M G; Hanke, T; Hughes, D A; Barclay, A N; Scholl, E; Hünig, T; Wright, M D

1995-08-01

The pan-leukocyte antigen CD53 is a member of the poorly understood transmembrane 4 superfamily (TM4SF) of cell membrane glycoproteins. CD53 is proposed to play a role in thymopoiesis, since rat CD53 is expressed on immature CD4-8-thymocytes and the functionally mature single-positive subset, but is largely absent from the intermediate CD4+8+ cells. We have characterized CD53 in the mouse through the production of two new monoclonal antibodies, MRC OX-79 and OX-80, which were raised against the RAW 264 cell line and screened on recombinant CD53 fusion proteins. The epitopes recognized by both antibodies are dependent on disulfide bonding and map to the major extracellular region of CD53, requiring the presence of a single threonine residue at position 154. Mouse CD53 has a molecular mass of 35-45 kDa and is expressed on virtually all peripheral leukocytes, but not on cells outside the lymphoid or myeloid lineages. CD53 expression distinguishes subpopulations of thymocytes in the mouse and resembles the expression pattern of rat CD53. Amongst the immature CD4-8-thymocytes, mouse CD53 is clearly detectable on the earliest CD44high25- subset, but down-regulated on the later CD44high25+, CD44low25+ and CD44low25- stages. Also, the subsequent transient TcR-/low CD4-8+ cells and most CD4+8+ thymocytes express little or no CD53. This is consistent with the idea that cells which are committed to enter the selectable CD4+8+ compartment switch off CD53. The effect of T cell receptor (TcR) engagement on the re-expression of CD53 on CD4+8+ thymocytes was studied both ex vivo and in vitro using F5 mice, transgenic for the H-2b/influenza nucleoprotein-peptide-specific TcR, back-crossed onto an H-2q or H-2b background of RAG-2-deficient mice. CD4+8+ thymocytes from non-selecting H-2q F5 mice are CD53 negative, but in vitro stimulation through the TcR dramatically induces CD53 expression. In contrast, a fraction of CD4+8+ thymocytes from positively selecting H-2b F5 transgenic mice express CD53. Therefore TcR engagement by selecting major histocompatibility complex peptide complexes, or surrogate ligands, induces CD53 expression on otherwise CD53-negative, non-selected CD4+8+ thymocytes. Whether CD53 itself participates as a signaling molecule in further stages of thymic selection is still a matter of speculation.

Inhibition of Ovarian Cancer Chemoresistance and Metastasis with Antagonists of Hyaluronan-CD44-CD147 Interactions

DTIC Science & Technology

2015-09-01

malignant and drug- resistant properties. This most likely occurs through assembly and/or stabilization of plasma membrane signaling complexes ...interactions of hyaluronan polymer with CD44 are necessary for stabilizing CD44-CD147 signaling complexes , and that small, monovalent, hyaluronan...transporter complexes (Ghatak et al., 2005; Grass et al., 2012; Grass et al., 2013; Qin et al., 2011; Slomiany et al., 2009a; Slomiany et al., 2009b

Self-renewal and circulating capacities of metastatic hepatocarcinoma cells required for collaboration between TM4SF5 and CD44

PubMed Central

Lee, Doohyung; Lee, Jung Weon

2015-01-01

Tumor metastasis involves circulating and tumor-initiating capacities of metastatic cancer cells. Hepatic TM4SF5 promotes EMT for malignant growth and migration. Hepatocellular carcinoma (HCC) biomarkers remain unexplored for metastatic potential throughout metastasis. Here, novel TM4SF5/CD44 interaction-mediated self-renewal and circulating tumor cell (CTC) capacities were mechanistically explored. TM4SF5-dependent sphere growth was correlated with CD133+, CD24-, ALDH activity, and a physical association between CD44 and TM4SF5. The TM4SF5/CD44 interaction activated c-Src/STAT3/ Twist1/ B mi1 signaling for spheroid formation, while disturbing the interaction, expression, or activity of any component in this signaling pathway inhibited spheroid formation. In serial xenografts of less than 5,000 cells/injection, TM4SF5-positive tumors exhibited locally-increased CD44 expression, suggesting tumor cell differentiation. TM4SF5-positive cells were identified circulating in blood 4 to 6 weeks after orthotopic liver-injection. Anti-TM4SF reagents blocked their metastasis to distal intestinal organs. Altogether, our results provide evidence that TM4SF5 promotes self-renewal and CTC properties supported by CD133+/TM4SF5+/CD44+(TM4SF5-bound)/ALDH+/ CD24- markers during HCC metastasis. [BMB Reports 2015; 48(3): 127-128] PMID:25772760

Assessing Specific Oligonucleotides and Small Molecule Antibiotics for the Ability to Inhibit the CRD-BP-CD44 RNA Interaction

PubMed Central

Thomsen, Dana; Lee, Chow H.

2014-01-01

Studies on Coding Region Determinant-Binding Protein (CRD-BP) and its orthologs have confirmed their functional role in mRNA stability and localization. CRD-BP is present in extremely low levels in normal adult tissues, but it is over-expressed in many types of aggressive human cancers and in neonatal tissues. Although the exact role of CRD-BP in tumour progression is unclear, cumulative evidence suggests that its ability to physically associate with target mRNAs is an important criterion for its oncogenic role. CRD-BP has high affinity for the 3′UTR of the oncogenic CD44 mRNA and depletion of CRD-BP in cells led to destabilization of CD44 mRNA, decreased CD44 expression, reduced adhesion and disruption of invadopodia formation. Here, we further characterize the CRD-BP-CD44 RNA interaction and assess specific antisense oligonucleotides and small molecule antibiotics for their ability to inhibit the CRD-BP-CD44 RNA interaction. CRD-BP has a high affinity for binding to CD44 RNA nts 2862–3055 with a Kd of 645 nM. Out of ten antisense oligonucleotides spanning nts 2862–3055, only three antisense oligonucleotides (DD4, DD7 and DD10) were effective in competing with CRD-BP for binding to 32P-labeled CD44 RNA. The potency of DD4, DD7 and DD10 in inhibiting the CRD-BP-CD44 RNA interaction in vitro correlated with their ability to specifically reduce the steady-state level of CD44 mRNA in cells. The aminoglycoside antibiotics neomycin, paramomycin, kanamycin and streptomycin effectively inhibited the CRD-BP-CD44 RNA interaction in vitro. Assessing the potential inhibitory effect of aminoglycoside antibiotics including neomycin on the CRD-BP-CD44 mRNA interaction in cells proved difficult, likely due to their propensity to non-specifically bind nucleic acids. Our results have important implications for future studies in finding small molecules and nucleic acid-based inhibitors that interfere with protein-RNA interactions. PMID:24622399

Assessing specific oligonucleotides and small molecule antibiotics for the ability to inhibit the CRD-BP-CD44 RNA interaction.

PubMed

King, Dustin T; Barnes, Mark; Thomsen, Dana; Lee, Chow H

2014-01-01

Studies on Coding Region Determinant-Binding Protein (CRD-BP) and its orthologs have confirmed their functional role in mRNA stability and localization. CRD-BP is present in extremely low levels in normal adult tissues, but it is over-expressed in many types of aggressive human cancers and in neonatal tissues. Although the exact role of CRD-BP in tumour progression is unclear, cumulative evidence suggests that its ability to physically associate with target mRNAs is an important criterion for its oncogenic role. CRD-BP has high affinity for the 3'UTR of the oncogenic CD44 mRNA and depletion of CRD-BP in cells led to destabilization of CD44 mRNA, decreased CD44 expression, reduced adhesion and disruption of invadopodia formation. Here, we further characterize the CRD-BP-CD44 RNA interaction and assess specific antisense oligonucleotides and small molecule antibiotics for their ability to inhibit the CRD-BP-CD44 RNA interaction. CRD-BP has a high affinity for binding to CD44 RNA nts 2862-3055 with a Kd of 645 nM. Out of ten antisense oligonucleotides spanning nts 2862-3055, only three antisense oligonucleotides (DD4, DD7 and DD10) were effective in competing with CRD-BP for binding to 32P-labeled CD44 RNA. The potency of DD4, DD7 and DD10 in inhibiting the CRD-BP-CD44 RNA interaction in vitro correlated with their ability to specifically reduce the steady-state level of CD44 mRNA in cells. The aminoglycoside antibiotics neomycin, paramomycin, kanamycin and streptomycin effectively inhibited the CRD-BP-CD44 RNA interaction in vitro. Assessing the potential inhibitory effect of aminoglycoside antibiotics including neomycin on the CRD-BP-CD44 mRNA interaction in cells proved difficult, likely due to their propensity to non-specifically bind nucleic acids. Our results have important implications for future studies in finding small molecules and nucleic acid-based inhibitors that interfere with protein-RNA interactions.

CD44 Promotes Inflammation and Extracellular Matrix Production During Arteriovenous Fistula Maturation.

PubMed

Kuwahara, Go; Hashimoto, Takuya; Tsuneki, Masayuki; Yamamoto, Kota; Assi, Roland; Foster, Trenton R; Hanisch, Jesse J; Bai, Hualong; Hu, Haidi; Protack, Clinton D; Hall, Michael R; Schardt, John S; Jay, Steven M; Madri, Joseph A; Kodama, Shohta; Dardik, Alan

2017-06-01

Arteriovenous fistulae (AVF) remain the optimal conduit for hemodialysis access but continue to demonstrate poor patency and poor rates of maturation. We hypothesized that CD44, a widely expressed cellular adhesion molecule that serves as a major receptor for extracellular matrix components, promotes wall thickening and extracellular matrix deposition during AVF maturation. AVF were created via needle puncture in wild-type C57BL/6J and CD44 knockout mice. CD44 mRNA and protein expression was increased in wild-type AVF. CD44 knockout mice showed no increase in AVF wall thickness (8.9 versus 26.8 μm; P =0.0114), collagen density, and hyaluronic acid density, but similar elastin density when compared with control AVF. CD44 knockout mice also showed no increase in vascular cell adhesion molecule-1 expression, intercellular adhesion molecule-1 expression, and monocyte chemoattractant protein-1 expression in the AVF compared with controls; there were also no increased M2 macrophage markers (transglutaminase-2: 81.5-fold, P =0.0015; interleukin-10: 7.6-fold, P =0.0450) in CD44 knockout mice. Delivery of monocyte chemoattractant protein-1 to CD44 knockout mice rescued the phenotype with thicker AVF walls (27.2 versus 14.7 μm; P =0.0306), increased collagen density (2.4-fold; P =0.0432), and increased number of M2 macrophages (2.1-fold; P =0.0335). CD44 promotes accumulation of M2 macrophages, extracellular matrix deposition, and wall thickening during AVF maturation. These data show the association of M2 macrophages with wall thickening during AVF maturation and suggest that enhancing CD44 activity may be a strategy to increase AVF maturation. © 2017 American Heart Association, Inc.

CD44 Promotes Inflammation and Extracellular Matrix Production During Arteriovenous Fistula Maturation

PubMed Central

Kuwahara, Go; Hashimoto, Takuya; Tsuneki, Masayuki; Yamamoto, Kota; Assi, Roland; Foster, Trenton R; Hanisch, Jesse J; Bai, Hualong; Hu, Haidi; Protack, Clinton D; Hall, Michael R; Schardt, John S; Jay, Steven M; Madri, Joseph A; Kodama, Shohta; Dardik, Alan

2017-01-01

Objective Arteriovenous fistulae (AVF) remain the optimal conduit for hemodialysis access but continue to demonstrate poor patency and poor rates of maturation. We hypothesized that CD44, a widely expressed cellular adhesion molecule that serves as a major receptor for extracellular matrix (ECM) components, promotes wall thickening and ECM deposition during AVF maturation. Approach and Results AVF were created via needle puncture in wild-type (WT) C57BL/6J and CD44 knockout (KO) mice. CD44 mRNA and protein expression was increased in WT AVF. CD44 KO mice showed no increase in AVF wall thickness (8.9 μm vs. 26.8 μm; P = 0.0114), collagen density, and hyaluronic acid density, but similar elastin density when compared to control AVF. CD44 KO mice also showed no increase in VCAM-1 expression, ICAM-1 expression and MCP-1 expression in the AVF compared to controls; there were also no increased M2 macrophage markers (TGM2: 81.5 fold, P = 0.0015; IL-10: 7.6 fold, P = 0.0450) in CD44 KO mice. Delivery of MCP-1 to CD44 KO mice rescued the phenotype with thicker AVF walls (27.2 μm vs. 14.7 μm; P = 0.0306), increased collagen density (2.4 fold; P = 0.0432), and increased number of M2 macrophages (2.1 fold; P = 0.0335). Conclusions CD44 promotes accumulation of M2 macrophages, ECM deposition and wall thickening during AVF maturation. These data show the association of M2 macrophages with wall thickening during AVF maturation and suggest that enhancing CD44 activity may be a strategy to increase AVF maturation. PMID:28450292

Uncovering the dual role of RHAMM as an HA receptor and a regulator of CD44 expression in RHAMM-expressing mesenchymal progenitor cells.

PubMed

Veiseh, Mandana; Leith, Sean J; Tolg, Cornelia; Elhayek, Sallie S; Bahrami, S Bahram; Collis, Lisa; Hamilton, Sara; McCarthy, James B; Bissell, Mina J; Turley, Eva

2015-01-01

The interaction of hyaluronan (HA) with mesenchymal progenitor cells impacts trafficking and fate after tissue colonization during wound repair and these events contribute to diseases such as cancer. How this interaction occurs is poorly understood. Using 10T½ cells as a mesenchymal progenitor model and fluorescent (F-HA) or gold-labeled HA (G-HA) polymers, we studied the role of two HA receptors, RHAMM and CD44, in HA binding and uptake in non-adherent and adherent mesenchymal progenitor (10T½) cells to mimic aspects of cell trafficking and tissue colonization. We show that fluorescent labeled HA (F-HA) binding/uptake was high in non-adherent cells but dropped over time as cells became increasingly adherent. Non-adherent cells displayed both CD44 and RHAMM but only function-blocking anti-RHAMM and not anti-CD44 antibodies significantly reduced F-HA binding/uptake. Adherent cells, which also expressed CD44 and RHAMM, primarily utilized CD44 to bind to F-HA since anti-CD44 but not anti-RHAMM antibodies blocked F-HA uptake. RHAMM overexpression in adherent 10T½ cells led to increased F-HA uptake but this increased binding remained CD44 dependent. Further studies showed that RHAMM-transfection increased CD44 mRNA and protein expression while blocking RHAMM function reduced expression. Collectively, these results suggest that cellular microenvironments in which these receptors function as HA binding proteins differ significantly, and that RHAMM plays at least two roles in F-HA binding by acting as an HA receptor in non-attached cells and by regulating CD44 expression and display in attached cells. Our findings demonstrate adhesion-dependent mechanisms governing HA binding/ uptake that may impact development of new mesenchymal cell-based therapies.

The microRNA miR-34a inhibits prostate cancer stem cells and metastasis by directly repressing CD44.

PubMed

Liu, Can; Kelnar, Kevin; Liu, Bigang; Chen, Xin; Calhoun-Davis, Tammy; Li, Hangwen; Patrawala, Lubna; Yan, Hong; Jeter, Collene; Honorio, Sofia; Wiggins, Jason F; Bader, Andreas G; Fagin, Randy; Brown, David; Tang, Dean G

2011-02-01

Cancer stem cells (CSCs), or tumor-initiating cells, are involved in tumor progression and metastasis. MicroRNAs (miRNAs) regulate both normal stem cells and CSCs, and dysregulation of miRNAs has been implicated in tumorigenesis. CSCs in many tumors--including cancers of the breast, pancreas, head and neck, colon, small intestine, liver, stomach, bladder and ovary--have been identified using the adhesion molecule CD44, either individually or in combination with other marker(s). Prostate CSCs with enhanced clonogenic and tumor-initiating and metastatic capacities are enriched in the CD44(+) cell population, but whether miRNAs regulate CD44(+) prostate cancer cells and prostate cancer metastasis remains unclear. Here we show, through expression analysis, that miR-34a, a p53 target, was underexpressed in CD44(+) prostate cancer cells purified from xenograft and primary tumors. Enforced expression of miR-34a in bulk or purified CD44(+) prostate cancer cells inhibited clonogenic expansion, tumor regeneration, and metastasis. In contrast, expression of miR-34a antagomirs in CD44(-) prostate cancer cells promoted tumor development and metastasis. Systemically delivered miR-34a inhibited prostate cancer metastasis and extended survival of tumor-bearing mice. We identified and validated CD44 as a direct and functional target of miR-34a and found that CD44 knockdown phenocopied miR-34a overexpression in inhibiting prostate cancer regeneration and metastasis. Our study shows that miR-34a is a key negative regulator of CD44(+) prostate cancer cells and establishes a strong rationale for developing miR-34a as a novel therapeutic agent against prostate CSCs.

Uncovering the dual role of RHAMM as an HA receptor and a regulator of CD44 expression in RHAMM-expressing mesenchymal progenitor cells

PubMed Central

Veiseh, Mandana; Leith, Sean J.; Tolg, Cornelia; Elhayek, Sallie S.; Bahrami, S. Bahram; Collis, Lisa; Hamilton, Sara; McCarthy, James B.; Bissell, Mina J.; Turley, Eva

2015-01-01

The interaction of hyaluronan (HA) with mesenchymal progenitor cells impacts trafficking and fate after tissue colonization during wound repair and these events contribute to diseases such as cancer. How this interaction occurs is poorly understood. Using 10T½ cells as a mesenchymal progenitor model and fluorescent (F-HA) or gold-labeled HA (G-HA) polymers, we studied the role of two HA receptors, RHAMM and CD44, in HA binding and uptake in non-adherent and adherent mesenchymal progenitor (10T½) cells to mimic aspects of cell trafficking and tissue colonization. We show that fluorescent labeled HA (F-HA) binding/uptake was high in non-adherent cells but dropped over time as cells became increasingly adherent. Non-adherent cells displayed both CD44 and RHAMM but only function-blocking anti-RHAMM and not anti-CD44 antibodies significantly reduced F-HA binding/uptake. Adherent cells, which also expressed CD44 and RHAMM, primarily utilized CD44 to bind to F-HA since anti-CD44 but not anti-RHAMM antibodies blocked F-HA uptake. RHAMM overexpression in adherent 10T½ cells led to increased F-HA uptake but this increased binding remained CD44 dependent. Further studies showed that RHAMM-transfection increased CD44 mRNA and protein expression while blocking RHAMM function reduced expression. Collectively, these results suggest that cellular microenvironments in which these receptors function as HA binding proteins differ significantly, and that RHAMM plays at least two roles in F-HA binding by acting as an HA receptor in non-attached cells and by regulating CD44 expression and display in attached cells. Our findings demonstrate adhesion-dependent mechanisms governing HA binding/ uptake that may impact development of new mesenchymal cell-based therapies. PMID:26528478

[Proliferative capacity of mesenchymal stem cells from human fetal bone marrow and their ability to differentiate into the derivative cell types of three embryonic germ layers].

PubMed

Wang, Yue-Chun; Zhang, Yuan

2008-06-25

Strong proliferative capacity and the ability to differentiate into the derivative cell types of three embryonic germ layers are the two important characteristics of embryonic stem cells. To study whether the mesenchymal stem cells from human fetal bone marrow (hfBM-MSCs) possess these embryonic stem cell-like biological characteristics, hfBM-MSCs were isolated from bone barrows and further purified according to the different adherence of different kinds of cells to the wall of culture flask. The cell cycle of hfBM-MSCs and MSC-specific surface markers such as CD29, CD44, etc were identified using flow cytometry. The expressions of human telomerase reverse transcriptase (hTERT), the embryonic stem cell-specific antigens, such as Oct4 and SSEA-4 were detected with immunocytochemistry at the protein level and were also tested by RT-PCR at the mRNA level. Then, hfBM-MSCs were induced to differentiate toward neuron cells, adipose cells, and islet B cells under certain conditions. It was found that 92.3% passage-4 hfBM-MSCs and 96.1% passage-5 hfBM-MSCs were at G(0)/G(1) phase respectively. hfBM-MSCs expressed CD44, CD106 and adhesion molecule CD29, but not antigens of hematopoietic cells CD34 and CD45, and almost not antigens related to graft-versus-host disease (GVHD), such as HLA-DR, CD40 and CD80. hfBM-MSCs expressed the embryonic stem cell-specific antigens such as Oct4, SSEA-4, and also hTERT. Exposure of these cells to various inductive agents resulted in morphological changes towards neuron-like cells, adipose-like cells, and islet B-like cells and they were tested to be positive for related characteristic markers. These results suggest that there are plenty of MSCs in human fetal bone marrow, and hfBM-MSCs possess the embryonic stem cell-like biological characteristics, moreover, they have a lower immunogenic nature. Thus, hfBM-MSCs provide an ideal source for tissue engineering and cellular therapeutics.

Biological Features of Human Bone Marrow Stromal Cells (hBMSC) Cultured with Animal Protein-Free Medium-Safety and Efficacy of Clinical Use for Neurotransplantation.

PubMed

Shichinohe, Hideo; Kuroda, Satoshi; Sugiyama, Taku; Ito, Masaki; Kawabori, Masahito; Nishio, Mitsufumi; Takeda, Yukari; Koike, Takao; Houkin, Kiyohiro

2011-09-01

The donor cell culture in animal serum-free medium is quite important for the clinical application of cell transplantation therapy. This study was aimed to test the hypothesis that the human bone marrow stromal cells (hBMSC) expanded with fetal calf serum (FCS)-free, platelet lysate (PL)-containing medium retain their biological features favoring central nervous system regeneration. The hBMSC were cultured with 5% PL or 10% FCS. Their phenotypes were analyzed with flow cytometry, and their production of growth factors was quantified with enzyme-linked immunosorbent assay. Their capacity of neural differentiation was verified by immunocytochemistry. There was no significant difference in morphology and cell surface marker between the hBMSC-FCS and hBMSC-PL. Both of them were positive for CD44, CD90, CD105, and CD166 and were negative for CD34, CD45, and CD271. The production of human brain-derived neurotrophic factor, human hepatocyte growth factor, human β-nerve growth factor, and human platelet-derived growth factor-BB did not differ between the two groups, although the hBMSC-PL produced significantly more amount of TGF-β1 than the hBMSC-FCS. There was no significant difference in their in vitro differentiation into the neurons and astrocytes between the two groups. The hBMSC expanded with PL-containing medium retain their biological capacity of neural differentiation and neuroprotection. The PL may be a clinically valuable and safe substitute for FCS in expanding the hBMSC for cell therapy.

R-spondin1/Wnt-enhanced Ascl2 autoregulation controls the self-renewal of colorectal cancer progenitor cells.

PubMed

Ye, Jun; Liu, Shanxi; Shang, Yangyang; Chen, Haoyuan; Wang, Rongquan

2018-06-25

The Wnt signaling pathway controls stem cell identity in the intestinal epithelium and cancer stem cells (CSCs). The transcription factor Ascl2 (Wnt target gene) is fate decider of intestinal cryptic stem cells and colon cancer stem cells. It is unclear how Wnt signaling is translated into Ascl2 expression and keeping the self-renewal of CRC progenitor cells. We showed that the exogenous Ascl2 in colorectal cancer (CRC) cells activated the endogenous Ascl2 expression via a direct autoactivatory loop, including Ascl2 binding to its own promoter and further transcriptional activation. Higher Ascl2 expression in human CRC cancerous tissues led to greater enrichment in Ascl2 immunoprecipitated DNA within the Ascl2 promoter in the CRC cancerous sample than the peri-cancerous mucosa. Ascl2 binding to its own promoter and inducing further transcriptional activation of the Ascl2 gene was predominant in the CD133 + CD44 + CRC population. R-spondin1/Wnt activated Ascl2 expression dose-dependently in the CD133 + CD44 + CRC population, but not in the CD133 - CD44 - CRC population, which was caused by differences in Ascl2 autoregulation under R-spondin1/Wnt activation. R-spondin1/Wnt treatment in the CD133 + CD44 + or CRC CD133 - CD44 - populations exerted a different pattern of stemness maintenance, which was defined by alterations of the mRNA levels of stemness-associated genes, the protein expression levels (Bmi1, C-myc, Oct-4 and Nanog) and tumorsphere formation. The results indicated that Ascl2 autoregulation formed a transcriptional switch that was enhanced by Wnt signaling in the CD133 + CD44 + CRC population, thus conferring their self-renewal.

Notch1 inhibition alters the CD44hi/CD24lo population and reduces the formation of brain metastases from breast cancer.

PubMed

McGowan, Patricia M; Simedrea, Carmen; Ribot, Emeline J; Foster, Paula J; Palmieri, Diane; Steeg, Patricia S; Allan, Alison L; Chambers, Ann F

2011-07-01

Brain metastasis from breast cancer is an increasingly important clinical problem. Here we assessed the role of CD44(hi)/CD24(lo) cells and pathways that regulate them, in an experimental model of brain metastasis. Notch signaling (mediated by γ-secretase) has been shown to contribute to maintenance of the cancer stem cell (CSC) phenotype. Cells sorted for a reduced stem-like phenotype had a reduced ability to form brain metastases compared with unsorted or CD44(hi)/CD24(lo) cells (P < 0.05; Kruskal-Wallis). To assess the effect of γ-secretase inhibition, cells were cultured with DAPT and the CD44/CD24 phenotypes quantified. 231-BR cells with a CD44(hi)/CD24(lo) phenotype was reduced by about 15% in cells treated with DAPT compared with DMSO-treated or untreated cells (P = 0.001, ANOVA). In vivo, mice treated with DAPT developed significantly fewer micro- and macrometastases compared with vehicle treated or untreated mice (P = 0.011, Kruskal-Wallis). Notch1 knockdown reduced the expression of CD44(hi)/CD24(lo) phenotype by about 20%. In vitro, Notch1 shRNA resulted in a reduction in cellular growth at 24, 48, and 72 hours time points (P = 0.033, P = 0.002, and P = 0.009, ANOVA) and about 60% reduction in Matrigel invasion was observed (P < 0.001, ANOVA). Cells transfected with shNotch1 formed significantly fewer macrometastases and micrometastases compared with scrambled shRNA or untransfected cells (P < 0.001; Kruskal-Wallis). These data suggest that the CSC phenotype contributes to the development of brain metastases from breast cancer, and this may arise in part from increased Notch activity. ©2011 AACR.

Screening and Identification of a Phage Display Derived Peptide That Specifically Binds to the CD44 Protein Region Encoded by Variable Exons.

PubMed

Zhang, Dan; Jia, Huan; Li, Weiming; Hou, Yingchun; Lu, Shaoying; He, Shuixiang

2016-01-01

CD44, especially the isoforms with variable exons (CD44v), is a promising biomarker for the detection of cancer. To develop a CD44v-specific probe, we screened a 7-mer phage peptide library against the CD44v3-v10 protein using an improved subtractive method. The consensus sequences with the highest frequency (designated CV-1) emerged after four rounds of panning. The binding affinity and specificity of the CV-1 phage and the synthesized peptide for the region of CD44 encoded by the variable exons were confirmed using enzyme-linked immunosorbent assay and competitive inhibition assays. Furthermore, the binding of the CV-1 probe to gastric cancer cells and tissues was validated using immunofluorescence and immunohistochemistry assays. CV-1 sensitively and specifically bound to CD44v on cancer cells and tissues. Thus, CV-1 has the potential to serve as a promising probe for cancer molecular imaging and target therapy. © 2015 Society for Laboratory Automation and Screening.

Evaluation of CD44 and CD133 as markers of liver cancer stem cells in Egyptian patients with HCV-induced chronic liver diseases versus hepatocellular carcinoma

PubMed Central

Rozeik, Mohammed Saeed; Hammam, Olfat Ali; Ali, Ali Ibrahim; Magdy, Mona; Khalil, Heba; Anas, Amgad; Abo el Hassan, Ahmed Abdelaleem; Rahim, Ali Abdel; El-Shabasy, Ahmed Ibrahim

2017-01-01

Background Cancer stem cells (CSCs) play a critical role in tumor development, progression, metastasis and recurrence. Aim To evaluate hepatic expression of CD44 and CD133 in Egyptian patients with HCV-induced chronic liver diseases and hepatocellular carcinomas (HCCs), and to assess its correlation with inflammatory activity scores, stages of fibrosis (in chronic hepatitis with or without cirrhosis) and grades of HCC. Methods This prospective case-control study was conducted on eighty subjects who attended the Tropical Diseases Department, Al-Azhar University Hospital, and in collaboration with Theodor Bilharz Research Institute (2014–2016). They were divided as follows: A) Control healthy group: Ten individuals with serologically negative HCV-Ab and HBsAg, and histopathologically normal liver, B) Seventy patients subdivided into 3 groups; Twenty subjects each, as: HCV-Ab+ non-cirrhotic, HCV-Ab+ cirrhotic and HCC. Necroinflammatory activity and fibrosis in non-neoplastic liver biopsies were scored according to the METAVIR scoring system. CD44 and CD133 immunostaining was evaluated in all groups semi-quantitatively using H score. Statistical analysis was performed by SPSS version 22, using independent-samples t-test. Results Our study showed a significant increase of mean CD44 & CD133 expression values with disease progression among the groups (p<0.05). Their expressions increased significantly with the inflammatory activity scores and stages of fibrosis, reaching the highest values in A3F4 score compared to A1F1 (p<0.05). Moreover, there was a significant increase of their expressions across HCC grades (p<0.05), however with no significant correlation with focal lesions size. Conclusion CSCs clusters exhibiting CD133+ and/or CD44+ profiles were identified in chronic hepatitis, liver cirrhosis and HCC. CD133 and CD44 expressions significantly corresponded to the increased inflammatory activity, fibrosis stages and higher tumor grades. Therefore, evaluation of CD44 and CD133 expression profiles as CSCs markers in non-neoplastic liver and HCCs can help in development of novel therapeutic agents for HCC targeting and prevention. PMID:28894525

An immunohistochemical analysis of canine haemangioma and haemangiosarcoma.

PubMed

Sabattini, S; Bettini, G

2009-01-01

The aim of the present study was to investigate immunohistochemically aspects of the biology of canine endothelial neoplasia. Forty samples of canine cutaneous and visceral haemangiosarcoma (HSA), 29 samples of cutaneous and visceral haemangioma (HA) and 10 control samples of granulation tissue (GT) were labelled with antisera specific for vimentin, smooth muscle actin, von Willebrand factor (vWF), CD117 (KIT), vascular endothelial growth factor receptor-3 (VEGFR-3), vascular endothelial growth factor-C (VEGFC) and CD44. Further antisera were employed to determine the level of cellular proliferation (MIB-1 index) and toluidine blue staining was used to detect populations of tumour-infiltrating mast cells (MCs). There was greater expression of CD117, VEGFR-3 and CD44 in HSA than in HA, suggesting that these proteins might be suitable targets for the future development of novel therapeutic approaches to canine HSA. Marked infiltration of MC was detected in HA, suggesting a possible role for these cells in the pathogenesis of benign vascular neoplasia in the dog.

Breast cancer stem cell-like cells generated during TGFβ-induced EMT are radioresistant.

PubMed

Konge, Julie; Leteurtre, François; Goislard, Maud; Biard, Denis; Morel-Altmeyer, Sandrine; Vaurijoux, Aurélie; Gruel, Gaetan; Chevillard, Sylvie; Lebeau, Jérôme

2018-05-04

Failure of conventional antitumor therapy is commonly associated with cancer stem cells (CSCs), which are often defined as inherently resistant to radiation and chemotherapeutic agents. However, controversy about the mechanisms involved in the radiation response remains and the inherent intrinsic radioresistance of CSCs has also been questioned. These discrepancies observed in the literature are strongly associated with the cell models used. In order to clarify these contradictory observations, we studied the radiosensitivity of breast CSCs using purified CD24 -/low /CD44 + CSCs and their corresponding CD24 + /CD44 low non-stem cells. These cells were generated after induction of the epithelial-mesenchymal transition (EMT) by transforming growth factor β (TGFβ) in immortalized human mammary epithelial cells (HMLE). Consequently, these 2 cellular subpopulations have an identical genetic background, their differences being related exclusively to TGFβ-induced cell reprogramming. We showed that mesenchymal CD24 -/low /CD44 + CSCs are more resistant to radiation compared with CD24 + /CD44 low parental cells. Cell cycle distribution and free radical scavengers, but not DNA repair efficiency, appeared to be intrinsic determinants of cellular radiosensitivity. Finally, for the first time, we showed that reduced radiation-induced activation of the death receptor pathways (FasL, TRAIL and TNF-α) at the transcriptional level was a key causal event in the radioresistance of CD24 -/low / CD44+ cells acquired during EMT.

Intracellular targeting of CD44+ cells with self-assembling, protein only nanoparticles.

PubMed

Pesarrodona, Mireia; Ferrer-Miralles, Neus; Unzueta, Ugutz; Gener, Petra; Tatkiewicz, Witold; Abasolo, Ibane; Ratera, Imma; Veciana, Jaume; Schwartz, Simó; Villaverde, Antonio; Vazquez, Esther

2014-10-01

CD44 is a multifunctional cell surface protein involved in proliferation and differentiation, angiogenesis and signaling. The expression of CD44 is up-regulated in several types of human tumors and particularly in cancer stem cells, representing an appealing target for drug delivery in the treatment of cancer. We have explored here several protein ligands of CD44 for the construction of self-assembling modular proteins designed to bind and internalize target cells. Among five tested ligands, two of them (A5G27 and FNI/II/V) drive the formation of protein-only, ring-shaped nanoparticles of about 14 nm that efficiently bind and penetrate CD44(+) cells by an endosomal route. The potential of these newly designed nanoparticles is evaluated regarding the need of biocompatible nanostructured materials for drug delivery in CD44-linked conditions. Copyright © 2014 Elsevier B.V. All rights reserved.

Clinical significance of CD44 expression in children with hepatoblastoma.

PubMed

Cai, H-Y; Yu, B; Feng, Z-C; Qi, X; Wei, X-J

2015-10-27

The aim of this study was to investigate the expression of CD44 and its clinical significance in children suffering from hepatoblastoma (HB). CD44 expression was detected with immunohistochemistry staining in 30 samples from hepatoblastoma children and 10 normal liver tissue samples from normal children. The data obtained was statistically analyzed using the chi-square test, using the SPSS (v.11.0) software. The rate of CD44 expression was significantly higher (66.7%) in hepatoblastoma tissues than in normal liver tissues (χ(2) = 4.848, P < 0.05). The rate of CD44 expression was significantly higher in children with stage III or IV hepatoblastoma (83.3%) than that in children with stage I and II hepatoblastoma (χ(2) ＝ 5.625, P < 0.05) (41.7%). Therefore, CD44 expression might play an important role in the pathogenesis, progression, and prognosis of HB in children.

Properties Of Passivant Films On HgCdTe - Interaction With The Substrate

NASA Astrophysics Data System (ADS)

Davis, G. D.; Sun, T. S.; Buchner, S. P.; Byer, N. E.

1981-12-01

Two commonly used passivants of Hg0.8Cd0.2Te, the anodic oxide and ZnS, have been studied by x-ray photoelectron spectroscopy combined with ion sputtering. Chemical depth profiles of anodic oxide films of 360 to 1600 A showed that the oxide composition is constant with depth and independent of oxide thickness. Chemical shifts and line shape analysis of the Cd M45N45N45 Auger transition in the oxide, CdO, Cd(OH)2, and CdTeO3 demonstrate that CdTeO3 is the major constituent of the anodic oxide. The oxide composi-tion is interpreted as 44% CdTeO3, 29% CdTe2O5, 17% HgTeO3, and 10% HgTe2O5. Anodization of HgCdTe depletes the semiconductor of 30% - 40% of its Hg near the interface. The spatial extent of this Hg depletion is a function of oxide thickness for thin oxides (<1000 A) but is a constant (150-200 A) for thick films. No significant change in the Cd concentration is seen. A ZnS film deposited on a chemically etched sample forms a graded interface of a (ZnHgCd)Te alloy. In this case, no Hg depletion is seen. Deposi-tion of ZnS on an anodized substrate in high vacuum leads to a reaction of the Zn with the residual 02 in the chamber to form ZnO on the anodic oxide before the ZnS. The ZnO then diffuses throughout the anodic oxide.

[Dectection of G3BP and CD44v6 in the tissues of laryngeal squamous cell carcinoma and their clinical significance].

PubMed

Luo, Dahu; Lou, Weihua

2017-07-01

Objective To study the expressions of RNA-binding Ras-GAP SH3 binding protein (G3BP) and tumor stem cell marker CD44v6 in laryngeal squamous cell carcinoma and their correlations with angiogenesis. Methods We collected the cancer tissues and corresponding paracancerous tissues from 56 patients with laryngeal squamous cell carcinoma. The expressions of G3BP and CD44v6 proteins were detected by Western blotting in cancer tissues and corresponding paracancerous tissues; the expressions of G3BP, CD44v6 and vascular endothelial growth factor A (VEGF-A) were tested by immunohistochemistry. Thereafter, we compared the positive expression rates of G3BP and CD44v6 between in cancer tissues and in normal tissues, analyzed the correlations between the expressions of G3BP, CD44v6 and the laryngeal squamous cell carcinoma features as well as their correlations with microvessel density (MVD) that was determined by FVIIIAg immunohistochemistry. Results Western blotting showed that the expressions of G3BP and CD44v6 proteins in the laryngeal squamous cell carcinoma were higher than those in the paracancerous tissues. Immunohistochemistry showed that compared with the paracancerous tissues, G3BP, CD44v6 and VEGF-A expressions (the positive rates are 58.9%, 53.6%, 46.4%, respectively) were higher in cancer tissues. The positive rates of G3BP and CD44v6 in cancer tissues were related with the clinical stage, recurrence or metastasis, and lymph node metastasis of laryngeal squamous cell carcinoma, but had nothing to do with patients' age and tumor size. Pearson correlation analysis showed the expressions of both G3BP and CD44v6 were positively correlated with VEGF-A (r=0.741, r=0.756). MVD values were significantly higher in the G3BP and CD44v6 positive cases than in paracancerous tissues, but there was no difference in MVD between those without G3BP and CD44v6 positive expressions and the paracancerous tissues. Conclusion The positive expression rates of G3BP and CD44v6 in laryngeal squamous cell carcinoma tissues are very high, and they have a close relationship with the clinical prognosis. They may raise the VEGF-A expression so as to promote angiogenesis, and then accelerate the development of the laryngeal squamous cell carcinoma.

Comparison of EpCAMhighCD44+ cancer stem cells with EpCAMhighCD44- tumor cells in colon cancer by single-cell sequencing.

PubMed

Liu, Mingshan; Di, Jiabo; Liu, Yang; Su, Zhe; Jiang, Beihai; Wang, Zaozao; Su, Xiangqian

2018-03-26

Cancer stem cells (CSCs) are considered to be responsible for tumorigenesis and cancer relapse. EpCAM high CD44 + tumor cells are putative colorectal CSCs that express high levels of stem cell genes, while the EpCAM high CD44 - population mostly contains differentiated tumor cells (DTCs). This study aims to determine whether single CSC (EpCAM high CD44 + ) and DTC (EpCAM high CD44 - ) can be distinguished in terms of somatic copy number alterations (SCNAs). We applied fluorescence-activated cell sorting to isolate the CD45 - EpCAM high CD44 + and CD45 - EpCAM high CD44 - populations from two primary colon tumors, on which low-coverage single-cell whole-genome sequencing (WGS) was then performed ∼0.1x depth. We compared the SCNAs of the CSCs and DTCs at single-cell resolution. In total, 47 qualified single cells of the two populations underwent WGS. The single-cell SCNA profiles showed that there were obvious SCNAs in both the CSCs and DTCs of each patient, and each patient had a specific copy number alteration pattern. Hierarchical clustering and correlation analysis both showed that the SCNA profiles of CSCs and DTCs from the same patient had similar SCNA pattern, while there were regional differences in the CSCs and DTCs in certain patient. SCNAs of CSCs in the same patient were highly reproducible. Our data suggest that major SCNAs occurred at an early stage and were inherited steadily. The similarity of ubiquitous SCNAs between the CSCs and DTCs might have arisen from lineage differentiation. CSCs from the same patient had reproducible SCNA profiles, indicating that gain or loss in certain chromosome is required for colon cancer development.

Salivary-soluble CD44 levels in smokers and non-smokers with chronic periodontitis: a pilot study.

PubMed

Ghallab, Noha; Shaker, Olfat

2010-05-01

Smoking is the most important environmental risk factor for periodontal disease. Elevated levels of serum-soluble CD44 (sCD44) have been detected in smokers and also have been recognized as a diagnostic marker in some smoking-induced diseases. The present study investigates the salivary sCD44 profiles of smokers and non-smokers with and without chronic periodontitis in response to scaling and root planing (SRP). The study included 44 subjects divided into two groups: 22 patients with chronic periodontitis and 22 periodontally healthy subjects. Both groups were equally subdivided into smokers (n = 11) and non-smokers (n = 11). Plaque index, gingival index, probing depth, and clinical attachment level were recorded only for chronic periodontitis patients. Salivary samples were collected from all 44 patients at baseline and after 1 month of SRP from the 22 chronic periodontitis patients. Assay for salivary sCD44 was carried out by enzyme-linked immunosorbent assay. Baseline salivary sCD44 profiles were significantly higher when smokers were compared to non-smokers in both chronic periodontitis patients and the control subjects (P <0.001) with the highest levels recorded in smokers within the chronic periodontitis group. There was a significant decline in salivary sCD44 levels after treatment in the chronic periodontitis group for both smokers and non-smokers (P <0.01); however, the difference between groups was insignificant. Salivary sCD44 might be considered a biomarker of periodontal destruction in smokers and non-smokers. The research opens the door to further research into a role for CD44 as a diagnostic marker for periodontitis.

CD133 expression is not restricted to stem cells, and both CD133+ and CD133– metastatic colon cancer cells initiate tumors

PubMed Central

Shmelkov, Sergey V.; Butler, Jason M.; Hooper, Andrea T.; Hormigo, Adilia; Kushner, Jared; Milde, Till; St. Clair, Ryan; Baljevic, Muhamed; White, Ian; Jin, David K.; Chadburn, Amy; Murphy, Andrew J.; Valenzuela, David M.; Gale, Nicholas W.; Thurston, Gavin; Yancopoulos, George D.; D’Angelica, Michael; Kemeny, Nancy; Lyden, David; Rafii, Shahin

2008-01-01

Colon cancer stem cells are believed to originate from a rare population of putative CD133+ intestinal stem cells. Recent publications suggest that a small subset of colon cancer cells expresses CD133, and that only these CD133+ cancer cells are capable of tumor initiation. However, the precise contribution of CD133+ tumor-initiating cells in mediating colon cancer metastasis remains unknown. Therefore, to temporally and spatially track the expression of CD133 in adult mice and during tumorigenesis, we generated a knockin lacZ reporter mouse (CD133lacZ/+), in which the expression of lacZ is driven by the endogenous CD133 promoters. Using this model and immunostaining, we discovered that CD133 expression in colon is not restricted to stem cells; on the contrary, CD133 is ubiquitously expressed on differentiated colonic epithelium in both adult mice and humans. Using Il10–/–CD133lacZ mice, in which chronic inflammation in colon leads to adenocarcinomas, we demonstrated that CD133 is expressed on a full gamut of colonic tumor cells, which express epithelial cell adhesion molecule (EpCAM). Similarly, CD133 is widely expressed by human primary colon cancer epithelial cells, whereas the CD133– population is composed mostly of stromal and inflammatory cells. Conversely, CD133 expression does not identify the entire population of epithelial and tumor-initiating cells in human metastatic colon cancer. Indeed, both CD133+ and CD133– metastatic tumor subpopulations formed colonospheres in in vitro cultures and were capable of long-term tumorigenesis in a NOD/SCID serial xenotransplantation model. Moreover, metastatic CD133– cells form more aggressive tumors and express typical phenotypic markers of cancer-initiating cells, including CD44 (CD44+CD24–), whereas the CD133+ fraction is composed of CD44lowCD24+ cells. Collectively, our data suggest that CD133 expression is not restricted to intestinal stem or cancer-initiating cells, and during the metastatic transition, CD133+ tumor cells might give rise to the more aggressive CD133– subset, which is also capable of tumor initiation in NOD/SCID mice. PMID:18497886

Skeletal myogenic differentiation of human urine-derived cells as a potential source for skeletal muscle regeneration.

PubMed

Chen, Wei; Xie, Minkai; Yang, Bin; Bharadwaj, Shantaram; Song, Lujie; Liu, Guihua; Yi, Shanhong; Ye, Gang; Atala, Anthony; Zhang, Yuanyuan

2017-02-01

Stem cells are regarded as possible cell therapy candidates for skeletal muscle regeneration. However, invasive harvesting of those cells can cause potential harvest-site morbidity. The goal of this study was to assess whether human urine-derived stem cells (USCs), obtained through non-invasive procedures, can differentiate into skeletal muscle linage cells (Sk-MCs) and potentially be used for skeletal muscle regeneration. In this study, USCs were harvested from six healthy individuals aged 25-55. Expression profiles of cell-surface markers were assessed by flow cytometry. To optimize the myogenic differentiation medium, we selected two from four different types of myogenic differentiation media to induce the USCs. Differentiated USCs were identified with myogenic markers by gene and protein expression. USCs were implanted into the tibialis anterior muscles of nude mice for 1 month. The results showed that USCs displayed surface markers with positive staining for CD24, CD29, CD44, CD73, CD90, CD105, CD117, CD133, CD146, SSEA-4 and STRO-1, and negative staining for CD14, CD31, CD34 and CD45. After myogenic differentiation, a change in morphology was observed from 'rice-grain'-like cells to spindle-shaped cells. The USCs expressed specific Sk-MC transcripts and protein markers (myf5, myoD, myosin, and desmin) after being induced with different myogenic culture media. Implanted cells expressed Sk-MC markers stably in vivo. Our findings suggest that USCs are able to differentiate into the Sk-MC lineage in vitro and after being implanted in vivo. Thus, they might be a potential source for cell injection therapy in the use of skeletal muscle regeneration. Copyright © 2014 John Wiley & Sons, Ltd. Copyright © 2014 John Wiley & Sons, Ltd.

CD44 gene vaccination for insulin-dependent diabetes mellitus in non-obese diabetic mice.

PubMed

Weiss, Lola; Botero-Anug, Ana Maria; Hand, Carla; Slavin, Shimon; Naor, David

2008-01-01

Standard CD44 and its alternatively spliced variants were found to be associated with the metastatic potential of tumor cells and with cell migration of autoimmune inflammatory cells, including cells involved in experimental insulin-dependent diabetes mellitus. To investigate whether induction of anti-CD44 immune reactivity, through cDNA vaccination, could attenuate IDDM in a transfer model of NOD mice. Our vaccination technique involved the insertion of CD44s or CD44v cDNA into a silicone tube filled with a 2.5 cm long segment of hydroxylated-polyvinyl acetate wound dressing sponge (forming a virtual lymph node) which was implanted under the skin of male NOD recipients reconstituted with diabetogenic spleen cells of female NOD donors. The VLN were implanted 20 days before and 3 days after cell transfer. In contrast to control groups of recipient mice, recipients vaccinated with VLN loaded with CD44v or CD44s cDNAs developed resistance to IDDM almost to the same extent. Our results suggest that the gene vaccination effect was mediated by anti-CD44 antibody rather than by cellular immunity. Histopathological examinations revealed a significant protection of pancreatic islets in the DNA-vaccinated recipients, whereas the islets of control recipients of diabetogenic cells were almost totally destroyed. These findings may open new opportunities for IDDM therapy in the future.

Effects of CD44 and E-cadherin overexpression on the proliferation, adhesion and invasion of ovarian cancer cells.

PubMed

Mao, Meiya; Zheng, Xiaojiao; Jin, Bohong; Zhang, Fubin; Zhu, Linyan; Cui, Lining

2017-12-01

CD44 is a prognostic indicator of shorter survival time in ovarian cancer. E-cadherin fragmentation promotes the progression of ovarian cancer. However, the effects of CD44 and E-cadherin overexpression on ovarian cancer cells have remained elusive. The present study aimed to investigate the effects of overexpression of CD44 and E-cadherin on cell proliferation, adhesion and invasion of SKOV-3 and OVCAR-3 ovarian cancer cells. Overexpression of CD44 and E-cadherin was achieved by transfecting SKOV-3 and OVCAR-3 cells with viruses carrying the CD44 or E-cadherin gene, respectively. Expression of CD44 and E-cadherin was detected by western blot analysis. The proliferation of SKOV-3 and OVCAR-3 cells was measured by a Cell Counting Kit-8 at 0, 24 and 48 h after viral transfection. The adhesion ability of SKOV-3 and OVCAR-3 cells to the endothelial layer was detected. A Transwell invasion assay was utilized to assess the invasion ability of the cells. Overexpression of CD44 and E-cadherin in SKOV-3 and OVCAR-3 cells was confirmed by western blot. Compared with the blank or negative control groups, the CD44 overexpression groups of SKOV-3 and OVCAR-3 cells exhibited an increased cell proliferation rate at 24 and 48 h, whereas overexpression of E-cadherin did not alter the proliferation of these cells. Furthermore, compared with the blank and negative control groups, the cell adhesion and invasion ability in the CD44 overexpression groups of SKOV-3 and OVCAR-3 cells was markedly higher. There were no significant differences in adhesion ability between the E-cadherin overexpression group and the blank/negative control group. Of note, overexpression of E-cadherin decreased the invasive ability of SKOV-3 and OVCAR-3 cells. In conclusion, Overexpression of CD44 increased the proliferation, adhesion and invasion of ovarian cancer cells, while overexpression of E-cadherin decreased the invasion of ovarian cancer cells.

Kaempferol regulates OPN-CD44 pathway to inhibit the atherogenesis of apolipoprotein E deficient mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xiao, Hong-Bo, E-mail: xhbzhb@yahoo.com; Lu, Xiang-Yang; Sun, Zhi-Liang

Recent studies show that osteopontin (OPN) and its receptor cluster of differentiation 44 (CD44) are two pro-inflammatory cytokines contributing to the development of atherosclerosis. The objective of this study was to explore the inhibitory effect of kaempferol, a naturally occurring flavonoid compound, on atherogenesis and the mechanisms involved. The experiments were performed in aorta and plasma from C57BL/6J control and apolipoprotein E-deficient (ApoE{sup -/-}) mice treated or not with kaempferol (50 or 100 mg/kg, intragastrically) for 4 weeks. Kaempferol treatment decreased atherosclerotic lesion area, improved endothelium-dependent vasorelaxation, and increased the maximal relaxation value concomitantly with decrease in the half-maximum effectivemore » concentration, plasma OPN level, aortic OPN expression, and aortic CD44 expression in ApoE{sup -/-} mice. In addition, treatment with kaempferol also significantly decreased reactive oxygen species production in mice aorta. The present results suggest that kaempferol regulates OPN-CD44 pathway to inhibit the atherogenesis of ApoE{sup -/-} mice. -- Graphical abstract: Kaempferol regulates OPN-CD44 pathway to inhibit the atherogenesis of ApoE{sup -/-} mice. Highlights: Black-Right-Pointing-Pointer OPN-CD44 pathway plays a critical role in the development of atherosclerosis. Black-Right-Pointing-Pointer We examine lesion area, OPN and CD44 changes after kaempferol treatment. Black-Right-Pointing-Pointer Kaempferol treatment decreased atherosclerotic lesion area in ApoE{sup -/-} mice. Black-Right-Pointing-Pointer Kaempferol treatment decreased aortic OPN and CD44 expressions in ApoE{sup -/-} mice. Black-Right-Pointing-Pointer Kaempferol regulates OPN-CD44 pathway to inhibit the atherogenesis.« less

Biocompatibility of quantum dots (CdSe/ZnS ) in human amniotic membrane-derived mesenchymal stem cells in vitro.

PubMed

Wang, Gongping; Zeng, Guangwei; Wang, Caie; Wang, Huasheng; Yang, Bo; Guan, Fangxia; Li, Dongpeng; Feng, Xiaoshan

2015-06-01

Amniotic membrane-derived mesenchymal stem cells (hAM-dMSCs) are a potential source of mesenchymal stem cells which could be used to repair skin damage. The use of mesenchymal stem cells to repair skin damage requires safe, effective and biocompatible agents to evaluate the effectiveness of the result. Quantum dots (QDs) composed of CdSe/ZnS are semiconductor nanocrystals with broad excitation and narrow emission spectra, which have been considered as a new chemical and fluorescent substance for non-invasively labeling different cells in vitro and in vivo. This study investigated the cytotoxic effects of QDs on hAM-dMSCs at different times following labeling. Using 0.75, 1.5 and 3.0 μL between quantum dots, labeled human amniotic mesenchymal stem cells were collected on days 1, 2 and 4 and observed morphological changes, performed an MTT cell growth assay and flow cytometry for mesenchymal stem cells molecular markers. Quantum dot concentration 0.75 μg/mL labeled under a fluorescence microscope, cell morphology was observed, The MTT assay showed cells in the proliferative phase. Flow cytometry expression CD29, CD31, CD34, CD44, CD90, CD105 and CD106. Within a certain range of concentrations between quantum dots labeled human amniotic mesenchymal stem cells has good biocompatibility.

Porcine uterus contains a population of mesenchymal stem cells.

PubMed

Miernik, Katarzyna; Karasinski, Janusz

2012-02-01

The uterus has a remarkable ability of cycling remodeling throughout the reproductive life of the female. Recent findings in the human and mouse indicate that adult stem/progenitor cells may play a prominent role in the maintenance of uterine endometrial and myometrial homeostasis. We aimed to characterize the prospective stem/progenitor cells in the porcine uterus and establish a new model for uterine stem cell research. In this study, we demonstrated that cells isolated from porcine uterus have capacity for in vitro differentiation into adipogenic and osteogenic lineages and express the mesenchymal stem cell (MSC) markers CD29, CD44, CD144, CD105, and CD140b as revealed by RT-PCR. Moreover, we showed that some cells isolated from the porcine uterus when cultured at low density produce large clones with an efficiency of 0.035%. Simultaneously, they were negative for hematopoietic stem cell markers such as CD34 and CD45. Low expression of nestin, which is specific for neural stem cells and various progenitor cells, was also detected. We conclude that the porcine uterus contains a small population of undifferentiated cells with MSC-like properties similar to human and mouse uteri.

FGF-2 signal promotes proliferation of cerebellar progenitor cells and their oligodendrocytic differentiation at early postnatal stage

DOE Office of Scientific and Technical Information (OSTI.GOV)

Naruse, Masae; Shibasaki, Koji; Ishizaki, Yasuki, E-mail: yasukiishizaki@gunma-u.ac.jp

The origins and developmental regulation of cerebellar oligodendrocytes are largely unknown, although some hypotheses of embryonic origins have been suggested. Neural stem cells exist in the white matter of postnatal cerebellum, but it is unclear whether these neural stem cells generate oligodendrocytes at postnatal stages. We previously showed that cerebellar progenitor cells, including neural stem cells, widely express CD44 at around postnatal day 3. In the present study, we showed that CD44-positive cells prepared from the postnatal day 3 cerebellum gave rise to neurospheres, while CD44-negative cells prepared from the same cerebellum did not. These neurospheres differentiated mainly into oligodendrocytesmore » and astrocytes, suggesting that CD44-positive neural stem/progenitor cells might generate oligodendrocytes in postnatal cerebellum. We cultured CD44-positive cells from the postnatal day 3 cerebellum in the presence of signaling molecules known as mitogens or inductive differentiation factors for oligodendrocyte progenitor cells. Of these, only FGF-2 promoted survival and proliferation of CD44-positive cells, and these cells differentiated into O4+ oligodendrocytes. Furthermore, we examined the effect of FGF-2 on cerebellar oligodendrocyte development ex vivo. FGF-2 enhanced proliferation of oligodendrocyte progenitor cells and increased the number of O4+ and CC1+ oligodendrocytes in slice cultures. These results suggest that CD44-positive cells might be a source of cerebellar oligodendrocytes and that FGF-2 plays important roles in their development at an early postnatal stage. - Highlights: • CD44 is expressed in cerebellar neural stem/progenitor cells at postnatal day 3 (P3). • FGF-2 promoted proliferation of CD44-positive progenitor cells from P3 cerebellum. • FGF-2 promoted oligodendrocytic differentiation of CD44-positive progenitor cells. • FGF-2 increased the number of oligodendrocytes in P3 cerebellar slice culture.« less

Challenging the roles of CD44 and lipolysis stimulated lipoprotein receptor in conveying Clostridium perfringens iota toxin cytotoxicity in breast cancer

PubMed Central

2014-01-01

Background Translational exploration of bacterial toxins has come to the forefront of research given their potential as a chemotherapeutic tool. Studies in select tissues have demonstrated that Clostridium perfringens iota toxin binds to CD44 and lipolysis stimulated lipoprotein receptor (LSR) cell-surface proteins. We recently demonstrated that LSR expression correlates with estrogen receptor positive breast cancers and that LSR signaling directs aggressive, tumor-initiating cell behaviors. Herein, we identify the mechanisms of iota toxin cytotoxicity in a tissue-specific, breast cancer model with the ultimate goal of laying the foundation for using iota toxin as a targeted breast cancer therapy. Methods In vitro model systems were used to determine the cytotoxic effect of iota toxin on breast cancer intrinsic subtypes. The use of overexpression and knockdown technologies confirmed the roles of LSR and CD44 in regulating iota toxin endocytosis and induction of cell death. Lastly, cytotoxicity assays were used to demonstrate the effect of iota toxin on a validated set of tamoxifen resistant breast cancer cell lines. Results Treatment of 14 breast cancer cell lines revealed that LSR+/CD44- lines were highly sensitive, LSR+/CD44+ lines were slightly sensitive, and LSR-/CD44+ lines were resistant to iota cytotoxicity. Reduction in LSR expression resulted in a significant decrease in toxin sensitivity; however, overexpression of CD44 conveyed toxin resistance. CD44 overexpression was correlated with decreased toxin-stimulated lysosome formation and decreased cytosolic levels of iota toxin. These findings indicated that expression of CD44 drives iota toxin resistance through inhibition of endocytosis in breast cancer cells, a role not previously defined for CD44. Moreover, tamoxifen-resistant breast cancer cells exhibited robust expression of LSR and were highly sensitive to iota-induced cytotoxicity. Conclusions Collectively, these data are the first to show that iota toxin has the potential to be an effective, targeted therapy for breast cancer. PMID:24990559

Challenging the roles of CD44 and lipolysis stimulated lipoprotein receptor in conveying Clostridium perfringens iota toxin cytotoxicity in breast cancer.

PubMed

Fagan-Solis, Katerina D; Reaves, Denise K; Rangel, M Cristina; Popoff, Michel R; Stiles, Bradley G; Fleming, Jodie M

2014-07-02

Translational exploration of bacterial toxins has come to the forefront of research given their potential as a chemotherapeutic tool. Studies in select tissues have demonstrated that Clostridium perfringens iota toxin binds to CD44 and lipolysis stimulated lipoprotein receptor (LSR) cell-surface proteins. We recently demonstrated that LSR expression correlates with estrogen receptor positive breast cancers and that LSR signaling directs aggressive, tumor-initiating cell behaviors. Herein, we identify the mechanisms of iota toxin cytotoxicity in a tissue-specific, breast cancer model with the ultimate goal of laying the foundation for using iota toxin as a targeted breast cancer therapy. In vitro model systems were used to determine the cytotoxic effect of iota toxin on breast cancer intrinsic subtypes. The use of overexpression and knockdown technologies confirmed the roles of LSR and CD44 in regulating iota toxin endocytosis and induction of cell death. Lastly, cytotoxicity assays were used to demonstrate the effect of iota toxin on a validated set of tamoxifen resistant breast cancer cell lines. Treatment of 14 breast cancer cell lines revealed that LSR+/CD44- lines were highly sensitive, LSR+/CD44+ lines were slightly sensitive, and LSR-/CD44+ lines were resistant to iota cytotoxicity. Reduction in LSR expression resulted in a significant decrease in toxin sensitivity; however, overexpression of CD44 conveyed toxin resistance. CD44 overexpression was correlated with decreased toxin-stimulated lysosome formation and decreased cytosolic levels of iota toxin. These findings indicated that expression of CD44 drives iota toxin resistance through inhibition of endocytosis in breast cancer cells, a role not previously defined for CD44. Moreover, tamoxifen-resistant breast cancer cells exhibited robust expression of LSR and were highly sensitive to iota-induced cytotoxicity. Collectively, these data are the first to show that iota toxin has the potential to be an effective, targeted therapy for breast cancer.

Phenotypic and Functional Characterization of Herpes Simplex Virus Glycoprotein B Epitope-Specific Effector and Memory CD8+ T Cells from Symptomatic and Asymptomatic Individuals with Ocular Herpes

PubMed Central

Khan, Arif A.; Srivastava, Ruchi; Spencer, Doran; Garg, Sumit; Fremgen, Daniel; Vahed, Hawa; Lopes, Patricia P.; Pham, Thanh T.; Hewett, Charlie; Kuang, Jasmine; Ong, Nicolas; Huang, Lei; Scarfone, Vanessa M.; Nesburn, Anthony B.

2015-01-01

ABSTRACT Herpes simplex virus 1 (HSV-1) glycoprotein B (gB)-specific CD8+ T cells protect mice from herpes infection and disease. However, whether and which HSV-1 gB-specific CD8+ T cells play a key role in the “natural” protection seen in HSV-1-seropositive healthy asymptomatic (ASYMP) individuals (who have never had clinical herpes disease) remain to be determined. In this study, we have dissected the phenotypes and the functions of HSV-1 gB-specific CD8+ T cells from HLA-A*02:01 positive, HSV-1 seropositive ASYMP and symptomatic (SYMP) individuals (with a history of numerous episodes of recurrent ocular herpes disease). We found the following. (i) Healthy ASYMP individuals maintained a significantly higher proportion of differentiated HSV-1 gB-specific effector memory CD8+ T cells (TEM cells) (CD45RAlow CCR7low CD44high CD62Llow). In contrast, SYMP patients had frequent less-differentiated central memory CD8+ T cells (TCM cells) (CD45RAlow CCR7high CD44low CD62Lhigh). (ii) ASYMP individuals had significantly higher proportions of multifunctional effector CD8+ T cells which responded mainly to gB342–350 and gB561–569 “ASYMP” epitopes, and simultaneously produced IFN-γ, CD107a/b, granzyme B, and perforin. In contrast, effector CD8+ T cells from SYMP individuals were mostly monofunctional and were directed mainly against nonoverlapping gB17–25 and gB183–191 “SYMP” epitopes. (iii) Immunization of an HLA-A*02:01 transgenic mouse model of ocular herpes with “ASYMP” CD8+ TEM cell epitopes, but not with “SYMP” CD8+ TCM cell epitopes, induced a strong CD8+ T cell-dependent protective immunity against ocular herpes infection and disease. Our findings provide insights into the role of HSV-specific CD8+ TEM cells in protection against herpes and should be considered in the development of an effective vaccine. IMPORTANCE A significantly higher proportion of differentiated and multifunctional HSV-1 gB-specific effector memory CD8+ T cells (TEM cells) (CD45RAlow CCR7low CD44high CD62Llow) were found in healthy ASYMP individuals who are seropositive for HSV-1 but never had any recurrent herpetic disease, while there were frequent less-differentiated and monofunctional central memory CD8+ T cells (TCM cells) (CD45RAlow CCR7high CD44low CD62Lhigh) in SYMP patients. Immunization with “ASYMP” CD8+ TEM cell epitopes, but not with “SYMP” CD8+ TCM cell epitopes, induced a strong protective HSV-specific CD8+ T cell response in HLA-A*02:01 transgenic mice. These findings are important for the development of a safe and effective T cell-based herpes vaccine. PMID:25609800

Hyaluronan functionalizing QDs as turn-on fluorescent probe for targeted recognition CD44 receptor

NASA Astrophysics Data System (ADS)

Zhou, Shang; Huo, Danqun; Hou, Changjun; Yang, Mei; Fa, Huanbao

2017-09-01

The recognition of tumor markers in living cancer cells has attracted increasing interest. In the present study, the turn-on fluorescence probe was designed based on the fluorescence of thiolated chitosan-coated CdTe QDs (CdTe/TCS QDs) quenched by hyaluronan, which could provide the low background signal for sensitive cellular imaging. This system is expected to offer specific recognition of CD44 receptor over other substances owing to the specific affinity of hyaluronan and CD44 receptor ( 8-9 kcal/mol). The probe is stable in aqueous and has little toxicity to living cells; thus, it can be utilized for targeted cancer cell imaging. The living lung cancer cell imaging experiments further demonstrate its value in recognizing cell-surface CD44 receptor with turn-on mode. In addition, the probe can be used to recognize and differentiate the subtypes of lung cancer cells based on the difference of CD44 expression on the surface of lung cancer cells. And, the western blot test further confirmed that the expression level of the CD44 receptor in lung cancer cells is different. Therefore, this probe may be potentially applied in recognizing lung cancer cells with higher contrast and sensitivity and provide new tools for cancer prognosis and therapy. [Figure not available: see fulltext.

No association of alcohol use and the risk of ulcerative colitis or Crohn's disease: data from a European Prospective cohort study (EPIC).

PubMed

Bergmann, M M; Hernandez, V; Bernigau, W; Boeing, H; Chan, S S M; Luben, R; Khaw, K-T; van Schaik, F; Oldenburg, B; Bueno-de-Mesquita, B; Overvad, K; Palli, D; Masala, G; Carbonnel, F; Boutron-Ruault, M-C; Olsen, A; Tjonneland, A; Kaaks, R; Katzke, V; Riboli, E; Hart, A R

2017-04-01

The role of long-term alcohol consumption for the risk of developing ulcerative colitis (UC) and Crohn's disease (CD) is unclear. For the first time, to prospectively assess the role of pre-disease alcohol consumption on the risk of developing UC or CD. Nested within the European Prospective Investigation into Cancer and Nutrition (EPIC-IBD), incident UC and CD cases and matched controls where included. At recruitment, participants completed validated food frequency and lifestyle questionnaires. Alcohol consumption was classified as either: non-use, former, light (⩽0.5 and 1 drink per week), below the recommended limits (BRL) (⩽1 and 2 drinks per day), moderate (⩽2.5 and 5 drinks per day), or heavy use (>2.5 and >5 drinks per day) for women and men, respectively; and was expressed as consumption at enrolment and during lifetime. Conditional logistic regression was applied adjusting for smoking and education, taking light users as the reference. Out of 262 451 participants in six countries, 198 UC incident cases/792 controls and 84 CD cases/336 controls were included. At enrolment, 8%/27%/32%/23%/11% UC cases and 7%/29%/40%/19%/5% CD cases were: non-users, light, BRL, moderate and heavy users, respectively. The corresponding figures for lifetime non-use, former, light, BRL, moderate and heavy use were: 3%/5%/23%/44%/19%/6% and 5%/2%/25%/44%/23%/1% for UC and CD cases, respectively. There were no associations between any categories of alcohol consumption and risk of UC or CD in the unadjusted and adjusted odds ratios. There was no evidence of associations between alcohol use and the odds of developing either UC or CD.

Mesenchymal stem cells in the Wharton's jelly of the human umbilical cord.

PubMed

Wang, Hwai-Shi; Hung, Shih-Chieh; Peng, Shu-Tine; Huang, Chun-Chieh; Wei, Hung-Mu; Guo, Yi-Jhih; Fu, Yu-Show; Lai, Mei-Chun; Chen, Chin-Chang

2004-01-01

The Wharton's jelly of the umbilical cord contains mucoid connective tissue and fibroblast-like cells. Using flow cytometric analysis, we found that mesenchymal cells isolated from the umbilical cord express matrix receptors (CD44, CD105) and integrin markers (CD29, CD51) but not hematopoietic lineage markers (CD34, CD45). Interestingly, these cells also express significant amounts of mesenchymal stem cell markers (SH2, SH3). We therefore investigated the potential of these cells to differentiate into cardiomyocytes by treating them with 5-azacytidine or by culturing them in cardiomyocyte-conditioned medium and found that both sets of conditions resulted in the expression of cardiomyocyte markers, namely N-cadherin and cardiac troponin I. We also showed that these cells have multilineage potential and that, under suitable culture conditions, are able to differentiate into cells of the adipogenic and osteogenic lineages. These findings may have a significant impact on studies of early human cardiac differentiation, functional genomics, pharmacological testing, cell therapy, and tissue engineering by helping to eliminate worrying ethical and technical issues.

PKI-587 and sorafenib alone and in combination on inhibition of liver cancer stem cell proliferation

PubMed Central

Gedaly, Roberto; Galuppo, Roberto; Musgrave, Yolanda; Angulo, Paul; Hundley, Jonathan; Shah, Malay; Daily, Michael F.; Chen, Changguo; Cohen, Donald A.; Spear, Brett T.; Evers, B. Mark

2015-01-01

Background Deregulated Ras/Raf/mitogen-activated protein kinase and PI3 K/AKT/mTOR signaling pathways are significant in hepatocellular carcinoma proliferation (HCC). In this study we evaluated differences in the antiproliferative effect of dual PI3 K/Akt/mTOR and Ras/Raf/mitogen-activated protein kinase inhibition of non liver cancer stem cell lines (PLC and HuH7) and liver cancer stem cell (LCSC) lines (CD133, CD44, CD24, and aldehyde dehydrogenase 1-positive cells). Materials and methods Flow cytometry was performed on the resulting tumors to identify the LCSC markers CD133, CD44, CD24, and aldehyde dehydrogenase 1. Methylthiazol tetrazolium assay was used to assess cellular proliferation. Finally, a Western blot assay was used to evaluate for inhibition of specific enzymes in these two signaling pathways. Results Using flow cytometry, we found that LCSC contain 64.4% CD133 + cells, 83.2% CD44 + cells, and 96.4% CD24 + cells. PKI-587 and sorafenib caused inhibiton of LCSC and HCC cell proliferation. PLC cells were more sensitive to PKI-587 than LCSC or Huh7 (P < 0.001). Interestingly, HuH7 cells were more sensitive to sorafenib than LCSC or PLC cells. Additionally, combination therapy with PKI-587 and sorafenib caused significantly more inhibition than monotherapy in HuH7, PLC, and LCSC. Using the methylthiazol tetrazolium assay, we found that the LCSC proliferation was inhibited with sorafenib monotherapy 39% at 5 μM (P < 0.001; n = 12) and 67% by PKI-587 at 0.1 μM (P = 0.002, n = 12) compared with control. The combination of PKI-587 and sorafenib, however, synergistically inhibited LCSC proliferation by 86% (P = 0.002; n = 12). Conclusions LCSC (CD133+, CD44+, CD24+) were able to develop very aggressive tumors with low cell concentrations at 4 to 6 wk. Cells CD133+, CD44+, CD24+ demonstrated at least moderate resistance to therapy in vitro. The combination of PKI-587 and sorafenib was better than either drug alone at inhibiting of LCSC and on HCC cell proliferation. PMID:23769634

PKI-587 and sorafenib alone and in combination on inhibition of liver cancer stem cell proliferation.

PubMed

Gedaly, Roberto; Galuppo, Roberto; Musgrave, Yolanda; Angulo, Paul; Hundley, Jonathan; Shah, Malay; Daily, Michael F; Chen, Changguo; Cohen, Donald A; Spear, Brett T; Evers, B Mark

2013-11-01

Deregulated Ras/Raf/mitogen-activated protein kinase and PI3 K/AKT/mTOR signaling pathways are significant in hepatocellular carcinoma proliferation (HCC). In this study we evaluated differences in the antiproliferative effect of dual PI3 K/Akt/mTOR and Ras/Raf/mitogen-activated protein kinase inhibition of non liver cancer stem cell lines (PLC and HuH7) and liver cancer stem cell (LCSC) lines (CD133, CD44, CD24, and aldehyde dehydrogenase 1-positive cells). Flow cytometry was performed on the resulting tumors to identify the LCSC markers CD133, CD44, CD24, and aldehyde dehydrogenase 1. Methylthiazol tetrazolium assay was used to assess cellular proliferation. Finally, a Western blot assay was used to evaluate for inhibition of specific enzymes in these two signaling pathways. Using flow cytometry, we found that LCSC contain 64.4% CD133 + cells, 83.2% CD44 + cells, and 96.4% CD24 + cells. PKI-587 and sorafenib caused inhibiton of LCSC and HCC cell proliferation. PLC cells were more sensitive to PKI-587 than LCSC or Huh7 (P < 0.001). Interestingly, HuH7 cells were more sensitive to sorafenib than LCSC or PLC cells. Additionally, combination therapy with PKI-587 and sorafenib caused significantly more inhibition than monotherapy in HuH7, PLC, and LCSC. Using the methylthiazol tetrazolium assay, we found that the LCSC proliferation was inhibited with sorafenib monotherapy 39% at 5 μM (P < 0.001; n = 12) and 67% by PKI-587 at 0.1 μM (P = 0.002, n = 12) compared with control. The combination of PKI-587 and sorafenib, however, synergistically inhibited LCSC proliferation by 86% (P = 0.002; n = 12). LCSC (CD133+, CD44+, CD24+) were able to develop very aggressive tumors with low cell concentrations at 4 to 6 wk. Cells CD133+, CD44+, CD24+, which demonstrated at least moderate resistance to therapy in vitro. The combination of PKI-587 and sorafenib was better than either drug alone at inhibiting of LCSC and on HCC cell proliferation. Copyright © 2013 Elsevier Inc. All rights reserved.

Expression of two isoforms of CD44 in human endometrium.

PubMed

Behzad, F; Seif, M W; Campbell, S; Aplin, J D

1994-10-01

The distribution of the cell-surface adhesion glycoprotein CD44 in human endometrium was examined by immunofluorescence using six monoclonal antibodies to epitopes common to all forms of the molecule, and by reverse transcription-polymerase chain reaction (RT-PCR). Immunoreactivity was observed throughout the menstrual cycle in stroma, vessels, glandular, and luminal epithelium. Variations in staining intensity were observed, especially in the epithelial compartment. CD44 was also expressed strongly by decidualized stromal cells of first-trimester pregnancy. No systematic variation of immunoreactivity was observed with stages of the normal cycle, but a fraction (25%) of the specimens lacked reactivity in the epithelium. To determine the molecular size of the epithelial isoform, an immunoprecipitation technique was developed using surface-radioiodinated, detergent-extracted glands. This indicated the presence at the cell surface of a single dominant CD44E species with an approximate molecular mass of 130 kDa. RT-PCR was used to investigate the isoforms present in whole endometrial tissue, isolated gland fragments, and Ishikawa endometrial carcinoma cells. Complementary DNA produced from total endometrial mRNA was PCR-amplified across the splice junction between exons 5 and 15. Transcripts corresponding to the hyaluronate receptor CD44H as well as a larger isoform were identified. CD44H was absent, or very scarce, in cDNA from purified gland epithelium. In contrast, Ishikawa cells expressed this form abundantly. The glands and Ishikawa cells also expressed CD44E containing sequences encoded by exons 12, 13, and 14. These data demonstrate the presence of CD44 in human endometrium and decidua, and show that different isoforms of CD44 are associated with tissue compartments in which different functional roles can be anticipated.

CD44 and ALDH1 immunoexpression as prognostic indicators of invasion and metastasis in oral squamous cell carcinoma.

PubMed

Ortiz, Rafael Carneiro; Lopes, Nathália Martins; Amôr, Nadia Ghinelli; Ponce, José Burgos; Schmerling, Cláudia Kliemann; Lara, Vanessa Soares; Moyses, Raquel Ajub; Rodini, Camila Oliveira

2018-05-23

Tumour metastasis has been associated with cancer stem cells, a small population with stem-like cells properties, higher rate of migration and metastatic potential compared to cells from the tumour bulk. Our aim was to evaluate the immunoexpression of the putative cancer stem cell biomarkers ALDH1 and CD44 in primary tumour and corresponding metastatic lymph nodes. Tumour tissue specimens (n=50) and corresponding metastatic lymph nodes (n=25) were surgically obtained from 50 patients with oral squamous cell carcinoma and submitted to immunohistochemistry. CD44 and ALDH1 were semi-quantitatively scored according to the proportion and intensity of positive cells within the invasive front and metastatic lymph nodes as a whole. A combined score was obtained by multiplying both parameters and later dichotomized into a final score classified as low (≤ 2) or high (> 2) immunoexpression. ALDH1 and CD44 immunoexpression was detected in both tumour sites, although the means of ALDH1 (P = 0.0985) and CD44 (P = 0.4220) cells were higher in metastasis compared to primary tumours. ALDH1 high was positively associated (P = 0.0184) with angiolymphatic invasion, while CD44 high was positively associated (P = 0.0181) with metastasis (N+). At multivariate analysis, CD44 significantly increased the odds of lymph node metastasis, regardless of T stage (OR=8,24; 1,64-65,64, p=0,0088). CD44 immunoexpression was a significant predictor of lymph node metastasis, while ALDH1 high immunostaining was associated with angiolymphatic invasion. Altogether, it suggests that immunoexpression of CD44 and ALDH1 links the cancer stem cell phenotype with oral squamous cell carcinoma invasion and metastasis. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

CD44v3 and VEGF-C expression and its relationship with lymph node metastasis in squamous cell carcinomas of the uterine cervix.

PubMed

Liu, Ye-Qing; Li, Hai-Feng; Han, Jing-Jing; Tang, Qiong-Lan; Sun, Qing; Huang, Zhi-Quan; Li, Hai-Gang

2014-01-01

To investigate the expression of CD44v3 and vascular endothelial growth factor-C (VEGF-C) and their relationship with lymph node metastasis in squamous cell carcinomas (SCC) of the uterine cervix. Expression of CD44v3 and VEGF-C was analyzed in 109 cases of cervical SCC by immunohistochemistry (IHC). The relationship was analyzed between expression and the patient age, histological differentiation, formation of tumor emboli in lymphoid vessels, lymph node metastasis, FIGO staging, and TNM classification. Expression rates for both CD44v3 and VEGF-C were 43.1% in cervical SCC. The cells with positive immunohistochemical staining of CD44v3 were distributed mainly around the keratin pearls in well differentiated carcinomas, but distributed diffusely in the moderately and poorly differentiated lesions. VEGF-C was found stained positively in most of the tumor cells. There were differences in expression between normal epithelium and atypical hyperplasia as well as carcinoma. Both CD44v3 and VEGF-C were found to be associated positively with lymph node metastasis and TNM classification (both p=0.000). Neither CD44v3 nor VEGF-C was found to be associated with patient age, histological differentiation, formation of tumor emboli in lymphoid vessels and FIGO staging. CD44v3 was found to be associated with VEGF-C positively (p=0.000). Abnormal expression of CD44v3 and VEGF-C is associated closely with the lymph node metastasis in cervical SCC, and these agents may cooperate in carcinogenesis and development of metastatic lesions.

CD133+CD54+CD44+ circulating tumor cells as a biomarker of treatment selection and liver metastasis in patients with colorectal cancer

PubMed Central

Wang, Cun; Huang, Qiaorong; Meng, Wentong; Yu, Yongyang; Yang, Lie; Peng, Zhihai; Hu, Jiankun; Li, Yuan; Mo, Xianming; Zhou, Zongguang

2016-01-01

Introduction Liver is the most common site of distant metastasis in colorectal cancer (CRC). Early diagnosis and appropriate treatment selection decides overall prognosis of patients. However, current diagnostic measures were basically imaging but not functional. Circulating tumor cells (CTCs) known as hold the key to understand the biology of metastatic mechanism provide a novel and auxiliary diagnostic strategy for CRC with liver metastasis (CRC-LM). Results The expression of CD133+ and CD133+CD54+CD44+ cellular subpopulations were higher in the peripheral blood of CRC-LM patients when compared with those without metastasis (P<0.001). Multivariate analysis proved the association between the expression of CD133+CD44+CD54+ cellular subpopulation and the existence of CRC-LM (P<0.001). The combination of abdominal CT/MRI, CEA and the CD133+CD44+CD54+ cellular subpopulation showed increased detection and discrimination rate for liver metastasis, with a sensitivity of 88.2% and a specificity of 92.4%. Meanwhile, it also show accurate predictive value for liver metastasis (OR=2.898, 95% C.I.1.374–6.110). Materials and Method Flow cytometry and multivariate analysis was performed to detect the expression of cancer initiating cells the correlation between cellular subpopulations and liver metastasis in patients with CRC. The receiver operating characteristic curves combined with the area under the curve were generated to compare the predictive ability of the cellular subpopulation for liver metastasis with current CT and MRI images. Conclusions The identification, expression and application of CTC subpopulations will provide an ideal cellular predictive marker for CRC liver metastasis and a potential marker for further investigation. PMID:27764803

[LincRNA-ROR functions as a ceRNA to regulate Oct4, Sox2, and Nanog expression by sponging miR-145 and its effect on biologic characteristics of colonic cancer stem cells].

PubMed

Yan, Z Y; Sun, X C

2018-04-08

Objective: To investigate the impact of lincRNA-ROR, a ceRNA by binding miR-145 on the expression of the downstream genes Oct4, Sox2 and Nanog, and related biological characteristics of colon cancer stem cells, and to elucidate the clinical significance of this molecular regulatory network. Methods: Fifty-two cases of colorectal cancer tissue and adjacent tissue were collected at Nanyang City Central Hospital and Nanyang Second Hospital, Henan Province, from 2014 to 2016. Real-time quantitative polymerase chain reaction (qPCR) was used to detect the expression of lincRNA-ROR and miR-145 in colorectal cancer tissue and isolated colon cancer cells. The correlation between the expression of lincRNA-ROR, miR-145 and the clinicopathologic features of colon cancer was performed. CD44(-)CD133(-) and CD44(+) CD133(+) cells were isolated from SW1116 by using flow cytometry. The expression of CD44, CD133, Oct4, Sox2, Nanog, lincRNA-ROR and miR-145 in cells were detected by qPCR. The relationship between lincRNA-ROR, miR-145, Oct4, Sox2 and Nanog was analyzed by bioinformatics, dual luciferase reporter assay, qPCR and Western blot. The effects of silencing lincRNA-ROR on the proliferation and chemosensitivity of colon cancer stem cells were detected by MTT, colony formation. Results: LincRNA-ROR was frequently up-regulated and inversely correlated with miR-145 down-regulation in the colon cancer specimens( P <0.05). LincRNA-ROR was related to tumor size, lymph node involvement and distant metastasis( P <0.05), and miR-145 was found related to tumor size and tumor location( P <0.05). CD44(+) CD133(+) cells were successfully isolated from SW1116 by flow cytometry. The levels of CD44, CD133, Oct4, Sox2, Nanog, lincRNA-ROR in CD44(+) CD133(+) cells were significantly increased, while miR-145 was decreased compared with CD44(-)CD133(-)cells( P <0.05). The levels of CD44, CD133, lnc-ROR in CD44(+) CD133(+) cells were significantly reduced upon cell adherence, while miR-145 was significantly increased( P <0.05). Bioinformatics analysis revealed that lincRNA-ROR shared miRNA response elements with core transcription factors Oct4, Sox2 and Nanog. MiR-145 significantly inhibited the expression of lincRNA-ROR, Oct4, Sox2 and Nanog. Silencing lincRNA-ROR significantly inhibited colon cancer stem cells proliferation and increased the sensitivity to chemotherapy. Conclusions: Linc-ROR functions as a key ceRNA to prevent core TFs, e. g., Oct4, Sox2, Nanog, from miR-145-mediated suppression in colon cancer stem cells and regulates cell proliferation and chemosensitivity.The data may provide insights into the pathophysiological interactions of the components of genetic networks in the development of colon cancer and may lead to new therapies in the future.

Is sphere assay useful for the identification of cancer initiating cells of the ovary?

PubMed

Martínez-Serrano, María José; Caballero-Baños, Miguel; Vilella, Ramon; Vidal, Laura; Pahisa, Jaume; Martínez-Roman, Sergio

2015-01-01

Current evidence suggests that the presence of tumor-initiating cells (TICs) in epithelial ovarian cancer (EOC) has a role in chemoresistance and relapse. Surface markers such as CD44(+)/CD24(-), CD117(+), and CD133(+) expression have been reported as potential markers for TICs related to ovarian cancer and tumorigenic cell lines. In this study, we have investigated if spheroid forms are TIC specific or whether they can also be produced by somatic stem cells from healthy tissue in vitro. In addition, we also investigated the specificity of surface markers to identify TICs from papillary serous EOC patients. Cells were obtained from fresh tumors from 10 chemotherapy-naive patients with EOC, and cells from ovarian and tubal epithelium were obtained from 5 healthy menopausal women undergoing surgery for benign pathology and cultured in standard and in selective medium. Cells forming nonadherent spheroids were considered TICs, and the adherent cells were considered as non-TIC-like. Percentages of CD24(+), CD44(+), CD117(+), CD133(+), and vascular endothelial growth factor receptor (VEGF-R)(+) cell surface markers were analyzed by flow cytometry. Four of 10 EOC cell tissues were excluded from the study. Tumor cells cultured in selective medium developed spheroid forms after 1 to 7 weeks in 5 of 6 EOC patients. No spheroid forms were observed in cultures of cells from healthy women. Unlike previously published data, low levels of CD24(+), CD44(+), CD117(+), and VEGF-R(+) expression were observed in spheroid cells, whereas expression of CD133(+) was moderate but higher in adherent cells from papillary serous EOC cells in comparison with adherent cells from controls. Papillary serous EOC contains TICs that form spheroids with low expression of CD44(+), CD24(+), CD117(+) and VEGF-R(+). Further research is required to find specific surface markers to identify papillary serous TICs.

Decreased expression of cell adhesion genes in cancer stem-like cells isolated from primary oral squamous cell carcinomas.

PubMed

Mishra, Amrendra; Sriram, Harshini; Chandarana, Pinal; Tanavde, Vivek; Kumar, Rekha V; Gopinath, Ashok; Govindarajan, Raman; Ramaswamy, S; Sadasivam, Subhashini

2018-05-01

The goal of this study was to isolate cancer stem-like cells marked by high expression of CD44, a putative cancer stem cell marker, from primary oral squamous cell carcinomas and identify distinctive gene expression patterns in these cells. From 1 October 2013 to 4 September 2015, 76 stage III-IV primary oral squamous cell carcinoma of the gingivobuccal sulcus were resected. In all, 13 tumours were analysed by immunohistochemistry to visualise CD44-expressing cells. Expression of CD44 within The Cancer Genome Atlas-Head and Neck Squamous Cell Carcinoma RNA-sequencing data was also assessed. Seventy resected tumours were dissociated into single cells and stained with antibodies to CD44 as well as CD45 and CD31 (together referred as Lineage/Lin). From 45 of these, CD44 + Lin - and CD44 - Lin - subpopulations were successfully isolated using fluorescence-activated cell sorting, and good-quality RNA was obtained from 14 such sorted pairs. Libraries from five pairs were sequenced and the results analysed using bioinformatics tools. Reverse transcription quantitative polymerase chain reaction was performed to experimentally validate the differential expression of selected candidate genes identified from the transcriptome sequencing in the same 5 and an additional 9 tumours. CD44 was expressed on the surface of poorly differentiated tumour cells, and within the The Cancer Genome Atlas-Head and Neck Squamous Cell Carcinoma samples, its messenger RNA levels were higher in tumours compared to normal. Transcriptomics revealed that 102 genes were upregulated and 85 genes were downregulated in CD44 + Lin - compared to CD44 - Lin - cells in at least 3 of the 5 tumours sequenced. The upregulated genes included those involved in immune regulation, while the downregulated genes were enriched for genes involved in cell adhesion. Decreased expression of PCDH18, MGP, SPARCL1 and KRTDAP was confirmed by reverse transcription quantitative polymerase chain reaction. Lower expression of the cell-cell adhesion molecule PCDH18 correlated with poorer overall survival in the The Cancer Genome Atlas-Head and Neck Squamous Cell Carcinoma data highlighting it as a potential negative prognostic factor in this cancer.

Hyaluronan receptors in the human ocular surface: a descriptive and comparative study of RHAMM and CD44 in tissues, cell lines and freshly collected samples.

PubMed

García-Posadas, Laura; Contreras-Ruiz, Laura; López-García, Antonio; Villarón Álvarez, Sonia; Maldonado, Miguel J; Diebold, Yolanda

2012-02-01

The purpose of this study was to demonstrate the presence of the receptor for hyaluronan-mediated motility (RHAMM) in human conjunctival epithelium and in two widely used cell lines from human corneal (HCE) and conjunctival (IOBA-NHC) epithelia. We compared the distribution of RHAMM proteins and mRNAs in human ocular surface tissues (corneal, limbal and conjunctival), HCE and IOBA-NHC cell lines, and corneal and conjunctival epithelia primary samples from healthy donors with the previously identified hyaluronan receptor CD44. We also aimed to determine if soluble CD44 (sCD44) was present in human tears, as it could have a role in the interaction of the tear fluid with hyaluronan. Protein expression was evaluated by Western blots and immunofluorescence microscopy. mRNA expression was evaluated by RT-PCR and Q-PCR. sCD44 was analyzed by ELISA in culture supernatants and in human tears. We describe the expression of RHAMM in human healthy conjunctiva and in HCE and IOBA-NHC cells at both protein and mRNA levels, and the presence of sCD44 in human tears. Furthermore, we detected CD44 and sCD44 expression variations in in vitro inflammatory conditions. This study also focused on the necessary caution with which the conclusions extracted from cell lines should be made, and in the great value of using primary samples as often as possible.

CD44 expression in curettage and postoperative specimens of endometrial cancer.

PubMed

Wojciechowski, Michał; Krawczyk, Tomasz; Śmigielski, Janusz; Malinowski, Andrzej

2015-02-01

Adhesive molecules like CD44 are well defined key players in the metastatic cascade in many cancers, including endometrial cancer. They could play a role of markers of invasion, metastasis and prognostic factors. The aim of the study is to assess a possible role of the CD44 as a marker of invasion in endometrial cancer, both at the moment of preoperative workup and final staging. Available for analysis were archival specimens of 51 patients who had underwent curettage and surgery between 2002 and 2007. An immunohistochemical study for CD44 expression was performed in curettage and postoperative specimens. Normal endometrium of 20 randomly chosen patients was used as a control group. In endometrial cancer the expression of CD44 was significantly more intensive than in normal endometrium. In postoperative specimens, the CD44 expression was weaker in serous than in endometrioid cancer. There was no significant correlation between the adhesion molecule expression and clinicopathological features: grade,depth of invasion, cervical involvement, serosal and adnexal involvement, lymph-vascular space involvement, lymph node and distant metastases nor FIGO stage. An increased expression of CD44 in endometrial cancer suggests its possible role in pathogenesis of this disease, however, it doesn't seem to be crucial. Different expression of the CD44 in endometrioid and papillary-serous type may reflect different pathogenesis of these types of cancer. No statistically proved relation between the investigated molecule expression and clinicopathological parameters suggests scepticism about its use in diagnostic process of endometrial cancer.

Extensive characterization of the immunophenotype and pattern of cytokine production by distinct subpopulations of normal human peripheral blood MHC II+/lineage− cells

PubMed Central

Almeida, J; Bueno, C; Alguero, M C; Sanchez, M L; Cañizo, M C; Fernandez, M E; Vaquero, J M; Laso, F J; Escribano, L; San Miguel, J F; Orfao, A

1999-01-01

Dendritic cells (DC) represent the most powerful professional antigen-presenting cells (APC) in the immune system. The aim of the present study was to analyse, on a single-cell basis by multiparametric flow cytometry with simultaneous four-colour staining and a two-step acquisition procedure, the immunophenotypic profile and cytokine production of DC from 67 normal whole peripheral blood (PB) samples. Two clearly different subsets of HLA-II+/lineage− were identified on the basis of their distinct phenotypic characteristics: one DC subset was CD33strong+ and CD123dim+ (0.16 ± 0.06% of the PB nucleated cells and 55.9 ± 11.9% of all PB DC) and the other, CD33dim+ and CD123strong+ (0.12 ± 0.04% of PB nucleated cells and 44.53 ± 11.5% of all PB DC). Moreover, the former DC subpopulation clearly showed higher expression of the CD13 myeloid-associated antigen, the CD29 and CD58 adhesion molecules, the CD2, CD5 and CD86 costimulatory molecules, the CD32 IgG receptor and the CD11c complement receptor. In addition, these cells showed stronger HLA-DR and HLA-DQ expression and a higher reactivity for the IL-6 receptor α-chain (CD126) and for CD38. In contrast, the CD123strong+/CD33dim+ DC showed a stronger reactivity for the CD4 and CD45RA molecules, whereas they did not express the CD58, CD5, CD11c and CD13 antigens. Regarding cytokine production, our results show that while the CD33strong+/CD123dim+ DC are able to produce significant amounts of inflammatory cytokines, such as IL-1β (97 ± 5% of positive cells), IL-6 (96 ± 1.1% of positive cells), IL-12 (81.5 ± 15.5% of positive cells) and tumour necrosis factor-alpha (TNF-α) (84 ± 22.1% of positive cells) as well as chemokines such as IL-8 (99 ± 1% of positive cells), the functional ability of the CD123strong+/CD33dim+ DC subset to produce cytokines under the same conditions was almost null. Our results therefore clearly show the presence of two distinct subsets of DC in normal human PB, which differ not only in their immunophenotype but also in their functionality, as regards cytokine production. PMID:10594557

A mesenchymal-like phenotype and expression of CD44 predict lack of apoptotic response to sorafenib in liver tumor cells.

PubMed

Fernando, Joan; Malfettone, Andrea; Cepeda, Edgar B; Vilarrasa-Blasi, Roser; Bertran, Esther; Raimondi, Giulia; Fabra, Àngels; Alvarez-Barrientos, Alberto; Fernández-Salguero, Pedro; Fernández-Rodríguez, Conrado M; Giannelli, Gianluigi; Sancho, Patricia; Fabregat, Isabel

2015-02-15

The multikinase inhibitor sorafenib is the only effective drug in advanced cases of hepatocellular carcinoma (HCC). However, response differs among patients and effectiveness only implies a delay. We have recently described that sorafenib sensitizes HCC cells to apoptosis. In this work, we have explored the response to this drug of six different liver tumor cell lines to define a phenotypic signature that may predict lack of response in HCC patients. Results have indicated that liver tumor cells that show a mesenchymal-like phenotype, resistance to the suppressor effects of transforming growth factor beta (TGF-β) and high expression of the stem cell marker CD44 were refractory to sorafenib-induced cell death in in vitro studies, which correlated with lack of response to sorafenib in nude mice xenograft models of human HCC. In contrast, epithelial-like cells expressing the stem-related proteins EpCAM or CD133 were sensitive to sorafenib-induced apoptosis both in vitro and in vivo. A cross-talk between the TGF-β pathway and the acquisition of a mesenchymal-like phenotype with up-regulation of CD44 expression was found in the HCC cell lines. Targeted CD44 knock-down in the mesenchymal-like cells indicated that CD44 plays an active role in protecting HCC cells from sorafenib-induced apoptosis. However, CD44 effect requires a TGF-β-induced mesenchymal background, since the only overexpression of CD44 in epithelial-like HCC cells is not sufficient to impair sorafenib-induced cell death. In conclusion, a mesenchymal profile and expression of CD44, linked to activation of the TGF-β pathway, may predict lack of response to sorafenib in HCC patients. © 2014 UICC.

Phenotypic and functional characterization of herpes simplex virus glycoprotein B epitope-specific effector and memory CD8+ T cells from symptomatic and asymptomatic individuals with ocular herpes.

PubMed

Khan, Arif A; Srivastava, Ruchi; Spencer, Doran; Garg, Sumit; Fremgen, Daniel; Vahed, Hawa; Lopes, Patricia P; Pham, Thanh T; Hewett, Charlie; Kuang, Jasmine; Ong, Nicolas; Huang, Lei; Scarfone, Vanessa M; Nesburn, Anthony B; Wechsler, Steven L; BenMohamed, Lbachir

2015-04-01

Herpes simplex virus 1 (HSV-1) glycoprotein B (gB)-specific CD8(+) T cells protect mice from herpes infection and disease. However, whether and which HSV-1 gB-specific CD8(+) T cells play a key role in the "natural" protection seen in HSV-1-seropositive healthy asymptomatic (ASYMP) individuals (who have never had clinical herpes disease) remain to be determined. In this study, we have dissected the phenotypes and the functions of HSV-1 gB-specific CD8(+) T cells from HLA-A*02:01 positive, HSV-1 seropositive ASYMP and symptomatic (SYMP) individuals (with a history of numerous episodes of recurrent ocular herpes disease). We found the following. (i) Healthy ASYMP individuals maintained a significantly higher proportion of differentiated HSV-1 gB-specific effector memory CD8(+) T cells (TEM cells) (CD45RA(low) CCR7(low) CD44(high) CD62L(low)). In contrast, SYMP patients had frequent less-differentiated central memory CD8(+) T cells (TCM cells) (CD45RA(low) CCR7(high) CD44(low) CD62L(high)). (ii) ASYMP individuals had significantly higher proportions of multifunctional effector CD8(+) T cells which responded mainly to gB342-350 and gB561-569 "ASYMP" epitopes, and simultaneously produced IFN-γ, CD107(a/b), granzyme B, and perforin. In contrast, effector CD8(+) T cells from SYMP individuals were mostly monofunctional and were directed mainly against nonoverlapping gB17-25 and gB183-191 "SYMP" epitopes. (iii) Immunization of an HLA-A*02:01 transgenic mouse model of ocular herpes with "ASYMP" CD8(+) TEM cell epitopes, but not with "SYMP" CD8(+) TCM cell epitopes, induced a strong CD8(+) T cell-dependent protective immunity against ocular herpes infection and disease. Our findings provide insights into the role of HSV-specific CD8(+) TEM cells in protection against herpes and should be considered in the development of an effective vaccine. A significantly higher proportion of differentiated and multifunctional HSV-1 gB-specific effector memory CD8(+) T cells (TEM cells) (CD45RA(low) CCR7(low) CD44(high) CD62L(low)) were found in healthy ASYMP individuals who are seropositive for HSV-1 but never had any recurrent herpetic disease, while there were frequent less-differentiated and monofunctional central memory CD8(+) T cells (TCM cells) (CD45RA(low) CCR7(high) CD44(low) CD62L(high)) in SYMP patients. Immunization with "ASYMP" CD8(+) TEM cell epitopes, but not with "SYMP" CD8(+) TCM cell epitopes, induced a strong protective HSV-specific CD8(+) T cell response in HLA-A*02:01 transgenic mice. These findings are important for the development of a safe and effective T cell-based herpes vaccine. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Differential Gene Expression Profiling of Functionally and Developmentally Distinct Human Prostate Epithelial Populations

PubMed Central

Liu, Haibo; Cadaneanu, Radu M; Lai, Kevin; Zhang, Baohui; Huo, Lihong; An, Dong Sun; Li, Xinmin; Lewis, Michael S; Garraway, Isla P

2015-01-01

BACKGROUND Human fetal prostate buds appear in the 10th gestational week as solid cords, which branch and form lumens in response to androgen 1. Previous in vivo analysis of prostate epithelia isolated from benign prostatectomy specimens indicated that Epcam+CD44−CD49fHi basal cells possess efficient tubule initiation capability relative to other subpopulations 2. Stromal interactions and branching morphogenesis displayed by adult tubule-initiating cells (TIC) are reminiscent of fetal prostate development. In the current study, we evaluated in vivo tubule initiation by human fetal prostate cells and determined expression profiles of fetal and adult epithelial subpopulations in an effort to identify pathways used by TIC. METHODS Immunostaining and FACS analysis based on Epcam, CD44, and CD49f expression demonstrated the majority (99.9%) of fetal prostate epithelial cells (FC) were Epcam+CD44− with variable levels of CD49f expression. Fetal populations isolated via cell sorting were implanted into immunocompromised mice. Total RNA isolation from Epcam+CD44−CD49fHi FC, adult Epcam+CD44−CD49fHi TIC, Epcam+CD44+CD49fHi basal cells (BC), and Epcam+CD44−CD49fLo luminal cells (LC) was performed, followed by microarray analysis of 19 samples using the Affymetrix Gene Chip Human U133 Plus 2.0 Array. Data was analyzed using Partek Genomics Suite Version 6.4. Genes selected showed >2-fold difference in expression and P < 5.00E-2. Results were validated with RT-PCR. RESULTS Grafts retrieved from Epcam+CD44− fetal cell implants displayed tubule formation with differentiation into basal and luminal compartments, while only stromal outgrowths were recovered from Epcam- fetal cell implants. Hierarchical clustering revealed four distinct groups determined by antigenic profile (TIC, BC, LC) and developmental stage (FC). TIC and BC displayed basal gene expression profiles, while LC expressed secretory genes. FC had a unique profile with the most similarities to adult TIC. Functional, network, and canonical pathway identification using Ingenuity Pathway Analysis Version 7.6 compiled genes with the highest differential expression (TIC relative to BC or LC). Many of these genes were found to be significantly associated with prostate tumorigenesis. CONCLUSIONS Our results demonstrate clustering gene expression profiles of FC and adult TIC. Pathways associated with TIC are known to be deregulated in cancer, suggesting a cell-of-origin role for TIC versus re-emergence of pathways common to these cells in tumorigenesis. Prostate 75: 764–776, 2015. © The Authors. The Prostate, published by Wiley Periodicals, Inc. PMID:25663004

Differential gene expression profiling of functionally and developmentally distinct human prostate epithelial populations.

PubMed

Liu, Haibo; Cadaneanu, Radu M; Lai, Kevin; Zhang, Baohui; Huo, Lihong; An, Dong Sun; Li, Xinmin; Lewis, Michael S; Garraway, Isla P

2015-05-01

Human fetal prostate buds appear in the 10th gestational week as solid cords, which branch and form lumens in response to androgen 1. Previous in vivo analysis of prostate epithelia isolated from benign prostatectomy specimens indicated that Epcam⁺ CD44⁻ CD49f(Hi) basal cells possess efficient tubule initiation capability relative to other subpopulations 2. Stromal interactions and branching morphogenesis displayed by adult tubule-initiating cells (TIC) are reminiscent of fetal prostate development. In the current study, we evaluated in vivo tubule initiation by human fetal prostate cells and determined expression profiles of fetal and adult epithelial subpopulations in an effort to identify pathways used by TIC. Immunostaining and FACS analysis based on Epcam, CD44, and CD49f expression demonstrated the majority (99.9%) of fetal prostate epithelial cells (FC) were Epcam⁺ CD44⁻ with variable levels of CD49f expression. Fetal populations isolated via cell sorting were implanted into immunocompromised mice. Total RNA isolation from Epcam⁺ CD44⁻ CD49f(Hi) FC, adult Epcam⁺ CD44⁻ CD49f(Hi) TIC, Epcam⁺ CD44⁺ CD49f(Hi) basal cells (BC), and Epcam⁺ CD44⁻ CD49f(Lo) luminal cells (LC) was performed, followed by microarray analysis of 19 samples using the Affymetrix Gene Chip Human U133 Plus 2.0 Array. Data was analyzed using Partek Genomics Suite Version 6.4. Genes selected showed >2-fold difference in expression and P < 5.00E-2. Results were validated with RT-PCR. Grafts retrieved from Epcam⁺ CD44⁻ fetal cell implants displayed tubule formation with differentiation into basal and luminal compartments, while only stromal outgrowths were recovered from Epcam- fetal cell implants. Hierarchical clustering revealed four distinct groups determined by antigenic profile (TIC, BC, LC) and developmental stage (FC). TIC and BC displayed basal gene expression profiles, while LC expressed secretory genes. FC had a unique profile with the most similarities to adult TIC. Functional, network, and canonical pathway identification using Ingenuity Pathway Analysis Version 7.6 compiled genes with the highest differential expression (TIC relative to BC or LC). Many of these genes were found to be significantly associated with prostate tumorigenesis. Our results demonstrate clustering gene expression profiles of FC and adult TIC. Pathways associated with TIC are known to be deregulated in cancer, suggesting a cell-of-origin role for TIC versus re-emergence of pathways common to these cells in tumorigenesis. © 2015 The Authors. The Prostate, published by Wiley Periodicals, Inc.

Lysosomal Degradation of CD44 Mediates Ceramide Nanoliposome-induced Anoikis and Diminished Extravasation in Metastatic Carcinoma Cells*

PubMed Central

Haakenson, Jeremy K.; Khokhlatchev, Andrei V.; Choi, Younhee J.; Linton, Samuel S.; Zhang, Pu; Zaki, Peter M.; Fu, Changliang; Cooper, Timothy K.; Manni, Andrea; Zhu, Junjia; Fox, Todd E.; Dong, Cheng; Kester, Mark

2015-01-01

The ceramide nanoliposome (CNL) has shown promise in being able to treat a variety of primary tumors. However, its potential for treating metastatic cancer remains unknown. In this study, we demonstrate that CNL increases anoikis while preventing cancer cell extravasation under both static and physiological fluid flow conditions. Mechanistically, CNL limits metastases by decreasing CD44 protein levels in human breast and pancreatic cancer cells via lysosomal degradation of CD44, independent of palmitoylation or proteasome targeting. siRNA down-regulation of CD44 mimics CNL-induced anoikis and diminished extravasation of cancer cells. Taken together, our data indicate that ceramide limits CD44-dependent cancer cell migration, suggesting that CNL could be used to prevent and treat solid tumor metastasis. PMID:25681441

Induction of resistance to diabetes in non-obese diabetic mice by targeting CD44 with a specific monoclonal antibody

PubMed Central

Weiss, Lola; Slavin, Shimon; Reich, Shoshana; Cohen, Patrizia; Shuster, Svetlana; Stern, Robert; Kaganovsky, Ella; Okon, Elimelech; Rubinstein, Ariel M.; Naor, David

2000-01-01

Inflammatory destruction of insulin-producing β cells in the pancreatic islets is the hallmark of insulin-dependent diabetes mellitus, a spontaneous autoimmune disease of non-obese diabetic mice resembling human juvenile (type I) diabetes. Histochemical analysis of diabetic pancreata revealed that mononuclear cells infiltrating the islets and causing autoimmune insulitis, as well as local islet cells, express the CD44 receptor; hyaluronic acid, the principal ligand of CD44, is detected in the islet periphery and islet endothelium. Injection of anti-CD44 mAb 1 hr before cell transfer of diabetogenic splenocytes and subsequently on alternate days for 4 weeks induced considerable resistance to diabetes in recipient mice, reflected by reduced insulitis. Contact sensitivity to oxazolone was not influenced by this treatment. A similar antidiabetic effect was observed even when the anti-CD44 mAb administration was initiated at the time of disease onset: i.e., 4–7 weeks after cell transfer. Administration of the enzyme hyaluronidase also induced appreciable resistance to insulin-dependent diabetes mellitus, suggesting that the CD44–hyaluronic acid interaction is involved in the development of the disease. These findings demonstrate that CD44-positive inflammatory cells may be a potential therapeutic target in insulin-dependent diabetes. PMID:10618410

CD44-tropic polymeric nanocarrier for breast cancer targeted rapamycin chemotherapy.

PubMed

Zhao, Yunqi; Zhang, Ti; Duan, Shaofeng; Davies, Neal M; Forrest, M Laird

2014-08-01

In contrast with the conventional targeting of nanoparticles to cancer cells with antibody or peptide conjugates, a hyaluronic acid (HA) matrix nanoparticle with intrinsic-CD44-tropism was developed to deliver rapamycin for localized CD44-positive breast cancer treatment. Rapamycin was chemically conjugated to the particle surface via a novel sustained-release linker, 3-amino-4-methoxy-benzoic acid. The release of the drug from the HA nanoparticle was improved by 42-fold compared to HA-temsirolimus in buffered saline. In CD44-positive MDA-MB-468 cells, using HA as drug delivery carrier, the cell viability was significantly decreased compared to free rapamycin and CD44-blocked controls. Rat pharmacokinetics showed that the area under the curve of HA nanoparticle formulation was 2.96-fold greater than that of the free drug, and the concomitant total body clearance was 8.82-fold slower. Moreover, in immunocompetent BALB/c mice bearing CD44-positive 4T1.2neu breast cancer, the rapamycin-loaded HA particles significantly improved animal survival, suppressed tumor growth and reduced the prevalence of lung metastasis. This study demonstrates increased efficiency of rapamycin delivery and consequential treatment effects in a breast cancer model by hyaluronic acid - L-rapamycin conjugates with intrinsic tropism for CD44-positive cells. Copyright © 2014 Elsevier Inc. All rights reserved.

Characterization of FaDu-R, a radioresistant head and neck cancer cell line, and cancer stem cells.

PubMed

Cho, Kwang-Jae; Park, Eun-Ji; Kim, Min-Sik; Joo, Young-Hoon

2018-06-01

The aim of this study was to evaluate the impact of CSC on insensitivity to radiotherapy in HNSCC. A radioresistant cell line, FaDu-R, was established using fractionated ionizing radiation. Cells with high and low CD44/ALDH activity were isolated. FaDu-R cells demonstrated significantly increased cell viability after radiation exposure compared with parental cells. CD44 high /ALDH high FaDu-R cells demonstrated significantly faster wound closure (p<0.05) and more efficient invasion (p<0.05) compared to the CD44 high /ALDH high FaDu cells or the CD44 low /ALDH low FaDu-R cells. There was a significant difference in tumor volume between the CD44 high /ALDH high FaDu-R cells and the CD44 high /ALDH high FaDu cells (p<0.05) as well as the CD44 low /ALDH low FaDu-R cells (p<0.05). Cancer stem cells (CSC) were associated with invasion and tumorigenesis in a radioresistant head and neck squamous cell carcinoma (HNSCC) cell line. This concept might help to improve the understanding of these mechanisms and to develop drugs that can overcome radioresistance during radiotherapy. Copyright © 2017 Elsevier B.V. All rights reserved.

Expression of CD44 3'-untranslated region regulates endogenous microRNA functions in tumorigenesis and angiogenesis.

PubMed

Jeyapalan, Zina; Deng, Zhaoqun; Shatseva, Tatiana; Fang, Ling; He, Chengyan; Yang, Burton B

2011-04-01

The non-coding 3'-untranslated region (UTR) plays an important role in the regulation of microRNA (miRNA) functions, since it can bind and inactivate multiple miRNAs. Here, we show the 3'-UTR of CD44 is able to antagonize cytoplasmic miRNAs, and result in the increased translation of CD44 and downstream target mRNA, CDC42. A series of cell function assays in the human breast cancer cell line, MT-1, have shown that the CD44 3'-UTR inhibits proliferation, colony formation and tumor growth. Furthermore, it modulated endothelial cell activities, favored angiogenesis, induced tumor cell apoptosis and increased sensitivity to Docetaxel. These results are due to the interaction of the CD44 3'-UTR with multiple miRNAs. Computational algorithms have predicted three miRNAs, miR-216a, miR-330 and miR-608, can bind to both the CD44 and CDC42 3'-UTRs. This was confirmed with luciferase assays, western blotting and immunohistochemical staining and correlated with a series of siRNA assays. Thus, the non-coding CD44 3'-UTR serves as a competitor for miRNA binding and subsequently inactivates miRNA functions, by freeing the target mRNAs from being repressed.

Expression of CD44 3′-untranslated region regulates endogenous microRNA functions in tumorigenesis and angiogenesis

PubMed Central

Jeyapalan, Zina; Deng, Zhaoqun; Shatseva, Tatiana; Fang, Ling; He, Chengyan; Yang, Burton B.

2011-01-01

The non-coding 3′-untranslated region (UTR) plays an important role in the regulation of microRNA (miRNA) functions, since it can bind and inactivate multiple miRNAs. Here, we show the 3′-UTR of CD44 is able to antagonize cytoplasmic miRNAs, and result in the increased translation of CD44 and downstream target mRNA, CDC42. A series of cell function assays in the human breast cancer cell line, MT-1, have shown that the CD44 3′-UTR inhibits proliferation, colony formation and tumor growth. Furthermore, it modulated endothelial cell activities, favored angiogenesis, induced tumor cell apoptosis and increased sensitivity to Docetaxel. These results are due to the interaction of the CD44 3′-UTR with multiple miRNAs. Computational algorithms have predicted three miRNAs, miR-216a, miR-330 and miR-608, can bind to both the CD44 and CDC42 3′-UTRs. This was confirmed with luciferase assays, western blotting and immunohistochemical staining and correlated with a series of siRNA assays. Thus, the non-coding CD44 3′-UTR serves as a competitor for miRNA binding and subsequently inactivates miRNA functions, by freeing the target mRNAs from being repressed. PMID:21149267

Expression of Bmi-1, P16, and CD44v6 in Uterine Cervical Carcinoma and Its Clinical Significance.

PubMed

Weng, Mei-Ying; Li, Lin; Feng, Shu-Ying; Hong, Shun-Jia

2012-03-01

Bmi-1, a putative proto-oncogene, is a core member of the polycomb gene family, which is expressed in many human tumors. The p16 protein negatively regulated cell proliferation, whereas CD44v6 is associated with proliferation as an important protein. Additionally, CD44v6 is an important nuclear antigen closely correlated to tumor metastasis. The present study aims to investigate the expression and significance of Bmi-1, p16, and CD44v6 in uterine cervical carcinoma (UCC). A total of 62 UCC, 30 cervical neoplasic, and 20 normal cervical mucosal tissues were used in the current study. The expression of Bmi-1, p16, and CD44v6 in these tissues was determined using immunohistochemical assay. The relationships among the expression of these indices, the clinicopathologic features of UCC, and the survival rate of UCC patients were also discussed. The correlation between Bmi-1 protein expression and p16 or CD44v6 protein in UCC was analyzed. The expression of Bmi-1, p16, and CD44v6 was significantly high in cervical carcinoma compared with that in the cervical neoplasia and normal colorectal mucosa (P<0.05). The over-expression of Bmi-1 protein in UCC was apparently related to the distant metastasis (P<0.01) and the tumor, nodes and metastasis-classification, i.e. the TNM staging, World Health Organization (P<0.05). Nevertheless, the positive expression of p16 protein in UCC was not significantly associated with the clinicopathologic features (P>0.05). The Kaplan-Meier survival analysis showed that the over-expression of Bmi-1 significantly decreased the survival rate of UCC patients (P<0.05). A strong correlation indicated that there was statistical significance between the expression of Bmi-1 and CD44V6 proteins in UCC (r=0.419, P=0.001). The over-expression of Bmi-1 and CD44v6 protein closely correlate to the tumorigenesis, metastasis, and prognosis of UCC. Bmi-1 and CD44v6 may be used to predict the prognosis of cervical carcinoma. Bmi-1 may indirectly regulate the expression of CD44v6 in UCC patients. The positive expression of p16 protein is possibly associated with the tumorigenesis, but not with the metastasis or prognosis of UCC.

Fate of cadmium at the soil-solution interface: a thermodynamic study as influenced by varying pH at South 24 Parganas, West Bengal, India.

PubMed

Karak, Tanmoy; Paul, Ranjit Kumar; Das, Sampa; Das, Dilip K; Dutta, Amrit Kumar; Boruah, Romesh K

2015-11-01

A study on the sorption kinetics of Cd from soil solution to soils was conducted to assess the persistence of Cd in soil solution as it is related to the leaching, bioavailability, and potential toxicity of Cd. The kinetics of Cd sorption on two non-contaminated alkaline soils from Canning (22° 18' 48.02″ N and 88° 39' 29.0″ E) and Lakshmikantapur (22° 06' 16.61″ N and 88° 19' 08.66″ E) of South 24 Parganas, West Bengal, India, were studied using conventional batch experiment. The variable soil suspension parameters were pH (4.00, 6.00, 8.18, and 9.00), temperatures (308, 318, and 328 K) and Cd concentrations (5-100 mg L(-1)). The average rate coefficient (kavg) and half-life (t1/2) values indicate that the persistence of Cd in soil solution is influenced by both temperature and soil suspension pH. The concentration of Cd in soil solution decreases with increase of temperature; therefore, Cd sorption on the soil-solution interface is an endothermic one. Higher pH decreases the t 1/2 of Cd in soil solution, indicating that higher pH (alkaline) is not a serious concern in Cd toxicity than lower pH (acidic). Based on the energy of activation (Ea) values, Cd sorption in acidic pH (14.76±0.29 to 64.45±4.50 kJ mol(-1)) is a surface control phenomenon and in alkaline pH (9.33±0.09 to 44.60±2.01 kJ mol(-1)) is a diffusion control phenomenon The enthalpy of activation (ΔH∓) values were found to be between 7.28 and 61.73 kJ mol(-1). Additionally, higher positive energy of activation (ΔG∓) values (46.82±2.01 to 94.47±2.36 kJ mol(-1)) suggested that there is an energy barrier for product formation.

Protein profile of basal prostate epithelial progenitor cells--stage-specific embryonal antigen 4 expressing cells have enhanced regenerative potential in vivo.

PubMed

Höfner, Thomas; Klein, Corinna; Eisen, Christian; Rigo-Watermeier, Teresa; Haferkamp, Axel; Sprick, Martin R

2016-04-01

The long-term propagation of basal prostate progenitor cells ex vivo has been very difficult in the past. The development of novel methods to expand prostate progenitor cells in vitro allows determining their cell surface phenotype in greater detail. Mouse (Lin(-)Sca-1(+) CD49f(+) Trop2(high)-phenotype) and human (Lin(-) CD49f(+) TROP2(high)) basal prostate progenitor cells were expanded in vitro. Human and mouse cells were screened using 242 anti-human or 176 antimouse monoclonal antibodies recognizing the cell surface protein profile. Quantitative expression was evaluated at the single-cell level using flow cytometry. Differentially expressed cell surface proteins were evaluated in conjunction with the known CD49f(+)/TROP2(high) phenotype of basal prostate progenitor cells and characterized by in vivo sandwich-transplantation experiments using nude mice. The phenotype of basal prostate progenitor cells was determined as CD9(+)/CD24(+)/CD29(+)/CD44(+)/CD47(+)/CD49f(+)/CD104(+)/CD147(+)/CD326(+)/Trop2(high) of mouse as well as human origin. Our analysis revealed several proteins, such as CD13, Syndecan-1 and stage-specific embryonal antigens (SSEAs), as being differentially expressed on murine and human CD49f(+) TROP2(+) basal prostate progenitor cells. Transplantation experiments suggest that CD49f(+) TROP2(high) SSEA-4(high) human prostate basal progenitor cells to be more potent to regenerate prostate tubules in vivo as compared with CD49f(+) TROP2(high) or CD49f(+) TROP2(high) SSEA-4(low) cells. Determination of the cell surface protein profile of functionally defined murine and human basal prostate progenitor cells reveals differentially expressed proteins that may change the potency and regenerative function of epithelial progenitor cells within the prostate. SSEA-4 is a candidate cell surface marker that putatively enables a more accurate identification of the basal PESC lineage. © 2016 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Human adipose-derived mesenchymal stem cells in vitro: evaluation of an optimal expansion medium preserving stemness.

PubMed

Baer, Patrick C; Griesche, Nadine; Luttmann, Werner; Schubert, Ralf; Luttmann, Arlette; Geiger, Helmut

2010-01-01

The potential of cultured adipose-derived stem cells (ASC) in regenerative medicine and new cell therapeutic concepts has been shown recently by many investigations. However, while the method of isolation of ASC from liposuction aspirates depending on plastic adhesion is well established, a standard expansion medium optimally maintaining the undifferentiated state has not been described. We cultured ASC in five commonly used culture media (two laboratory-made media and three commercially available media) and compared them with a standard medium. We analyzed the effects on cell morphology, proliferation, hepatocyte growth factor (HGF) expression, stem cell marker profile and differentiation potential. Proliferation was measured with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and a fluorescent assay. Release of HGF was assessed by an immunoassay. Expression of characteristic stem cell-related transcription factors and markers was evaluated by quantitative polymerase chain reaction (qPCR) (Nanog, Sox-2, Rex-1, nestin and Oct-4) and flow cytometry (CD44, CD73, CD90, CD105 and CD166), and differentiation was shown by adipogenic medium. The morphology and expansion of ASC were significantly affected by the media used, whereas none of the media influenced the ASC potential to differentiate into adipocytes. Furthermore, two of the media induced an increase in expression of transcription factors, an increased secretion of HGF and a decrease in CD105 expression. Culture of ASC in one of these two media before using the cells in cell therapeutic approaches may have a benefit on their regenerative potential.

Combining G-CSF with a blockade of adhesion strongly improves the reconstitutive capacity of mobilized hematopoietic progenitor cells.

PubMed

Christ, O; Kronenwett, R; Haas, R; Zöller, M

2001-03-01

Mobilization of hematopoietic progenitor cells is achieved mainly by application of growth factors and, more recently, by blockade of adhesion. In this report, we describe the advantages of a combined treatment with granulocyte colony-stimulating factor (G-CSF) and anti-VLA4 (CD49d)/anti-CD44 as compared to treatment with the individual components. Mobilization by intravenous injection of anti-CD44, anti-VLA4, or G-CSF was controlled in spleen and bone marrow with regard to frequencies of multipotential colony-forming unit (C-CFU), marrow repopulating ability, long-term reconstitution, recovery of myelopoiesis, and regain of immunocompetence. Mobilization by anti-CD44 had a strong effect on expansion of early progenitor cells in the bone marrow, while the recovery in the spleen was poor. In anti-CD49d-mobilized noncommitted and committed progenitors, progenitor expansion was less pronounced, but settlement in the spleen was quite efficient. Thus, anti-CD44 and anti-CD49d differently influenced mobilization. Accordingly, mobilization and recovery after transfer were improved by combining anti-CD44 with anti-CD49d treatment. Mobilization by G-CSF was most efficient with respect to recovery of progenitor cells in the spleen. However, when transferring G-CSF-mobilized cells, regain of immunocompetence was strongly delayed. This disadvantage could be overridden when progenitor cells were mobilized via blockade of adhesion and when expansion of these mobilized progenitor cells was supported by low-dose G-CSF only during the last 24 hours before transfer. Mobilization of pluripotent progenitor cells via antibody blockade of CD44 or CD49d or via G-CSF relies on distinct mechanisms. Therefore, the reconstitutive capacity of a transplant can be significantly improved by mobilization regimens combining antibody with low-dose G-CSF treatment.

[Increased expressions of peripheral PD-1+ lymphocytes and CD4+CD25+FOXP3+ T cells in gastric adenocarcinoma patients].

PubMed

Li, Hao; Li, Songyan; Hu, Shidong; Zou, Guijun; Hu, Zilong; Wei, Huahua; Wang, Yufeng; Du, Xiaohui

2017-01-01

Objective To detect the frequencies of peripheral programmed death-1 + (PD-1 + ) lymphocytes and CD4 + CD25 + FOXP3 + regulatory T cells in patients with gastric adenocarcinoma. Methods The study enrolled 29 patients with gastric adenocarcinoma and 29 age- and sex-matched healthy controls. Frequencies of PD-1 + lymphocytes and CD4 + CD25 + FOXP3 + regulatory T cells were detected using flow cytometry. Results The number of PD-1 + lymphocytes and CD4 + CD25 + FOXP3 + regulatory T cells in peripheral blood was higher in patients with gastric adenocarcinoma than that in the control group. Moreover, linear correlation analysis indicated a positive correlation between PD-1 expression and frequency of CD4 + CD25 + FOXP3 + regulatory T cells in peripheral blood of the patients. Conclusion Gastric adenocarcinoma patients present with increased PD-1 + lymphocytes and CD4 + CD25 + FOXP3 + regulatory T cells in the peripheral blood.

Association of Glioblastoma Multiforme Stem Cell Characteristics, Differentiation, and Microglia Marker Genes with Patient Survival

PubMed Central

Balz, Ellen; Herzog, Susann; Plantera, Laura; Vogelgesang, Silke; Seifert, Carolin; Bialke, Angela; Venugopal, Chitra; Singh, Sheila K.; Hoffmann, Wolfgang; Schroeder, Henry W. S.

2018-01-01

Patients with glioblastoma multiforme (GBM) are at high risk to develop a relapse despite multimodal therapy. Assumedly, glioma stem cells (GSCs) are responsible for treatment resistance of GBM. Identification of specific GSC markers may help to develop targeted therapies. Here, we performed expression analyses of stem cell (ABCG2, CD44, CD95, CD133, ELF4, Nanog, and Nestin) as well as differentiation and microglia markers (GFAP, Iba1, and Sparc) in GBM compared to nonmalignant brain. Furthermore, the role of these proteins for patient survival and their expression in LN18 stem-like neurospheres was analyzed. At mRNA level, ABCG2 and CD95 were reduced, GFAP was unchanged; all other investigated markers were increased in GBM. At protein level, CD44, ELF4, Nanog, Nestin, and Sparc were elevated in GBM, but only CD133 and Nestin were strongly associated with survival time. In addition, ABCG2 and GFAP expression was decreased in LN18 neurospheres whereas CD44, CD95, CD133, ELF4, Nanog, Nestin, and Sparc were upregulated. Altogether only CD133 and Nestin were associated with survival rates. This raises concerns regarding the suitability of the other target structures as prognostic markers, but makes both CD133 and Nestin candidates for GBM therapy. Nevertheless, a search for more specific marker proteins is urgently needed. PMID:29535786

Investigating Cell Surface Markers on Normal Hematopoietic Stem Cells in Three Different Niche Conditions

PubMed Central

Garg, Swati; Madkaikar, Manisha

2013-01-01

Hematopoietic stem cells are of therapeutic interest to the clinicians and researchers due to their promising assistance in management of malignant and inherited hematological conditions. Evaluation of cell surface markers using multiparametric flow cytometry is a well adapted qualitative measure of cells in question for many years. An artillery of these markers has been studied in hematological malignancies and related disorders. However, their role and differential expression on normal hematopoietic stem cells from clinically available sources is not always described carefully. In the present study, we attempted to evaluate expression of CD44, CD90, CD96 and CD123 in three clinically available sources of normal HSCs (Hematopoietic stem cells). Sources of HSCs in the present study involved umbilical cord blood (UCB), normal bone marrow (NBM) and bone marrow from idiopathic thrombocytopenic purpura (ITP) patients (IBM). CD44 is an important homing receptor while CD90 is involved in maintaining stem cell quiescent. CD96 is known to be leukemia specific marker and CD123 is involved in stem cell differentiation and survival. We observed a significant difference in expression CD44, CD90 and CD123 on normal HSCs derived from umbilical cord and ITP marrow. CD96 was highly expressed on HSCs obtained from ITP marrow. Investigating expression of these markers on normal HSCs in different niches will be helpful in correlating their function with niche condition and delineating their ‘abnormal’ expression from the normal. PMID:24386557

Investigating cell surface markers on normal hematopoietic stem cells in three different niche conditions.

PubMed

Garg, Swati; Madkaikar, Manisha; Ghosh, Kanjaksha

2013-11-01

Hematopoietic stem cells are of therapeutic interest to the clinicians and researchers due to their promising assistance in management of malignant and inherited hematological conditions. Evaluation of cell surface markers using multiparametric flow cytometry is a well adapted qualitative measure of cells in question for many years. An artillery of these markers has been studied in hematological malignancies and related disorders. However, their role and differential expression on normal hematopoietic stem cells from clinically available sources is not always described carefully. In the present study, we attempted to evaluate expression of CD44, CD90, CD96 and CD123 in three clinically available sources of normal HSCs (Hematopoietic stem cells). Sources of HSCs in the present study involved umbilical cord blood (UCB), normal bone marrow (NBM) and bone marrow from idiopathic thrombocytopenic purpura (ITP) patients (IBM). CD44 is an important homing receptor while CD90 is involved in maintaining stem cell quiescent. CD96 is known to be leukemia specific marker and CD123 is involved in stem cell differentiation and survival. We observed a significant difference in expression CD44, CD90 and CD123 on normal HSCs derived from umbilical cord and ITP marrow. CD96 was highly expressed on HSCs obtained from ITP marrow. Investigating expression of these markers on normal HSCs in different niches will be helpful in correlating their function with niche condition and delineating their 'abnormal' expression from the normal.

Core 1-derived O-glycans are essential E-selectin ligands on neutrophils.

PubMed

Yago, Tadayuki; Fu, Jianxin; McDaniel, J Michael; Miner, Jonathan J; McEver, Rodger P; Xia, Lijun

2010-05-18

Neutrophils roll on E-selectin in inflamed venules through interactions with cell-surface glycoconjugates. The identification of physiologic E-selectin ligands on neutrophils has been elusive. Current evidence suggests that P-selectin glycoprotein ligand-1 (PSGL-1), E-selectin ligand-1 (ESL-1), and CD44 encompass all glycoprotein ligands for E-selectin; that ESL-1 and CD44 use N-glycans to bind to E-selectin; and that neutrophils lacking core 2 O-glycans have partially defective interactions with E-selectin. These data imply that N-glycans on ESL-1 and CD44 and O-glycans on PSGL-1 constitute all E-selectin ligands, with neither glycan subset having a dominant role. The enzyme T-synthase transfers Gal to GalNAcalpha1-Ser/Thr to form the core 1 structure Galbeta1-3GalNAcalpha1-Ser/Thr, a precursor for core 2 and extended core 1 O-glycans that might serve as selectin ligands. Here, using mice lacking T-synthase in endothelial and hematopoietic cells, we found that E-selectin bound to CD44 and ESL-1 in lysates of T-synthase-deficient neutrophils. However, the cells exhibited markedly impaired rolling on E-selectin in vitro and in vivo, failed to activate beta2 integrins while rolling, and did not emigrate into inflamed tissues. These defects were more severe than those of neutrophils lacking PSGL-1, CD44, and the mucin CD43. Our results demonstrate that core 1-derived O-glycans are essential E-selectin ligands; that some of these O-glycans are on protein(s) other than PSGL-1, CD44, and CD43; and that PSGL-1, CD44, and ESL-1 do not constitute all glycoprotein ligands for E-selectin.

The influence of rat mesenchymal stem cell CD44 surface markers on cell growth, fibronectin expression, and cardiomyogenic differentiation on silk fibroin - Hyaluronic acid cardiac patches.

PubMed

Yang, Ming-Chia; Chi, Nai-Hsin; Chou, Nai-Kuan; Huang, Yi-You; Chung, Tze-Wen; Chang, Yu-Lin; Liu, Hwa-Chang; Shieh, Ming-Jium; Wang, Shoei-Shen

2010-02-01

Since MSCs contain an abundant of CD44 surface markers, it is of interesting to investigate whether CD44 on rat MSC (rMSCs) influenced cell growth, fibronectin expression and cardiomyogenic differentiation on new SF/HA cardiac patches. For this investigation, we examined the influences of rMSCs with or without a CD44-blockage treatment on the aforementioned issues after they were cultivated, and further induced by 5-aza on SF and SF/HA patches. The results showed that the relative growth rates of rMSCs cultured on cultural wells, SF/HA patches without or with a CD44-blockage treatment were 100%, 208.9+/-7.1 (%) or 48.4+/-6.0 (%) (n=3, for all), respectively, after five days of cultivations. Moreover, rMSCs cultivated on SF/HA patches highly promoted fibronectin expressions (e.g., 1.8x10(5)/cell, in fluorescent intensity) while cells with a CD44-blockage treatment markedly diminished the expressions (e.g., 1.1x10(4)/cell, in fluorescent intensity) on same patches. For investigating possible influences of CD44 surface markers of rMSCs on their cardiomyogenic differentiation, the expressions of specific cardiac genes of cells were examined by using real-time PCR analysis. The results indicated that 5-aza inducing rMSCs significantly promoted the expressions of Gata4, Nkx2.5, Tnnt2 and Actc1 genes (all, P<0.01 or better, n=3) on SF/HA patches compared with those expressions on SF patches and for cells with a CD44-blockage treatment on SF/HA patches. Furthermore, the intensity of the expressions of cardiotin and connexin 43 of 5-aza inducing rMSCs were markedly higher than those of cells with a CD44-blockage treatment after they were cultured on SF/HA patches. Through this study, we reported that CD44 surface markers of rMSCs highly influenced the proliferations, fibronectin expressions and cardiomyogenic differentiation of rMSCs cultivated on cardiac SF/HA patches.

Cyclophilin B induces integrin-mediated cell adhesion by a mechanism involving CD98-dependent activation of protein kinase C-delta and p44/42 mitogen-activated protein kinases.

PubMed

Melchior, Aurélie; Denys, Agnès; Deligny, Audrey; Mazurier, Joël; Allain, Fabrice

2008-02-01

Initially identified as a cyclosporin-A binding protein, cyclophilin B (CyPB) is an inflammatory mediator that induces adhesion of T lymphocytes to fibronectin, by a mechanism dependent on CD147 and alpha 4 beta 1 integrins. Recent findings have suggested that another cell membrane protein, CD98, may cooperate with CD147 to regulate beta1 integrin functions. Based on these functional relationships, we examined the contribution of CD98 in the pro-adhesive activity of CyPB, by utilizing the responsive promonocyte cell line THP-1. We demonstrated that cross-linking CD98 with CD98-AHN-18 antibody mimicked the responses induced by CyPB, i.e. homotypic aggregation, integrin-mediated adhesion to fibronectin and activation of p44/42 MAPK. Consistent with previous data, immunoprecipitation confirmed the existence of a heterocomplex wherein CD147, CD98 and beta1 integrins were associated. We then demonstrated that CyPB-induced cell adhesion and p44/42 MAPK activation were dependent on the participation of phosphoinositide 3-kinase and subsequent activation of protein kinase C-delta. Finally, silencing the expression of CD98 by RNA interference potently reduced CyPB-induced cell responses, thus confirming the role of CD98 in the pro-adhesive activity of CyPB. Altogether, our results support a model whereby CyPB induces integrin-mediated adhesion via interaction with a multimolecular unit formed by the association between CD147, CD98 and beta1 integrins.

CD21+ (B2 antigen+) cell decrement and CD4+CD29+ (helper-inducer) cell increment suggest an activation of cell immune reactivity in multiple sclerosis.

PubMed

Gambi, D; Porrini, A M; Giampietro, A; Macor, S

1991-08-01

Two-color flow cytometric analysis on peripheral blood lymphocytes of 35 untreated multiple sclerosis (MS) patients, 17 other medical disease (OMD) patients and 14 healthy control (HC) subjects was performed to evaluate the levels of different T and B cell subpopulations. In MS patients we observed an increase in CD4+CD29+ helper-inducer cells but this increase was not related to the different phases of the disease. We hypothesize that this change is related to the reduction of CD21+ cells expressing B2 antigen, a 140 kDa molecule disappearing after B cell activation. An increased level of CD4+CD45RA- (helper-inducer-like cells) and a reduction of CD4+CD29- (suppressor-inducer-like cells) were also present in our patients. These findings demonstrate an immune 'disequilibrium' in MS, which is linked with an increased level of CD25+ cells expressing the interleukin-2 (IL-2) receptor. IL-2, besides being a T cell growth factor, is also a B cell growth factor. These data let us hypothesize that an activation of the immune response is present in MS.

High-Throughput Sequencing of Islet-Infiltrating Memory CD4+ T Cells Reveals a Similar Pattern of TCR Vβ Usage in Prediabetic and Diabetic NOD Mice

PubMed Central

Marrero, Idania; Hamm, David E.; Davies, Joanna D.

2013-01-01

Autoreactive memory CD4+ T cells play a critical role in the development of type 1 diabetes, but it is not yet known how the clonotypic composition and TCRβ repertoire of the memory CD4+ T cell compartment changes during the transition from prediabetes to diabetes. In this study, we used high-throughput sequencing to analyze the TCRβ repertoire of sorted islet-infiltrating memory CD4+CD44high T cells in 10-week-old prediabetic and recently diabetic NOD mice. We show that most clonotypes of islet-infiltrating CD4+CD44high T cells were rare, but high-frequency clonotypes were significantly more common in diabetic than in prediabetic mice. Moreover, although the CD4+CD44high TCRβ repertoires were highly diverse at both stages of disease development, dominant use of TRBV1 (Vβ2), TRBV13-3 (Vβ8.1), and TRBV19 (Vβ6) was evident in both prediabetic and diabetic mice. Our findings strongly suggest that therapeutic targeting of cells specifically expressing the dominant TCRβ might reduce pancreatic infiltration in prediabetic mice and attenuate the progression to diabetes. PMID:24146886

Interaction between hyaluronan and CD44 in the development of dimethylnitrosamine-induced liver cirrhosis.

PubMed

Satoh, T; Ichida, T; Matsuda, Y; Sugiyama, M; Yonekura, K; Ishikawa, T; Asakura, H

2000-04-01

A significant increase in serum hyaluronan (HA) levels has been reported in patients with liver cirrhosis. This mechanism is not yet clear, and receptors for HA have not been characterized. In this study, we examined the expression of both HA and its receptors, CD44 and intercellular adhesion molecule-1 (ICAM-1), in dimethylnitrosamine-induced liver cirrhosis. Using biotinylated HA binding protein, HA was detected in the area of periportal fibrosis and around the sinusoidal wall where hepatic fibrosis was developing. Electron microscopy revealed that HA was localized on Ito cells and sinusoidal endothelial cells (SEC). Conversely, CD44, which was only expressed weakly in normal liver, was present in large amounts in cirrhotic liver. The distribution pattern of CD44 was similar to that of HA, however, CD44 was mainly localized on the infiltrating lymphocytes and Kupffer cells. Moreover, CD44 was detected on part of factor VIII-positive SEC. Intercellular adhesion molecule-1, another receptor for HA, was detected on the surface of hepatocytes and around the sinusoidal wall in cirrhotic liver, but its distribution was not accompanied by expression of HA. With respect to CD44 isoforms, the standard form m-RNA predominated in both normal and cirrhotic liver. Variant pMeta-1 mRNA was detected at low levels. An interaction between HA and CD44 may play a role in the recruitment of numerous infiltrating cells and HA accumulation in hepatic sinusoids. Together with phenotypic changes in the SEC, these results may lead to a disturbance in the elimination of HA during the progression of liver cirrhosis.

Radiobiological characteristics of cancer stem cells from esophageal cancer cell lines

PubMed Central

Wang, Jian-Lin; Yu, Jing-Ping; Sun, Zhi-Qiang; Sun, Su-Ping

2014-01-01

AIM: To study the cancer stem cell population in esophageal cancer cell lines KYSE-150 and TE-1 and identify whether the resulting stem-like spheroid cells display cancer stem cells and radiation resistance characteristics. METHODS: A serum-free medium (SFM) suspension was used to culture esophageal cancer stem cell lines and enrich the esophageal stem-like spheres. A reverse transcription polymerase chain reaction assay was used to detect stem cell gene expression in the spheroid cells. Radiosensitivity of stem-like spheres and parental cells were evaluated by clonogenic assays. Furthermore, different cells after different doses of irradiation were tested to evaluate the change in sphere formation, cell cycle and CD44+CD271+ expression of tumor stem-like spheroid cells using flow cytometry before and after irradiation. RESULTS: The cells were observed to generate an increased number of spheres in SFM with increasing cell passage. Radiation increased the rate of generation of stem-like spheres in both types of cells. The average survival fraction (SF2) of the cultured KYSE-150 compared with TE-1 stem-like spheres after 2 Gy of radiation was 0.81 ± 0.03 vs 0.87 ± 0.01 (P < 0.05), while the average SF2 of KYSE-150 compared with TE-1 parental cells was 0.69 ± 0.04 vs 0.80 ± 0.03, P < 0.05. In the esophageal parental cells, irradiation dose-dependently induced G2 arrest. Stem-like esophageal spheres were resistant to irradiation-induced G2 arrest without significant changes in the percentage population of irradiated stem-like cells. Under irradiation at 0, 4, and 8 Gy, the CD44+CD271+ cell percentage for KYSE150 parental cells was 1.08% ± 0.03% vs 1.29% ± 0.07% vs 1.11% ± 0.09%, respectively; the CD44+CD271+ cell percentage for TE1 parental cells was 1.16% ± 0.11% vs 0.97% ± 0.08% vs 1.45% ± 0.35%, respectively. The differences were not statistically significant. Under irradiation at 0, 4, and 8 Gy, the CD44+CD271+ cell percentage for KYSE-150 stem-like spheres was 35.83% ± 1.23% vs 44.9% ± 1.67% vs 57.77% ± 1.88%, respectively; the CD44+CD271+ cell percentage for TE1 stem-like spheres was 16.07% ± 0.91% vs 22.67% ± 1.12%, 16.07% ± 0.91% vs 33.27% ± 1.07%, respectively. The 4 and 8 Gy irradiated KYSE-150 and TE-1 stem-like spheres were compared with the 0 Gy irradiated group, and the differences were statistically significant (P < 0.05). CONCLUSION: The KYSE-150 and TE-1 stem-like spheres are more radioresistant than their parental cells which may suggest that cancer stem cells are related to radioresistance. PMID:25561796

Radiobiological characteristics of cancer stem cells from esophageal cancer cell lines.

PubMed

Wang, Jian-Lin; Yu, Jing-Ping; Sun, Zhi-Qiang; Sun, Su-Ping

2014-12-28

To study the cancer stem cell population in esophageal cancer cell lines KYSE-150 and TE-1 and identify whether the resulting stem-like spheroid cells display cancer stem cells and radiation resistance characteristics. A serum-free medium (SFM) suspension was used to culture esophageal cancer stem cell lines and enrich the esophageal stem-like spheres. A reverse transcription polymerase chain reaction assay was used to detect stem cell gene expression in the spheroid cells. Radiosensitivity of stem-like spheres and parental cells were evaluated by clonogenic assays. Furthermore, different cells after different doses of irradiation were tested to evaluate the change in sphere formation, cell cycle and CD44(+)CD271(+) expression of tumor stem-like spheroid cells using flow cytometry before and after irradiation. The cells were observed to generate an increased number of spheres in SFM with increasing cell passage. Radiation increased the rate of generation of stem-like spheres in both types of cells. The average survival fraction (SF2) of the cultured KYSE-150 compared with TE-1 stem-like spheres after 2 Gy of radiation was 0.81 ± 0.03 vs 0.87 ± 0.01 (P < 0.05), while the average SF2 of KYSE-150 compared with TE-1 parental cells was 0.69 ± 0.04 vs 0.80 ± 0.03, P < 0.05. In the esophageal parental cells, irradiation dose-dependently induced G2 arrest. Stem-like esophageal spheres were resistant to irradiation-induced G2 arrest without significant changes in the percentage population of irradiated stem-like cells. Under irradiation at 0, 4, and 8 Gy, the CD44(+)CD271(+) cell percentage for KYSE150 parental cells was 1.08% ± 0.03% vs 1.29% ± 0.07% vs 1.11% ± 0.09%, respectively; the CD44(+)CD271(+) cell percentage for TE1 parental cells was 1.16% ± 0.11% vs 0.97% ± 0.08% vs 1.45% ± 0.35%, respectively. The differences were not statistically significant. Under irradiation at 0, 4, and 8 Gy, the CD44(+)CD271(+) cell percentage for KYSE-150 stem-like spheres was 35.83% ± 1.23% vs 44.9% ± 1.67% vs 57.77% ± 1.88%, respectively; the CD44(+)CD271(+) cell percentage for TE1 stem-like spheres was 16.07% ± 0.91% vs 22.67% ± 1.12%, 16.07% ± 0.91% vs 33.27% ± 1.07%, respectively. The 4 and 8 Gy irradiated KYSE-150 and TE-1 stem-like spheres were compared with the 0 Gy irradiated group, and the differences were statistically significant (P < 0.05). The KYSE-150 and TE-1 stem-like spheres are more radioresistant than their parental cells which may suggest that cancer stem cells are related to radioresistance.

Aberrant lymphoid antigen expression in acute myeloid leukemia in Saudi Arabia.

PubMed

El-Sissy, Azza H; El-Mashari, May A; Bassuni, Wafaa Y; El-Swaayed, Aziza F

2006-09-01

Immunophenotyping improves both accuracy and reproducibility of acute leukemia classification and is considered particularly useful for identifying aberrant lineage association of acute leukemia, biphenotypic and bilineal acute leukemia, as well as monitoring minimal residual disease. Some immunophenotypes correlate with cytogenetic abnormalities and prognosis. Is to determine aberrant lymphoid antigen expression in Saudi acute myeloid leukemia (AML), correlate them with FAB subtypes, evaluate early surface markers CD7 and CD56, and to investigate the role of cytoplasmic CD79a (a B cell marker that is assigned a high score of 2.0 in the WHO classification). Thirty four newly diagnosed AML cases were included in this study, 47% showed aberrant lymphoid antigen expression. CD9 was the most frequently expressed lymphoid antigen (29.4%) followed by CD7 & CD19 (11.8%), CD4 (8.8%) and CD22 (2.9%). CD9 was expressed in 3/6 (50%) of M3 cases, CD7 was expressed in 11.8% and was mostly confined to FAB M1 and M2 and associated with immature antigens CD34, HLA-DR and TdT. CD56 was expressed in 7/34 (20.6%) cases, three of these cases (42.9%) belonged to the monocytic group. CD56 was also detected in 2 cases with 11q23 rearrangement. CD56 was expressed in 2/7 (28.6%) M2 cases, and was associated with t (8;21) (q22;q22) together with CD19. Co-expression of CD56 and CD7 was detected in 2.9% of the cases. CD79a was expressed in one case together with CD19, diagnosed as acute biphenotypic leukemia, and was associated with t(8;21) (q22;q22). Minimal residual disease in AML is very difficult to trace, detection of aberrant expression of lymphoid antigens will make it easier. The high score given to CD79a by EGIL is questionable based on cytogenetic classification.

Alternate Splicing of CD44 Messenger RNA in Prostate Cancer Growth

DTIC Science & Technology

2009-04-01

Higashi M, Kishi H, Hiwasa T, Koda K, Nakajima N, Harigaya K. CD44 signaling through focal adhesion kinase and its anti-apoptotic effect. FEBS Lett...Nakamura, S., Azuma, K., Ishii, G., Higashi, M., Kishi, H., Hiwasa, T., Koda , K., Nakajima, N. and Harigaya, K.: CD44 signaling through focal adhesion

Arthroscopic Harvest of Adipose-Derived Mesenchymal Stem Cells From the Infrapatellar Fat Pad.

PubMed

Dragoo, Jason L; Chang, Wenteh

2017-11-01

The successful isolation of adipose-derived mesenchymal stem cells (ADSCs) from the arthroscopically harvested infrapatellar fat pad (IFP) would provide orthopaedic surgeons with an autologous solution for regenerative procedures. To demonstrate the quantity and viability of the mesenchymal stem cell population arthroscopically harvested from the IFP as well as the surrounding synovium. Descriptive laboratory study. The posterior border of the IFP, including the surrounding synovial tissue, was harvested arthroscopically from patients undergoing anterior cruciate ligament reconstruction. Tissue was then collected in an AquaVage adipose canister, followed by fat fractionization using syringe emulsification and concentration with an AdiPrep device. In the laboratory, the layers of tissue were separated and then digested with 0.3% type I collagenase. The pelleted stromal vascular fraction (SVF) cells were then immediately analyzed for viability, mesenchymal cell surface markers by fluorescence-activated cell sorting, and clonogenic capacity. After culture expansion, the metabolic activity of the ADSCs was assessed by an AlamarBlue assay, and the multilineage differentiation capability was tested. The transition of surface antigens from the SVF toward expanded ADSCs at passage 2 was further evaluated. SVF cells were successfully harvested with a mean yield of 4.86 ± 2.64 × 10 5 cells/g of tissue and a mean viability of 69.03% ± 10.75%, with ages ranging from 17 to 52 years (mean, 35.14 ± 13.70 years; n = 7). The cultured ADSCs composed a mean 5.85% ± 5.89% of SVF cells with a mean yield of 0.33 ± 0.42 × 10 5 cells/g of tissue. The nonhematopoietic cells (CD45 - ) displayed the following surface antigens as a percentage of the viable population: CD44 + (52.21% ± 4.50%), CD73 + CD90 + CD105 + (19.20% ± 17.04%), and CD44 + CD73 + CD90 + CD105 + (15.32% ± 15.23%). There was also a significant increase in the expression of ADSC markers CD73 (96.97% ± 1.72%; P < .01), CD10 (84.47% ± 15.46%; P < .05), and CD166 (11.63% ± 7.84%; P < .005) starting at passage 2 compared with freshly harvested SVF cells. The clonogenic efficiency of SVF cells was determined at a mean 3.21% ± 1.52% for layer 1 and 1.51% ± 0.55% for layer 2. Differentiation into cartilage, fat, and bone tissue was demonstrated by tissue-specific staining and quantitative polymerase chain reaction. SVF cells from the IFP and adjacent synovial tissue were successfully harvested using an arthroscopic technique and produced ADSCs with surface markers that meet criteria for defined mesenchymal stem cells. An autologous source of stem cells can now be harvested using a simple arthroscopic technique that will allow orthopaedic surgeons easier access to progenitor cells for regenerative procedures.

Lysosomal degradation of CD44 mediates ceramide nanoliposome-induced anoikis and diminished extravasation in metastatic carcinoma cells.

PubMed

Haakenson, Jeremy K; Khokhlatchev, Andrei V; Choi, Younhee J; Linton, Samuel S; Zhang, Pu; Zaki, Peter M; Fu, Changliang; Cooper, Timothy K; Manni, Andrea; Zhu, Junjia; Fox, Todd E; Dong, Cheng; Kester, Mark

2015-03-27

The ceramide nanoliposome (CNL) has shown promise in being able to treat a variety of primary tumors. However, its potential for treating metastatic cancer remains unknown. In this study, we demonstrate that CNL increases anoikis while preventing cancer cell extravasation under both static and physiological fluid flow conditions. Mechanistically, CNL limits metastases by decreasing CD44 protein levels in human breast and pancreatic cancer cells via lysosomal degradation of CD44, independent of palmitoylation or proteasome targeting. siRNA down-regulation of CD44 mimics CNL-induced anoikis and diminished extravasation of cancer cells. Taken together, our data indicate that ceramide limits CD44-dependent cancer cell migration, suggesting that CNL could be used to prevent and treat solid tumor metastasis. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Differential cellular internalization of anti-CD19 and -CD22 immunotoxins results in different cytotoxic activity.

PubMed

Du, Xing; Beers, Richard; Fitzgerald, David J; Pastan, Ira

2008-08-01

B-cell malignancies routinely express surface antigens CD19 and CD22. Immunotoxins against both antigens have been evaluated, and the immunotoxins targeting CD22 are more active. To understand this disparity in cytotoxicity and guide the screening of therapeutic targets, we compared two immunotoxins, FMC63(Fv)-PE38-targeting CD19 and RFB4(Fv)-PE38 (BL22)-targeting CD22. Six lymphoma cell lines have 4- to 9-fold more binding sites per cell for CD19 than for CD22, but BL22 is 4- to 140-fold more active than FMC63(Fv)-PE38, although they have a similar cell binding affinity (Kd, approximately 7 nmol/L). In 1 hour, large amounts of BL22 are internalized (2- to 3-fold more than the number of CD22 molecules on the cell surface), whereas only 5.2% to 16.6% of surface-bound FMC63(Fv)-PE38 is internalized. The intracellular reservoir of CD22 decreases greatly after immunotoxin internalization, indicating that it contributes to the uptake of BL22. Treatment of cells with cycloheximide does not reduce the internalization of BL22. Both internalized immunotoxins are located in the same vesicles. Our results show that the rapid internalization of large amounts of BL22 bound to CD22 makes CD22 a better therapeutic target than CD19 for immunotoxins and probably for other immunoconjugates that act inside cells.

Exosomes Promote Ovarian Cancer Cell Invasion through Transfer of CD44 to Peritoneal Mesothelial Cells.

PubMed

Nakamura, Koji; Sawada, Kenjiro; Kinose, Yasuto; Yoshimura, Akihiko; Toda, Aska; Nakatsuka, Erika; Hashimoto, Kae; Mabuchi, Seiji; Morishige, Ken-Ichirou; Kurachi, Hirohisa; Lengyel, Ernst; Kimura, Tadashi

2017-01-01

Epithelial ovarian cancer (EOC) cells metastasize within the peritoneal cavity and directly encounter human peritoneal mesothelial cells (HPMC) as the initial step of metastasis. The contact between ovarian cancer cells and the single layer of mesothelial cells involves direct communications that modulate cancer progression but the mechanisms are unclear. One candidate mediating cell-cell communications is exosomes, 30-100 nm membrane vesicles of endocytic origin, through the cell-cell transfer of proteins, mRNAs, or microRNAs. Therefore, the goal was to mechanistically characterize how EOC-derived exosomes modulate metastasis. Exosomes from ovarian cancer cells were fluorescently labeled and cocultured with HPMCs which internalized the exosomes. Upon exosome uptake, HPMCs underwent a change in cellular morphology to a mesenchymal, spindle phenotype. CD44, a cell surface glycoprotein, was found to be enriched in the cancer cell-derived exosomes, transferred, and internalized to HPMCs, leading to high levels of CD44 in HPMCs. This increased CD44 expression in HPMCs promoted cancer invasion by inducing the HPMCs to secrete MMP9 and by cleaning the mesothelial barrier for improved cancer cell invasion. When CD44 expression was knocked down in cancer cells, exosomes had fewer effects on HPMCs. The inhibition of exosome release from cancer cells blocked CD44 internalization in HPMCs and suppressed ovarian cancer invasion. In ovarian cancer omental metastasis, positive CD44 expression was observed in those mesothelial cells that directly interacted with cancer cells, whereas CD44 expression was negative in the mesothelial cells remote from the invading edge. This study indicates that ovarian cancer-derived exosomes transfer CD44 to HPMCs, facilitating cancer invasion. Mechanistic insight from the current study suggests that therapeutic targeting of exosomes may be beneficial in treating ovarian cancer. Mol Cancer Res; 15(1); 78-92. ©2016 AACR. ©2016 American Association for Cancer Research.

Dose-escalation study for the targeting of CD44v+ cancer stem cells by sulfasalazine in patients with advanced gastric cancer (EPOC1205).

PubMed

Shitara, Kohei; Doi, Toshihiko; Nagano, Osamu; Imamura, Chiyo K; Ozeki, Takeshi; Ishii, Yuya; Tsuchihashi, Kenji; Takahashi, Shunji; Nakajima, Takako E; Hironaka, Shuichi; Fukutani, Miki; Hasegawa, Hiromi; Nomura, Shogo; Sato, Akihiro; Einaga, Yasuaki; Kuwata, Takeshi; Saya, Hideyuki; Ohtsu, Atsushi

2017-03-01

Cancer stem cells (CSCs) have enhanced mechanisms of protection from oxidative stress. A variant form of CD44 (CD44v), a major CSC marker, was shown to interact with xCT, a subunit of cystine-glutamate transporter, which maintains high levels of intracellular reduced glutathione (GSH) which defend the cell against oxidative stress. Sulfasalazine (SSZ) is an inhibitor of xCT and was shown to suppress the survival of CD44v-positive stem-like cancer cells both in vitro and in vivo. To find the dose of SSZ which can safely reduce the population of CD44v-positive cells in tumors, a dose-escalation study in patients with advanced gastric cancer was conducted. SSZ was given four times daily by oral administration with 2 weeks as one cycle. Tumor biopsies were obtained before and after 14 days of administration of SSZ to evaluate expression of CD44v and the intratumoral level of GSH. Eleven patients were enrolled and received a dosage from 8 to 12 g/day. Safety was confirmed up to a dosage of 12 g/day, which was considered the maximum tolerated dose. Among the eight patients with CD44v-positive cells in their pretreatment biopsy samples, the CD44v-positive cancer cell population appeared to be reduced in the posttreatment biopsy tissues of four patients. Intratumoral GSH levels were also decreased in two patients, suggesting biological effectiveness of SSZ at 8 g/day or greater. This is the first study of SSZ as an xCT inhibitor for targeting CSCs. Reduction of the levels of CD44v-positive cells and GSH was observed in some patients, consistent with the mode of action of SSZ in CSCs.

Triple negative breast tumors in African-American and Hispanic/Latina women are high in CD44+, low in CD24+, and have loss of PTEN.

PubMed

Wu, Yanyuan; Sarkissyan, Marianna; Elshimali, Yahya; Vadgama, Jaydutt V

2013-01-01

African-American women have higher mortality from breast cancer than other ethnic groups. The association between poor survival and differences with tumor phenotypes is not well understood. The purpose of this study is to assess the clinical significance of (1) Stem cell-like markers CD44 and CD24; (2) PI3K/Akt pathway associated targets PTEN, activation of Akt, and FOXO1; and (3) the Insulin-like growth factor-1 (IGF-I) and IGF binding protein-3 (IGFBP3) in different breast cancer subtypes, and compare the differences between African-American and Hispanic/Latina women who have similar social-economic-status. A total of N=318 African-American and Hispanic/Latina women, with clinically-annotated information within the inclusion criteria were included. Formalin fixed paraffin embedded tissues from these patients were tested for the different markers using immunohistochemistry techniques. Kaplan-Meier survival-curves and Cox-regression analyses were used to assess Relative Risk and Disease-Free-Survival (DFS). The triple-negative-breast-cancer (TNBC) receptor-subtype was more prevalent among premenopausal women, and the Hormonal Receptor (HR) positive subtype was most common overall. TNBC tumors were more likely to have loss of PTEN, express high Ki67, and have increased CD44+/CD24- expression. TNBC was also associated with higher plasma-IGF-I levels. HR-/HER2+ tumors showed high pAkt, decreased FOXO1, and high CD24+ expression. The loss of PTEN impacted DFS significantly in African Americans, but not in Hispanics/Latinas after adjusted for treatment and other tumor pathological factors. The CD44+/CD24- and CD24+/CD44- phenotypes decreased DFS, but were not independent predictors for DFS. HER2-positive and TNBC type of cancers continued to exhibit significant decrease in DFS after adjusting for the selected biomarkers and treatment. TNBC incidence is high among African-American and Hispanic/Latino women residing in South Los Angeles. Our study also shows for the first time that TNBC was significantly associated with PTEN loss, high Ki67 and the CD44+/CD24- phenotype. The loss of PTEN impacts DFS significantly in African Americans.

Functional characterization of mouse spinal cord infiltrating CD8+ lymphocytes

PubMed Central

Deb, Chandra; Howe, Charles L

2011-01-01

Understanding the immunopathogenesis of neuroimmunological diseases of the CNS requires a robust method for isolating and characterizing the immune effector cells that infiltrate the spinal cord in animal models. We have developed a simple and rapid isolation method that produces high yields of spinal cord infiltrating leukocytes from a single demyelinated spinal cord and which maintains high surface expression of key immunophenotyping antigens. Using this method and the Theiler’s virus model of chronic demyelination, we report the presence of spinal cord infiltrating acute effector CD8+ lymphocytes that are CD45hiCD44loCD62L− and a population of spinal cord infiltrating target effector memory CD8+ lymphocytes that are CD45hiCD44hiCD62L−. These cells respond robustly to ex vivo stimulation by producing interferon γ but do not exhibit specificity for Theiler’s virus in a cytotoxicity assay. We conclude that target-derived lymphocytes in a mouse model of chronic spinal cord demyelination may have unique functional specificities. PMID:19596449

CD44 increases the efficiency of distant metastasis of breast cancer

PubMed Central

McFarlane, Suzanne; Coulter, Jonathan A.; Tibbits, Paul; O'Grady, Anthony; McFarlane, Cheryl; Montgomery, Nicola; Hill, Ashleigh; McCarthy, Helen O.; Young, Leonie S.; Kay, Elaine W.; Isacke, Clare M.; Waugh, David J.J.

2015-01-01

Metastasis is the predominant cause of death from cancer yet we have few biomarkers to predict patients at increased risk of metastasis and are unable to effectively treat disseminated disease. Analysis of 448 primary breast tumors determined that expression of the hylauronan receptor CD44 associated with high grade (p = 0.046), ER- (p = 0.001) and PR-negative tumors (p = 0.029), and correlated with increased distant recurrence and reduced disease-free survival in patients with lymph-node positive or large tumors. To determine its functional role in distant metastasis, CD44 was knocked-down in MDA-MB-231 cells using two independent shRNA sequences. Loss of CD44 attenuated tumor cell adhesion to endothelial cells and reduced cell invasion but did not affect proliferation in vitro. To verify the importance of CD44 to post-intravasation events, tumor formation was assessed by quantitative in vivo imaging and post-mortem tissue analysis following an intra-cardiac injection of transfected cells. CD44 knock-down increased survival and decreased overall tumor burden at multiple sites, including the skeleton in vivo. We conclude that elevated CD44 expression on tumour cells within the systemic circulation increases the efficiency of post-intravasation events and distant metastasis in vivo, consistent with its association with increased distant recurrence and reduced disease-free survival in patients. PMID:25888636

Self-Assembled Polymeric Micelles Based on Hyaluronic Acid-g-Poly(d,l-lactide-co-glycolide) Copolymer for Tumor Targeting

PubMed Central

Son, Gyung Mo; Kim, Hyun Yul; Ryu, Je Ho; Chu, Chong Woo; Kang, Dae Hwan; Park, Su Bum; Jeong, Young-IL

2014-01-01

Graft copolymer composed hyaluronic acid (HA) and poly(d,l-lactide-co-glycolide) (PLGA) (HAgLG) was synthesized for antitumor targeting via CD44 receptor of tumor cells. The carboxylic end of PLGA was conjugated with hexamethylenediamine (HMDA) to have amine end group in the end of chain (PLGA-amine). PLGA-amine was coupled with carboxylic acid of HA. Self-assembled polymeric micelles of HAgLG have spherical morphologies and their sizes were around 50–200 nm. Doxorubicin (DOX)-incorporated polymeric micelles were prepared by dialysis procedure. DOX was released over 4 days and its release rate was accelerated by the tumoric enzyme hyaluronidase. To assess targetability of polymeric micelles, CD44-positive HepG2 cells were employed treated with fluorescein isothiocyanate (FITC)-labeled polymeric micelles. HepG2 cells strongly expressed green fluorescence at the cell membrane and cytosol. However, internalization of polymeric micelles were significantly decreased when free HA was pretreated to block the CD44 receptor. Furthermore, the CD44-specific anticancer activity of HAgLG polymeric micelles was confirmed using CD44-negative CT26 cells and CD44-positive HepG2 cells. These results indicated that polymeric micelles of HaLG polymeric micelles have targetability against CD44 receptor of tumor cells. We suggest HAgLG polymeric micelles as a promising candidate for specific drug targeting. PMID:25216338

Immunophenotyping and activation status of maternal peripheral blood leukocytes during pregnancy and labour, both term and preterm.

PubMed

Zhang, Jianhong; Shynlova, Oksana; Sabra, Sally; Bang, Annie; Briollais, Laurent; Lye, Stephen J

2017-10-01

The onset of labour in rodents and in humans is associated with physiological inflammation which is manifested by infiltration of activated maternal peripheral leukocytes (mPLs) into uterine tissues. Here, we used flow cytometry to immunophenotype mPLs throughout gestation and labour, both term and preterm. Peripheral blood was collected from non-pregnant women and pregnant women in the 1st, 2nd and 3rd trimesters. Samples were also collected from women in active labour at term (TL) or preterm (PTL) and compared with women term not-in-labour (TNIL) and preterm not-in-labour (PTNIL). Different leukocyte populations were identified by surface markers such as CD45, CD14, CD15, CD3, CD4, CD8, CD19 and CD56. Their activation status was measured by the expression levels of CD11b, CD44, CD55, CD181 and CD192 proteins. Of all circulating CD45+ leukocytes, we detected significant increases in CD15+ granulocytes (i) in pregnant women versus non-pregnant; (ii) in TL women versus TNIL and versus pregnant women in the 1st/2nd/3rd trimester; (iii) in PTL women versus PTNIL. TL was characterized by (iv) increased expressions of CD11b, CD55 and CD192 on granulocytes; (v) increased mean fluorescent intensity (MFI) of CD55 and CD192 on monocytes; (vi) increased CD44 MFI on CD3+ lymphocytes as compared to late gestation. In summary, we have identified sub-populations of mPLs that are specifically activated in association with gestation (granulocytes) or with the onset of labour (granulocytes, monocytes and lymphocytes). Additionally, beta regression analysis created a set of reference values to rank this association between immune markers of pregnancy and to identify activation status with potential prognostic and diagnostic capability. © 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Expression of Bmi-1, P16, and CD44v6 in Uterine Cervical Carcinoma and Its Clinical Significance

PubMed Central

Weng, Mei-ying; Li, Lin; Feng, Shu-ying; Hong, Shun-jia

2012-01-01

Objective Bmi-1, a putative proto-oncogene, is a core member of the polycomb gene family, which is expressed in many human tumors. The p16 protein negatively regulated cell proliferation, whereas CD44v6 is associated with proliferation as an important protein. Additionally, CD44v6 is an important nuclear antigen closely correlated to tumor metastasis. The present study aims to investigate the expression and significance of Bmi-1, p16, and CD44v6 in uterine cervical carcinoma (UCC). Methods A total of 62 UCC, 30 cervical neoplasic, and 20 normal cervical mucosal tissues were used in the current study. The expression of Bmi-1, p16, and CD44v6 in these tissues was determined using immunohistochemical assay. The relationships among the expression of these indices, the clinicopathologic features of UCC, and the survival rate of UCC patients were also discussed. The correlation between Bmi-1 protein expression and p16 or CD44v6 protein in UCC was analyzed. Results The expression of Bmi-1, p16, and CD44v6 was significantly high in cervical carcinoma compared with that in the cervical neoplasia and normal colorectal mucosa (P<0.05). The over-expression of Bmi-1 protein in UCC was apparently related to the distant metastasis (P<0.01) and the tumor, nodes and metastasis-classification, i.e. the TNM staging, World Health Organization (P<0.05). Nevertheless, the positive expression of p16 protein in UCC was not significantly associated with the clinicopathologic features (P>0.05). The Kaplan–Meier survival analysis showed that the over-expression of Bmi-1 significantly decreased the survival rate of UCC patients (P<0.05). A strong correlation indicated that there was statistical significance between the expression of Bmi-1 and CD44V6 proteins in UCC (r=0.419, P=0.001). Conclusions The over-expression of Bmi-1 and CD44v6 protein closely correlate to the tumorigenesis, metastasis, and prognosis of UCC. Bmi-1 and CD44v6 may be used to predict the prognosis of cervical carcinoma. Bmi-1 may indirectly regulate the expression of CD44v6 in UCC patients. The positive expression of p16 protein is possibly associated with the tumorigenesis, but not with the metastasis or prognosis of UCC. PMID:23691455

Evaluation of rapid progressors in HIV infection as an extreme phenotype.

PubMed

Olson, Ashley D; Guiguet, Marguerite; Zangerle, Robert; Gill, John; Perez-Hoyos, Santiago; Lodi, Sara; Ghosn, Jade; Dorrucci, Maria; Johnson, Anne; Sannes, Mette; Moreno, Santiago; Porter, Kholoud

2014-09-01

Rapid CD4 cell loss represents an HIV phenotype used to identify causal variants of accelerated disease progression. The optimal rate and threshold for identifying this extreme phenotype in recently infected individuals is unclear. Using a cohort of patients with known dates of HIV-1 seroconversion (SC), CASCADE (Concerted Action on SeroConversion on AIDS and Death in Europe), we identified proportions experiencing nadir CD4 cell levels within 1 year of SC, and assessed their mean AIDS-free survival time at 10-year follow-up and hazard of AIDS/death, compared with those whose CD4 remained >500 cells per cubic millimeter. Follow-up was censored at December 31, 1996 to avoid bias due to combination antiretroviral therapy initiation. Of 4876 individuals, 2.8%, 7.3%, and 24.9% experienced ≥1 CD4 <100, 200, and 350 cells per cubic millimeter, respectively, within 1 year of SC. Minimum CD4 levels of 30, 166, 231, and 506 cells per cubic millimeter were experienced during this period by 1%, 5%, 10%, and 50% of individuals, respectively. Mean (95% confidence interval) AIDS-free survival at 10 years follow-up was 2.9 (2.3 to 3.6), 5.5 (5.0 to 6.1), 6.7 (6.5 to 7.0), 7.4 (7.2 to 7.6), and 8.1 (7.9 to 8.3), for those with minimum counts ≤100, 100-200, 200-350, 350-500, >500 cells per cubic millimeter, respectively. Using counts of >500 cells per cubic millimeter as reference, the hazard ratios (95% confidence interval) of AIDS/death were 15.0 (11.9 to 18.9), 3.6 (2.9 to 4.5), 2.1 (1.8 to 2.4), and 1.5 (1.3 to 1.7), respectively. The hazard ratio increased to 37.5 (26.5 to 53.1) when a minimum CD4 count <100 was confirmed within 1 year of SC. At least 1 CD4 ≤100 cells per cubic millimeter within the first year of SC identifies a rare group of individuals at high risk of disease progression and could form the basis for defining the rapid progressor phenotype.

Autophagy regulates cisplatin-induced stemness and chemoresistance via the upregulation of CD44, ABCB1 and ADAM17 in oral squamous cell carcinoma.

PubMed

Naik, Prajna Paramita; Mukhopadhyay, Subhadip; Panda, Prashanta Kumar; Sinha, Niharika; Das, Chandan Kanta; Mishra, Rajakishore; Patil, Shankargouda; Bhutia, Sujit Kumar

2018-02-01

We inspected the relevance of CD44, ABCB1 and ADAM17 in OSCC stemness and deciphered the role of autophagy/mitophagy in regulating stemness and chemoresistance. A retrospective analysis of CD44, ABCB1 and ADAM17 with respect to the various clinico-pathological factors and their correlation was analysed in sixty OSCC samples. Furthermore, the stemness and chemoresistance were studied in resistant oral cancer cells using sphere formation assay, flow cytometry and florescence microscopy. The role of autophagy/mitophagy was investigated by transient transfection of siATG14, GFP-LC3, tF-LC3, mKeima-Red-Mito7 and Western blot analysis of autophagic and mitochondrial proteins. In OSCC, high CD44, ABCB1 and ADAM17 expressions were correlated with higher tumour grades and poor differentiation and show significant correlation in their co-expression. In vitro and OSCC tissue double labelling confirmed that CD44 + cells co-expresses ABCB1 and ADAM17. Further, cisplatin (CDDP)-resistant FaDu cells displayed stem-like features and higher CD44, ABCB1 and ADAM17 expression. Higher autophagic flux and mitophagy were observed in resistant FaDu cells as compared to parental cells, and inhibition of autophagy led to the decrease in stemness, restoration of mitochondrial proteins and reduced expression of CD44, ABCB1 and ADAM17. The CD44 + /ABCB1 + /ADAM17 + expression in OSCC is associated with stemness and chemoresistance. Further, this study highlights the involvement of mitophagy in chemoresistance and autophagic regulation of stemness in OSCC. © 2017 John Wiley & Sons Ltd.

Duodenal intraepithelial T lymphocytes in patients with functional dyspepsia

PubMed Central

Gargala, Gilles; Lecleire, Stéphane; François, Arnaud; Jacquot, Serge; Déchelotte, Pierre; Ballet, Jean Jacques; Favennec, Loic; Ducrotté, Philippe

2007-01-01

AIM: To quantify the intraepithelial lymphocytes (IELs) and to document the membrane expression of CD4, CD8, TCRγδ and adhesion and/or activation-associated molecules (CD103, CD28, CD44, CD69, HLA-DR, CD95/Fas) in the duodenal mucosa of patients with functional dyspepsia (FD) in order to provide arguments for an immunological process in FD. METHODS: Twenty-six FD patients according to Rome II criteria (20 were H pylori negative) were studied and compared to 12 healthy adults. IELs were isolated from five duodenal biopsy samples, then quantified by microscopy and flow cytometry while the membrane phenotypes were determined by cytofluorometry. RESULTS: Duodenal histological examination was normal. In H pylori negative patients, the number of IELs was not different from that in healthy controls. Median percentage expression of CD4, CD8, or TCRγδ and CD103, CD44, CD28, CD69 on CD3+ IELs, among the adhesion/activation associated molecules tested, was not different from that in healthy controls. In contrast, the median percentage expression of CD95/Fas [22 (9-65) vs 45 (19-88), P = 0.03] and HLA-DR expressing CD3+ IELs [4 (0-30) vs 13 (4-42), P = 0.04] was significantly lower in the H pylori negative FD group than in healthy controls, respectively. The number of IELs was significantly greater in H pylori positive FD patients than in healthy controls [median ratiofor 100 enterocytes 27.5 (6.7-62.5) vs 10.8 (3-33.3), P = 0.02] due to a higher number of CD8+ CD3+ IELs. CONCLUSION: In H pylori negative FD patients, the phenotypic characterization of IELs suggests that we cannot exclude a role of IELs in FD. PMID:17511033

CD44 Gene Polymorphisms in Breast Cancer Risk and Prognosis: A Study in North Indian Population

PubMed Central

Tulsyan, Sonam; Agarwal, Gaurav; Lal, Punita; Agrawal, Sushma; Mittal, Rama Devi; Mittal, Balraj

2013-01-01

Background Cell surface biomarker CD44 plays an important role in breast cancer cell growth, differentiation, invasion, angiogenesis and tumour metastasis. Therefore, we aimed to investigate the role of CD44 gene polymorphisms in breast cancer risk and prognosis in North Indian population. Materials & Methods A total of 258 breast cancer patients and 241 healthy controls were included in the case-control study for risk prediction. According to RECIST, 114 patients who received neo-adjuvant chemotherapy were recruited for the evaluation of breast cancer prognosis. We examined the association of tagging SNP (rs353639) of Hapmap Gujrati Indians in Houston (GIH population) in CD44 gene along with a significant reported SNP (rs13347) in Chinese population by genotyping using Taqman allelic discrimination assays. Statistical analysis was done using SPSS software, version 17. In-silico analysis for prediction of functional effects was done using F-SNP and FAST-SNP. Results No significant association of both the genetic variants of the CD44 gene polymorphisms was found with breast cancer risk. On performing univariate analysis with clinicopathological characteristics and treatment response, we found significant association of genotype (CT+TT) of rs13347 polymorphism with earlier age of onset (P = 0.029, OR = 0.037). However, significance was lost in multivariate analysis. For rs353639 polymorphism, significant association was seen with clinical tumour size, both at the genotypic (AC+CC) (P = 0.039, OR = 3.02) as well as the allelic (C) (P = 0.042, OR = 2.87) levels. On performing multivariate analysis, increased significance of variant genotype (P = 0.017, OR = 4.29) and allele (P = 0.025, OR = 3.34) of rs353639 was found with clinical tumour size. In-silico analysis using F-SNP, showed altered transcriptional regulation for rs353639 polymorphism. Conclusions These findings suggest that CD44 rs353639 genetic variants may have significant effect in breast cancer prognosis. However, both the polymorphisms- rs13347 and rs353639 had no effect on breast cancer susceptibility. PMID:23940692

Evaluation of bone marrow specimens with acute myelogenous leukemia for CD34, CD15, CD117, and myeloperoxidase.

PubMed

Dunphy, C H; Polski, J M; Evans, H L; Gardner, L J

2001-08-01

Immunophenotyping of bone marrow (BM) specimens with acute myelogenous leukemia (AML) may be performed by flow cytometric (FC) or immunohistochemical (IH) techniques. Some markers (CD34, CD15, and CD117) are available for both techniques. Myeloperoxidase (MPO) analysis may be performed by enzyme cytochemical (EC) or IH techniques. To determine the reliability of these markers and MPO by these techniques, we designed a study to compare the results of analyses of these markers and MPO by FC (CD34, CD15, and CD117), EC (MPO), and IH (CD34, CD15, CD117, and MPO) techniques. Twenty-nine AMLs formed the basis of the study. These AMLs all had been immunophenotyped previously by FC analysis; 27 also had had EC analysis performed. Of the AMLs, 29 had BM core biopsies and 26 had BM clots that could be evaluated. The paraffin blocks of the 29 BM core biopsies and 26 BM clots were stained for CD34, CD117, MPO, and CD15. These results were compared with results by FC analysis (CD34, CD15, and CD117) and EC analysis (MPO). Immunodetection of CD34 expression in AML had a similar sensitivity by FC and IH techniques. Immunodetection of CD15 and CD117 had a higher sensitivity by FC analysis than by IH analysis. Detection of MPO by IH analysis was more sensitive than by EC analysis. There was no correlation of French-American-British (FAB) subtype of AML with CD34 or CD117 expression. Expression of CD15 was associated with AMLs with a monocytic component. Myeloperoxidase reactivity by IH analysis was observed in AMLs originally FAB subtyped as M0. CD34 can be equally detected by FC and IH techniques. CD15 and CD117 are better detected by FC analysis and MPO is better detected by IH analysis.

Incomplete Recovery of CD4 count, CD4 Percentage, and CD4/CD8 ratio in HIV-Infected Patients on Long-Term Antiretroviral Therapy with Suppressed Viremia.

PubMed

Mutoh, Yoshikazu; Nishijima, Takeshi; Inaba, Yosuke; Tanaka, Noriko; Kikuchi, Yoshimi; Gatanaga, Hiroyuki; Oka, Shinichi

2018-03-02

The extent and duration of long-term recovery of CD4 count, CD4%, and CD4/CD8 ratio after initiation of combination antiretroviral therapy (cART) in patients with suppressed viral load are largely unknown. HIV-1 infected patients who started cART between January 2004 and January 2012 and showed persistent viral suppression (<200 copies/mL) for at least 4 years were followed up at AIDS Clinical Center, Tokyo. Change point analysis was used to determine the time point where CD4 count recovery shows a plateau, and linear mixed model was applied to estimate CD4 count at the change point. Data of 752 patients were analyzed [93% males, median age 38, median baseline CD4 count 172/µL (IQR, 62-253), CD4% 13.8% (IQR, 7.7-18.5), and CD4/8 ratio 0.23 (IQR, 0.12-0.35)]. The median follow-up period was 81.2 months and 91 (12.1%) patients were followed for >10 years. Change point analysis showed that CD4 count, CD4%, and CD4/CD8 ratio, continued to increase until 78.6, 62.2, and 64.3 months, respectively, with adjusted mean of 590 /µL (95%CI 572-608), 29.5% (29-30.1), and 0.89 (0.86-0.93), respectively, at the change point. Although 73.8% of the study patients achieved CD4 count ≥500 /μL, 48.2% of the patients with baseline CD4 count <100 /μL did not achieve CD4 count ≥500 /μL. Neither CD4% nor CD4/CD8 ratio normalized in a majority of patients. The results showed lack of normalization of CD4 count, CD4%, and CD4/CD8 ratio to the levels seen in healthy individuals even after long-term successful cART in patients with suppressed viral load.

A putative mesenchymal stem cells population isolated from adult human testes.

PubMed

Gonzalez, R; Griparic, L; Vargas, V; Burgee, K; Santacruz, P; Anderson, R; Schiewe, M; Silva, F; Patel, A

2009-08-07

Mesenchymal stem cells (MSCs) isolated from several adult human tissues are reported to be a promising tool for regenerative medicine. In order to broaden the array of tools for therapeutic application, we isolated a new population of cells from adult human testis termed gonadal stem cells (GSCs). GSCs express CD105, CD166, CD73, CD90, STRO-1 and lack hematopoietic markers CD34, CD45, and HLA-DR which are characteristic identifiers of MSCs. In addition, GSCs express pluripotent markers Oct4, Nanog, and SSEA-4. GSCs propagated for at least 64 population doublings and exhibited clonogenic capability. GSCs have a broad plasticity and the potential to differentiate into adipogenic, osteogenic, and chondrogenic cells. These studies demonstrate that GSCs are easily obtainable stem cells, have growth kinetics and marker expression similar to MSCs, and differentiate into mesodermal lineage cells. Therefore, GSCs may be a valuable tool for therapeutic applications.

Characterization of pancreatic stem cells derived from adult human pancreas ducts by fluorescence activated cell sorting.

PubMed

Lin, Han-Tso; Chiou, Shih-Hwa; Kao, Chung-Lan; Shyr, Yi-Ming; Hsu, Chien-Jen; Tarng, Yih-Wen; Ho, Larry L-T; Kwok, Ching-Fai; Ku, Hung-Hai

2006-07-28

To isolate putative pancreatic stem cells (PSCs) from human adult tissues of pancreas duct using serum-free, conditioned medium. The characterization of surface phenotype of these PSCs was analyzed by flow cytometry. The potential for pancreatic lineage and the capability of beta-cell differentiation in these PSCs were evaluated as well. By using serum-free medium supplemented with essential growth factors, we attempted to isolate the putative PSCs which has been reported to express nestin and pdx-1. The Matrigel(TM) was employed to evaluate the differential capacity of isolated cells. Dithizone staining, insulin content/secretion measurement, and immunohistochemistry staining were used to monitor the differentiation. Fluorescence activated cell sorting (FACS) was used to detect the phenotypic markers of putative PSCs. A monolayer of spindle-like cells was cultivated. The putative PSCs expressed pdx-1 and nestin. They were also able to differentiate into insulin-, glucagon-, and somatostatin-positive cells. The spectrum of phenotypic markers in PSCs was investigated; a similarity was revealed when using human bone marrow-derived stem cells as the comparative experiment, such as CD29, CD44, CD49, CD50, CD51, CD62E, PDGFR-alpha, CD73 (SH2), CD81, CD105(SH3). In this study, we successfully isolated PSCs from adult human pancreatic duct by using serum-free medium. These PSCs not only expressed nestin and pdx-1 but also exhibited markers attributable to mesenchymal stem cells. Although work is needed to elucidate the role of these cells, the application of these PSCs might be therapeutic strategies for diabetes mellitus.

Syndecan-1/CD147 association is essential for cyclophilin B-induced activation of p44/42 mitogen-activated protein kinases and promotion of cell adhesion and chemotaxis.

PubMed

Pakula, Rachel; Melchior, Aurélie; Denys, Agnès; Vanpouille, Christophe; Mazurier, Joël; Allain, Fabrice

2007-05-01

Many of the biological functions attributed to cell surface proteoglycans are dependent on the interaction with extracellular mediators through their heparan sulphate (HS) moieties and the participation of their core proteins in signaling events. A class of recently identified inflammatory mediators is secreted cyclophilins, which are mostly known as cyclosporin A-binding proteins. We previously demonstrated that cyclophilin B (CyPB) triggers chemotaxis and integrin-mediated adhesion of T lymphocytes mainly of the CD4+/CD45RO+ phenotype. These activities are related to interactions with two types of binding sites, CD147 and cell surface HS. Here, we demonstrate that CyPB-mediated adhesion of CD4+/CD45RO+ T cells is related to p44/42 mitogen-activated protein kinase (MAPK) activation by a mechanism involving CD147 and HS proteoglycans (HSPG). Although HSPG core proteins are represented by syndecan-1, -2, -4, CD44v3 and betaglycan in CD4+/CD45RO+ T cells, we found that only syndecan-1 is physically associated with CD147. The intensity of the heterocomplex increased in response to CyPB, suggesting a transient enhancement and/or stabilization in the association of CD147 to syndecan-1. Pretreatment with anti-syndecan-1 antibodies or knockdown of syndecan-1 expression by RNA interference dramatically reduced CyPB-induced p44/p42 MAPK activation and consequent migration and adhesion, supporting the model in which syndecan-1 serves as a binding subunit to form the fully active receptor of CyPB. Altogether, our findings provide a novel example of a soluble mediator in which a member of the syndecan family plays a critical role in efficient interaction with signaling receptors and initiation of cellular responses.

Rapid generation of functional hepatocyte-like cells from human adipose-derived stem cells.

PubMed

Fu, Yanli; Deng, Jie; Jiang, Qingyuan; Wang, Yuan; Zhang, Yujing; Yao, Yunqi; Cheng, Fuyi; Chen, Xiaolei; Xu, Fen; Huang, Meijuan; Yang, Yang; Zhang, Shuang; Yu, Dechao; Zhao, Robert Chunhua; Wei, Yuquan; Deng, Hongxin

2016-08-05

Liver disease is a major cause of death worldwide. Orthotropic liver transplantation (OLT) represents the only effective treatment for patients with liver failure, but the increasing demand for organs is unfortunately so great that its application is limited. Hepatocyte transplantation is a promising alternative to OLT for the treatment of some liver-based metabolic disorders or acute liver failure. Unfortunately, the lack of donor livers also makes it difficult to obtain enough viable hepatocytes for hepatocyte-based therapies. Currently, a fundamental solution to this key problem is still lacking. Here we show a novel non-transgenic protocol that facilitates the rapid generation of functional induced hepatocytes (iHeps) from human adipose-derived stem cells (hADSCs), providing a source of available cells for autologous hepatocytes to treat liver disease. We used collagenase digestion to isolate hADSCs. The surface marker was detected by flow cytometry. The multipotential differentiation potency was detected by induction into adipocytes, osteocytes, and chondrocytes. Passage 3-7 hADSCs were induced into iHeps using an induction culture system composed of small molecule compounds and cell factors. Primary cultured hADSCs presented a fusiform or polygon appearance that became fibroblast-like after passage 3. More than 95 % of the cells expressed the mesenchymal cell markers CD29, CD44, CD166, CD105, and CD90. hADSCs possessed multipotential differentiation towards adipocytes, osteocytes, and chondrocytes. We rapidly induced hADSCs into iHeps within 10 days in vitro; the cellular morphology changed from fusiform to close-connected cubiform, which was similar to hepatocytes. After induction, most of the iHeps co-expressed albumin and alpha-1 antitrypsin; they also expressed mature hepatocyte special genes and achieved the basic functions of hepatocyte. Moreover, iHep transplantation could improve the liver function of acute liver-injured NPG mice and prolong life. We isolated highly purified hADSCs and rapidly induced them into functional hepatocyte-like cells within 10 days. These results provide a source of available cells for autologous hepatocytes to treat liver disease.

Incidence and phenotype of inflammatory bowel disease based on results from the Asia-pacific Crohn's and colitis epidemiology study.

PubMed

Ng, Siew C; Tang, Whitney; Ching, Jessica Y; Wong, May; Chow, Chung Mo; Hui, A J; Wong, T C; Leung, Vincent K; Tsang, Steve W; Yu, Hon Ho; Li, Mo Fong; Ng, Ka Kei; Kamm, Michael A; Studd, Corrie; Bell, Sally; Leong, Rupert; de Silva, H Janaka; Kasturiratne, Anuradhani; Mufeena, M N F; Ling, Khoon Lin; Ooi, Choon Jin; Tan, Poh Seng; Ong, David; Goh, Khean L; Hilmi, Ida; Pisespongsa, Pises; Manatsathit, Sathaporn; Rerknimitr, Rungsun; Aniwan, Satimai; Wang, Yu Fang; Ouyang, Qin; Zeng, Zhirong; Zhu, Zhenhua; Chen, Min Hu; Hu, Pin Jin; Wu, Kaichun; Wang, Xin; Simadibrata, Marcellus; Abdullah, Murdani; Wu, Justin Cy; Sung, Joseph J Y; Chan, Francis K L

2013-07-01

Inflammatory bowel diseases (IBD) are becoming more common in Asia, but epidemiologic data are lacking. The Asia-Pacific Crohn's and Colitis Epidemiology Study aimed to determine the incidence and phenotype of IBD in 8 countries across Asia and in Australia. We performed a prospective, population-based study of IBD incidence in predefined catchment areas, collecting data for 1 year, starting on April 1, 2011. New cases were ascertained from multiple overlapping sources and entered into a Web-based database. Cases were confirmed using standard criteria. Local endoscopy, pathology, and pharmacy records were searched to ensure completeness of case capture. We identified 419 new cases of IBD (232 of ulcerative colitis [UC], 166 of Crohn's disease [CD], and 21 IBD-undetermined). The crude annual overall incidence values per 100,000 individuals were 1.37 for IBD in Asia (95% confidence interval: 1.25-1.51; 0.76 for UC, 0.54 for CD, and 0.07 for IBD-undetermined) and 23.67 in Australia (95% confidence interval: 18.46-29.85; 7.33 for UC, 14.00 for CD, and 2.33 for IBD-undetermined). China had the highest incidence of IBD in Asia (3.44 per 100,000 individuals). The ratios of UC to CD were 2.0 in Asia and 0.5 in Australia. Median time from symptom onset to diagnosis was 5.5 months (interquartile range, 1.4-15 months). Complicated CD (stricturing, penetrating, or perianal disease) was more common in Asia than Australia (52% vs 24%; P = .001), and a family history of IBD was less common in Asia (3% vs 17%; P < .001). We performed a large-scale population-based study and found that although the incidence of IBD varies throughout Asia, it is still lower than in the West. IBD can be as severe or more severe in Asia than in the West. The emergence of IBD in Asia will result in the need for specific health care resources, and offers a unique opportunity to study etiologic factors in developing nations. Copyright © 2013 AGA Institute. Published by Elsevier Inc. All rights reserved.

Verification of ALDH Activity as a Biomarker in Colon Cancer Stem Cells-Derived HT-29 Cell Line.

PubMed

Khorrami, Samaneh; Zavaran Hosseini, Ahmad; Mowla, Seyed Javad; Malekzadeh, Reza

2015-10-01

Recent evidence has suggested that epithelial cancers including colorectal cancer (CRC) have driven by a small population of self-renewing, multi-potent cells termed cancer stem cells (CSCs) which could be responsible for recurrence of cancer. Aldehyde dehydrogenase 1 (ALDH1) activity has used as a functional stem cell biomarker to isolate CSCs in different cancers such as colorectal cancer. The main aim of this research was to determine the utility of ALDH1 activity along with CD44 and EPCAM in identifying stem cell-like cells in human HT-29 colonic adenocarcinoma cell line. In this experimental study, colon CSCs biomarkers including CD44, EPCAM and ALDH1 in colonospheres and parent cells have analyzed by flow cytometry. The expression levels of stemness genes in spheroid and parental cells have investigated using SYBR Green real-time PCR. In addition, in vivo xenografts assay has performed to determine tumorigenic potential of tumor spheroid cells in nude mice. According to results, over 92% of spheroids were CD44+/EpCAM+, while parent cells only have expressed 38% of CD44/EpCAM biomarkers (P < 0.001). Controversially, ALDH activity was about 2-fold higher in the parent cells than spheroid cells (P < 0.05). In comparison with the parental cells, expression levels of ''stemness'' genes, like Sox2, Oct4, Nanog, C-myc, and Klf4 have significantly increased in colonosphere cells (P < 0.05). Further, administration of 2500 spheroids could be sufficient to initiate tumor growth in nude mice, while 1x106 of parental cells has needed to form tumor. For the first time, we have shown that colonospheres with low ALDH1 activity has indicated increased tumorigenic potential and stemness properties. So, it hasn't seemed that ALDH1 could become a useful biomarker to identify CSCs population in HT-29 cell line.

Lymphocyte integrin expression differences between SIRS and sepsis patients.

PubMed

Heffernan, D S; Monaghan, S F; Ayala, Alfred

2017-11-01

Systemic Inflammatory Response Syndrome (SIRS) and sepsis remain leading causes of death. Despite many similarities, the two entities are very distinct clinically and immunologically. T-Lymphocytes play a key pivotal role in the pathogenesis and ultimately outcome following both SIRS and sepsis. Integrins are essential in the trafficking and migration of lymphocytes. They also serve vital roles in efficient wound healing and clearance of infections. Here, we investigate whether integrin expression, specifically β1 (CD29) and β2 (CD18), are disrupted in SIRS and sepsis, and assess differences in integrin expression between these two critically ill clinical categories. T-Lymphocytes were isolated from whole blood collected from ICU patients exhibiting SIRS or sepsis. Samples were analyzed for CD18 (β2) and CD29 (β1) on CD3 + T cells through flow cytometry. Septic patients were stratified into either exclusively abdominal or non-abdominal sources of sepsis. CD18 was almost ubiquitously expressed on CD3 + T cells irrespective of clinical condition. However, CD29 (β1 integrin) was lowest in SIRS patients (20.4% of CD3 + T cells) when compared with either septic patients (35.5%) or healthy volunteers (54.1%). Furthermore, there was evidence of compartmentalization in septic patients, where abdominal sources had a greater percentage of CD3 + CD29 + T cells (41.7%) when compared with those with non-abdominal sources (29.5%). Distinct differences in T-cell integrin expression exists between patients in SIRS versus sepsis, as well as relative to the source of sepsis. Further work is needed to understand cause and effect relative to the progression from SIRS into sepsis.

CD44 mediated hyaluronan adhesion of Toxoplasma gondii-infected leukocytes.

PubMed

Hayashi, Takeshi; Unno, Akihiro; Baba, Minami; Ohno, Tamio; Kitoh, Katsuya; Takashima, Yasuhiro

2014-04-01

Toxoplasma gondii is an obligate intracellular apicomplexan parasite that infects humans and animals. Ingested parasites cross the intestinal epithelium, invade leukocytes and are then disseminated to peripheral organs. However, the mechanism of extravasation of the infected leukocytes remains poorly understood. In this study, we demonstrate that T. gondii-invaded human and mouse leukocytes express higher level of CD44, a ligand of hyaluronan (HA), and its expression on myeloid and non-myeloid leukocytes causes T. gondii-invaded human and mouse leukocyte to adhere to HA more effectively than non-invaded leukocytes. The specific adherence of parasite-invaded leukocytes was inhibited by anti CD44 antibody. Leukocytes of CD44 knockout mice did not show parasite-invaded leukocyte specific adhesion. Our results indicate that parasite-invaded leukocytes, regardless of whether myeloid or not, gain higher ability to adhere to HA than non-invaded leukocytes, via upregulation of CD44 expression and/or selective invasion to CD44 highly expressing cells. The difference in ability to adhere to HA between parasite-invaded cells and non-invaded neighboring cells might facilitate effective delivery of parasite-invaded leukocytes to the HA-producing endothelial cell surface and/or HA-rich extra cellular matrix. © 2013.

Group A Streptococcus tissue invasion by CD44-mediated cell signalling

NASA Astrophysics Data System (ADS)

Cywes, Colette; Wessels, Michael R.

2001-12-01

Streptococcus pyogenes (also known as group A Streptococcus, GAS), the agent of streptococcal sore throat and invasive soft-tissue infections, attaches to human pharyngeal or skin epithelial cells through specific recognition of its hyaluronic acid capsular polysaccharide by the hyaluronic-acid-binding protein CD44 (refs 1, 2). Because ligation of CD44 by hyaluronic acid can induce epithelial cell movement on extracellular matrix, we investigated whether molecular mimicry by the GAS hyaluronic acid capsule might induce similar cellular responses. Here we show that CD44-dependent GAS binding to polarized monolayers of human keratinocytes induced marked cytoskeletal rearrangements manifested by membrane ruffling and disruption of intercellular junctions. Transduction of the signal induced by GAS binding to CD44 on the keratinocyte surface involved Rac1 and the cytoskeleton linker protein ezrin, as well as tyrosine phosphorylation of cellular proteins. Studies of bacterial translocation in two models of human skin indicated that cell signalling triggered by interaction of the GAS capsule with CD44 opened intercellular junctions and promoted tissue penetration by GAS through a paracellular route. These results support a model of host cytoskeleton manipulation and tissue invasion by an extracellular bacterial pathogen.

Mesenchymal stem cells do not suppress lymphoblastic leukemic cell line proliferation.

PubMed

Mousavi Niri, Neda; Jaberipour, Mansooreh; Razmkhah, Mahboobeh; Ghaderi, Abbas; Habibagahi, Mojtaba

2009-12-01

Several studies have demonstrated the immunosuppresive effects of mesenchymal stem cells (MSCs) in allogeneic or mitogenic interactions. Cell-cell contact inhibition and secretion of suppressive soluble factors have been suggested in this regard. To investigate if adipose derived MSCs could inhibit Jurkat lymphoblastic leukemia T cell proliferation during coculture. Adherent cells with the ability of cellular growth were isolated from normal adipose tissues. Initial characterization of growing cells by flow cytometry suggested their mesenchymal stem cell characteristics. Cells were maintained in culture and used during third to fifth culture passages. Jurkat or allogeneic peripheral blood mononuclear cells (PBMCs) were labeled with carboxy fluorescein diacetate succinimidyl ester and cocultured with increasing doses of MSCs or MSC culture supernatant. Proliferation of PBMCs or Jurkat cells under these conditions was assessed by flow cytometry after 2 and 3 days of coculture, respectively. Results showed the expression of CD105, CD166 and CD44, and the absence of CD45, CD34 and CD14 on the surface of MSC like cells. Moreover, initial differentiation studies showed the potential of cell differentiation into hepatocytes. Comparison of Jurkat cell proliferation in the presence and absence of MSCs showed no significant difference, with 70% of cells displaying signs of at least one cell division. Similarly, the highest concentration of MSC culture supernatant (50% vol/vol) had no significant effect on Jurkat cell proliferation (p>0.6). The same MSC lots significantly suppressed the allogeneic PHA activated PBMCs under similar culture conditions. Using Jurkat cells as a model of leukemia T cells, our results indicated an uncertainty about the suppressive effect of MSCs and their inhibitory metabolites on tumor or leukemia cell proliferation. Additional systematic studies with MSCs of different sources are needed to fully characterize the immunological properties of MSCs before planning clinical applications.

Selective resistance of CD44hi T cells to p53-dependent cell death results in persistence of immunologic memory after total body irradiation.

PubMed

Yao, Zhenyu; Jones, Jennifer; Kohrt, Holbrook; Strober, Samuel

2011-10-15

Our previous studies showed that treatment of mice with total body irradiation (TBI) or total lymphoid tissue irradiation markedly changes the balance of residual T cell subsets to favor CD4(+)CD44(hi) NKT cells because of the differential resistance of the latter subset to cell death. The object of the current study was to further elucidate the changed balance and mechanisms of differential radioresistance of T cell subsets after graded doses of TBI. The experimental results showed that CD4(+) T cells were markedly more resistant than CD8(+) T cells, and CD44(hi) T cells, including NKT cells and memory T cells, were markedly more resistant than CD44(lo) (naive) T cells. The memory T cells immunized to alloantigens persisted even after myeloablative (1000 cGy) TBI and were able to prevent engraftment of bone marrow transplants. Although T cell death after 1000 cGy was prevented in p53(-/-) mice, there was progressive T cell death in p53(-/-) mice at higher doses. Although p53-dependent T cell death changed the balance of subsets, p53-independent T cell death did not. In conclusion, resistance of CD44(hi) T cells to p53-dependent cell death results in the persistence of immunological memory after TBI and can explain the immune-mediated rejection of marrow transplants in sensitized recipients.

Passage-dependent morphological and phenotypical changes of a canine histiocytic sarcoma cell line (DH82 cells).

PubMed

Heinrich, Franziska; Contioso, Vanessa Bono; Stein, Veronika M; Carlson, Regina; Tipold, Andrea; Ulrich, Reiner; Puff, Christina; Baumgärtner, Wolfgang; Spitzbarth, Ingo

2015-01-15

DH82 cells represent a permanent macrophage cell line isolated from a dog with histiocytic sarcoma (HS) and are commonly used in various fields of research upon infection and cancer, respectively. Despite its frequent use, data on cell surface antigen expression of this cell line are fragmentary and in part inconsistent. We therefore aimed at a detailed morphological and antigenic characterization of DH82 cells with respect to passage-dependent differences. Cellular morphology of early (≤ 13) and late (≥ 66) passages of DH82 cells was evaluated via scanning electron microscopy. Moreover, cells were labelled with 10 monoclonal antibodies directed against CD11c, CD14, CD18, CD44, CD45, CD80, CD86, MHC-I, MHC-II, and ICAM-1 for flow cytometric analysis. Early passage cells were characterized by round cell bodies with abundant small cytoplasmic projections whereas later passages exhibited a spindle-shaped morphology with large processes. The percentage of CD11c-, CD14-, CD18-, CD45-, and CD80 positive cells significantly decreased in late passages whereas the expression of CD44, CD86, MHC-I, MHC-II and ICAM-1 remained unchanged. DH82 cells represent a remarkably heterogeneous cell line with divergent antigenic and morphologic properties. The present findings have important implications for future studies, which should consider distinct characteristics with regard to the used passage. Copyright © 2014 Elsevier B.V. All rights reserved.

Prognostic value of CD8CD45RO tumor infiltrating lymphocytes in patients with extrahepatic cholangiocarcinoma

PubMed Central

Kim, Richard; Coppola, Domenico; Wang, Emilie; Chang, Young Doo; Kim, Yuhree; Anaya, Daniel; Kim, Dae Won

2018-01-01

Cholangiocarcinoma is a malignancy arising from the biliary tract epithelial cells with poor prognosis. Tumor infiltrating lymphocytes (TIL)s and programmed cell death receptor ligand 1 (PD-L1) have a prognostic impact in various solid tumors. We aimed to investigate TILs and PD-L1 expression and their clinical relevance in cholangiocarcinoma. Tumor samples from 44 patients with resected and histologically verified extrahepatic cholangiocarcinoma were evaluated for CD8, CD45RO and PD-L1 expression, and their correlations with clinicopathological data and survival data were analyzed. Total 44 extrahepatic cholangiocarcinoma tissues were evaluated. CD8+ tumor infiltrating lymphocytes (TIL)s were observed in 30 (68%) tumors. Among them, 14 had CD8+CD45RO+ TILs. PD-L1 was expressed on cancer cells in 10 (22.7%) tumors in 34 evaluable extrahepatic cholangiocarciniomas. The presence of CD8+ TILs or CD8+CD45RO+ TILs was not associated with clinical staging or tumor differentiation. Extrahepatic cholangiocarcinoma with CD8+CD45RO+ TILs had longer overall survival (OS) on univariate (P = 0.013) and multivariate (P = 0.012) analysis. Neither CD8+TIL nor PD-L1 expression on cancer cells correlated significantly with OS. These results add to the understanding of the clinical features associated with CD8 TILs and PD-L1 expression in extrahepatic cholangiocarcinoma, and they support the potential rationale of using PD-1 blockade immunotherapy in cholangiocarcinoma.

MET Signaling Mediates Intestinal Crypt-Villus Development, Regeneration, and Adenoma Formation and Is Promoted by Stem Cell CD44 Isoforms.

PubMed

Joosten, Sander P J; Zeilstra, Jurrit; van Andel, Harmen; Mijnals, R Clinton; Zaunbrecher, Joost; Duivenvoorden, Annet A M; van de Wetering, Marc; Clevers, Hans; Spaargaren, Marcel; Pals, Steven T

2017-10-01

Resistance of metastatic human colorectal cancer cells to drugs that block epidermal growth factor (EGF) receptor signaling could be caused by aberrant activity of other receptor tyrosine kinases, activating overlapping signaling pathways. One of these receptor tyrosine kinases could be MET, the receptor for hepatocyte growth factor (HGF). We investigated how MET signaling, and its interaction with CD44 (a putative MET coreceptor regulated by Wnt signaling and highly expressed by intestinal stem cells [ISCs] and adenomas) affects intestinal homeostasis, regeneration, and adenoma formation in mini-gut organoids and mice. We established organoid cultures from ISCs stimulated with HGF or EGF and assessed intestinal differentiation by immunohistochemistry. Mice with total epithelial disruption of MET (Ah Cre /Met fl/fl /LacZ) or ISC-specific disruption of MET (Lgr5 Creert2 /Met fl/fl /LacZ) and control mice (Ah Cre /Met +/+ /LacZ, Lgr5 Creert2 /Met +/+ /LacZ) were exposed to 10 Gy total body irradiation; intestinal tissues were collected, and homeostasis and regeneration were assessed by immunohistochemistry. We investigated adenoma organoid expansion stimulated by HGF or EGF using adenomas derived from Lgr5 Creert2 /Met fl/fl /Apc fl/fl and Lgr5 Creert2 /Met +/+ /Apc fl/fl mice. The same mice were evaluated for adenoma prevalence and size. We also quantified adenomas in Ah Cre /Met fl/fl /Apc fl/+ mice compared with Ah Cre /Met +/+ /Apc fl/+ control mice. We studied expansion of organoids generated from crypts and adenomas, stimulated by HGF or EGF, that were derived from mice expressing different CD44 splice variants (Cd44 +/+ , Cd44 -/- , Cd44 s/s , or Cd44 v4-10/v4-10 mice). Crypts incubated with EGF or HGF expanded into self-organizing mini-guts with similar levels of efficacy and contained all differentiated cell lineages. MET-deficient mice did not have defects in intestinal homeostasis. Total body irradiation reduced numbers of proliferating crypts in Ah Cre /Met fl/fl /LacZ mice. Lgr5 Creert2 /Met fl/fl /LacZ mice had impaired regeneration of MET-deficient ISCs. Adenoma organoids stimulated with EGF or HGF expanded to almost twice the size of nonstimulated organoids. MET-deficient adenoma organoids did not respond to HGF stimulation, but did respond to EGF. ISC-specific disruption of Met (Lgr5 Creert2 /Met fl/fl /Apc fl/fl mice) caused a twofold increase in apoptosis in microadenomas, resulting in an approximately 50% reduction of microadenoma numbers and significantly reduced average adenoma size. Total epithelial disruption of Met (Ah Cre /Met fl/fl /Apc fl/+ mice) resulted in an approximate 50% reduction in (micro)adenoma numbers. Intestinal crypts from Cd44 -/- mice did not expand to the same extent as crypts from Cd44 +/+ mice on stimulation with HGF, but had the same response to EGF. The negative effect on HGF-mediated growth was overcome by expression of CD44v4-10, but not by CD44s. Similarly, HGF-mediated expansion of adenoma organoids required CD44v4-10. In studies of intestinal organoid cultures and mice with inducible deletion of MET, we found HGF receptor signaling to regulate intestinal homeostasis and regeneration, as well as adenoma formation. These activities of MET are promoted by the stem cell CD44 isoform CD44v4-10. Our findings provide rationale for targeting signaling via MET and CD44 during anti-EGF receptor therapy of patients with colorectal cancer or in patients resistant to EGF receptor inhibitors. Copyright © 2017 AGA Institute. Published by Elsevier Inc. All rights reserved.

Cadmium-1,4-cyclohexanedicarboxylato coordination polymers bearing different di-alkyl-2,2'-bipyridines: syntheses, crystal structures and photoluminescence studies.

PubMed

Rosales-Vázquez, Luis D; Sánchez-Mendieta, Víctor; Dorazco-González, Alejandro; Martínez-Otero, Diego; García-Orozco, Iván; Morales-Luckie, Raúl A; Jaramillo-Garcia, Jonathan; Téllez-López, Antonio

2017-09-26

Four coordination polymers have been synthesized using self-assembly solution reactions under ambient conditions, reacting Cd(ii) ions with 1,4-cyclohexanedicarboxylic acid in the presence of different 2,2'-bipyridine co-ligands: {[Cd(H 2 O)(e,a-cis-1,4-chdc)(2,2'-bpy)]·H 2 O} n (1); [Cd 2 (H 2 O) 2 (e,a-cis-1,4-chdc) 2 (4,4'-dmb) 2 ] n (2); {[Cd(e,a-cis-1,4-chdc)(5,5'-dmb)]·H 2 O·CH 3 OH} n (3) and {[Cd(e,e-trans-1,4-chdc)(4,4'-dtbb)]·CH 3 OH} n (4), where 1,4-chdc = 1,4-cyclohexanedicarboxylato, 2,2'-bpy = 2,2'-bipyridine, 4,4'-dmb = 4,4'-dimethyl-2,2'-bipyridine, 5,5'-dmb = 5,5'-dimethyl-2,2'-bipyridine and 4,4'-dtbb = 4,4'-di-tert-butyl-2,2'-bipyridine. Crystallographic studies show that compound 1 has a 1D structure propagating along the crystallographic b-axis; the Cd ion in 1 is six-coordinated with a distorted-octahedral coordination sphere. Compound 2 has two crystallographic different Cd ions and both are six-coordinated with a distorted-octahedral coordination sphere. Compound 3 exhibits a seven-coordinated Cd ion having a distinctive distorted-monocapped trigonal prismatic geometry. In compound 4, the Cd ion is also seven-coordinated in a distorted monocapped octahedral geometry. Compounds 2, 3 and 4 possess rhombic-shaped dinuclear units (Cd 2 O 2 ) as nodes to generate larger cycles made up of four dinuclear units, a Cd 4 motif, bridged by four 1,4-chdc ligands, accomplishing, thus, 2D structures. Remarkably, in compound 4 the 1,4-chdc ligand conformation changes to the equatorial, equatorial trans, unlike the other compounds where the bridging ligand conformation is the more typical equatorial, axial cis. The solid state luminescence properties of 1-4 were investigated; polymers 3 and 4 exhibited a strong blue emission (λ em = 410-414 nm) compared to 1 and 2; structure-related photoluminescence is attributed to the degree of hydration of the compounds. Furthermore, Cd-polymer 3 suspended in acetone allows the fluorescence selective sensing of acetonitrile over common organic solvents such as alcohols and DMF, based on turn-on fluorescence intensity with a limit of 53 μmol L -1 .

[Analysis of expression of cancer stem cell-related markers in orbital adenoid cystic carcinoma].

PubMed

Lin, Ting-ting; Zhu, Li-min; He, Yan-jin; Zhang, Hong

2011-08-01

To observe the expression and distribution of CD44, CD133, and ABCG2 in orbital adenoid cystic carcinoma (ACC) and investigate their correlations with pathological type and prognosis. Two steps method of immunohistochemical staining was employed in 33 cases of paraffin embedded surgical specimens of human orbital ACC, 5 cases of recurrence samples, 3 cases of an excised lacrimal gland caused by neither inflammation nor tumor diseases, and 6 cases of xenograft tumors in nude mice. A retrospective analysis was performed on the clinical material of these patients, which were collected from Jan. 1991 to Mar. 2009. The positive rate of CD44 was 54.5% (18/33), with 76.9% (10/13) in solid type and 40.0% (8/20) in adeno-tubiform type. There was no statistically significant difference between them (P = 0.072). In solid type the positive expression cells were often located at the marginal part of the cancer nest. In the adeno-tubiform type, positive cells were often located at the outer layer of the tubiform structure (myoepithelial cells). CD44 was also expressed in normal tissues. The positive rate of CD133 was 57.6% (19/33), with 76.9% (10/13) in solid type and 45.0% (9/20) in adeno-tubiform type. There was no significant difference between them (P = 0.087). CD133 antigen was expressed in either the cytoplasm or nucleus, or expressed in both the cytoplasm and nucleus. The positive rate of ABCG2 was 21.2% (7/33), with 30.77% (4/13) in solid type and 15.0% (3/20) in adeno-tubiform type. There was no significant difference between them (P = 0.393). Many positive cells surrounded the vessels in tumor tissues. There were no significant differences between different prognosis groups of these surface phenotypes. The correlative analysis results of three surface phenotypes showed that CD44(+) cells have positive correlation with CD133(+) cells (Spearman, r(s) = 0.416, P = 0.016). In six transplanted tumors of nude mice, the number of positive cases for CD44(+), CD133(+) and ABCG2(+) was 1, 1 and 4 cases, respectively. The expression of CD44, CD133 and ABCG2 in ACC may influence the progress of ACC. However, they cannot be used as the markers for the evaluation of the prognosis of this tumor.

Delayed Expansion and Contraction of CD8+ T Cell Response during Infection with Virulent Salmonella typhimurium1

PubMed Central

Luu, Rachel A.; Gurnani, Komal; Dudani, Renu; Kammara, Rajagopal; van Faassen, Henk; Sirard, Jean-Claude; Krishnan, Lakshmi; Sad, Subash

2014-01-01

Ag presentation to CD8+ T cells often commences immediately after infection, which facilitates their rapid expansion and control of infection. Subsequently, the primed cells undergo rapid contraction. We report that this paradigm is not followed during infection with virulent Salmonella enterica, serovar Typhimurium (ST), an intracellular bacterium that replicates within phagosomes of infected cells. Although susceptible mice die rapidly (~7 days), resistant mice (129×1SvJ) harbor a chronic infection lasting ~60–90 days. Using rOVA-expressing ST (ST-OVA), we show that T cell priming is considerably delayed in the resistant mice. CD8+ T cells that are induced during ST-OVA infection undergo delayed expansion, which peaks around day 21, and is followed by protracted contraction. Initially, ST-OVA induces a small population of cycling central phenotype (CD62LhighIL-7RαhighCD44high) CD8+ T cells. However, by day 14–21, majority of the primed CD8+ T cells display an effector phenotype (CD62LlowIL-7RαlowCD44high). Subsequently, a progressive increase in the numbers of effector memory phenotype cells (CD62LlowIL-7RαhighCD44high) occurs. This differentiation program remained unchanged after accelerated removal of the pathogen with antibiotics, as majority of the primed cells displayed an effector memory phenotype even at 6 mo postinfection. Despite the chronic infection, CD8+ T cells induced by ST-OVA were functional as they exhibited killing ability and cytokine production. Importantly, even memory CD8+ T cells failed to undergo rapid expansion in response to ST-OVA infection, suggesting a delay in T cell priming during infection with virulent ST-OVA. Thus, phagosomal lifestyle may allow escape from host CD8+ T cell recognition, conferring a survival advantage to the pathogen. PMID:16849458

Disease severity in familial cases of IBD.

PubMed

Andreu, M; Márquez, L; Domènech, E; Gisbert, J P; García, V; Marín-Jiménez, I; Peñalva, M; Gomollón, F; Calvet, X; Merino, O; Garcia-Planella, E; Vázquez-Romero, N; Esteve, M; Nos, P; Gutiérrez, A; Vera, I; Cabriada, J L; Martín, M D; Cañas-Ventura, A; Panés, J

2014-03-01

Phenotypic traits of familial IBD relative to sporadic cases are controversial, probably related to limited statistical power of published evidence. To know if there are phenotype differences between familial and sporadic IBD, evaluating the prospective Spanish registry (ENEIDA) with 11,983 cases. 5783 patients (48.3%) had ulcerative colitis (UC) and 6200 (51.7%) Crohn's disease (CD). Cases with one or more 1st, 2nd or 3rd degree relatives affected by UC/CD were defined as familial case. In UC and CD, familial cases compared with sporadic cases had an earlier disease onset (UC: 33 years [IQR 25-44] vs 37 years [IQR 27-49]; p<0.0001); (CD: 27 years [IQR 21-35] vs 29 years [IQR 22-40]; p<0.0001), higher prevalence of extraintestinal immune-related manifestations (EIMs) (UC: 17.2% vs 14%; p=0.04); (CD: 30.1% vs 23.6%; p<0.0001). Familial CD had higher percentage of ileocolic location (42.7% vs 51.8%; p=0.0001), penetrating behavior (21% vs 17.6%; p=0.01) and perianal disease (32% vs 27.1%; p=0.003). Differences are not influenced by degree of consanguinity. When a sufficiently powered cohort is evaluated, familial aggregation in IBD is associated to an earlier disease onset, more EIMs and more severe phenotype in CD. This feature should be taken into account at establishing predictors of disease course. © 2013.

Establishment and Characterization of Immortalized Human Amniotic Epithelial Cells

PubMed Central

Zhou, Kaixuan; Koike, Chika; Yoshida, Toshiko; Okabe, Motonori; Fathy, Moustafa; Kyo, Satoru; Kiyono, Tohru; Saito, Shigeru

2013-01-01

Abstract Human amniotic epithelial cells (HAEs) have a low immunogenic profile and possess potent immunosuppressive properties. HAEs also have several characteristics similar to stem cells, and they are discarded after parturition. Thus, they could potentially be used in cell therapy with fewer ethical problems. HAEs have a short life, so our aim is to establish and characterize immortalized human amniotic epithelial cells (iHAEs). HAEs were introduced with viral oncogenes E6/E7 and with human telomerase reverse transcriptase (hTERT) to create iHAEs. These iHAEs have proliferated around 200 population doublings (PDs) for at least 12 months. High expression of stem cell markers (Oct 3/4, Nanog, Sox2, Klf4) and epithelial markers (CK5, CK18) were detected by immunohistochemistry and reverse transcription polymerase chain reaction (RT-PCR). These iHAEs were expanded in ultra-low-attachment dishes to form spheroids similarly to epithelial stem/precursor cells. High expression of mesenchymal (CD44, CD73, CD90, CD105) and somatic (CD24, CD29, CD271, Nestin) stem cell markers was detected by flow cytometry. The iHAEs showed adipogenic, osteogenic, neuronal, and cardiac differentiation abilities. In conclusion, the immortalization of HAEs with the characteristics of stem cells has been established, allowing these iHAEs to become useful for cell therapy and regenerative medicine. PMID:23298399

CD10/neutral endopeptidase 24.11 in developing human fetal lung. Patterns of expression and modulation of peptide-mediated proliferation.

PubMed

Sunday, M E; Hua, J; Torday, J S; Reyes, B; Shipp, M A

1992-12-01

The cell membrane-associated enzyme CD10/neutral endopeptidase 24.11 (CD10/NEP) functions in multiple organ systems to downregulate responses to peptide hormones. Recently, CD10/NEP was found to hydrolyze bombesin-like peptides (BLP), which are mitogens for normal bronchial epithelial cells and small cell lung carcinomas. Growth of BLP-responsive small cell lung carcinomas was potentiated by CD10/NEP inhibition, implicating CD10/NEP in regulation of BLP-mediated tumor growth. BLP are also likely to participate in normal lung development because high BLP levels are found in fetal lung, and bombesin induces proliferation and maturation of human fetal lung in organ cultures and murine fetal lung in utero. To explore potential roles for CD10/NEP in regulating peptide-mediated human fetal lung development, we have characterized temporal and cellular patterns of CD10/NEP expression and effects of CD10/NEP inhibition in organ cultures. Peak CD10/NEP transcript levels are identified at 11-13 wk gestation by Northern blots and localized to epithelial cells and mesenchyme of developing airways by in situ hybridization. CD10/NEP immunostaining is most intense in undifferentiated airway epithelium. In human fetal lung organ cultures, inhibition of CD10/NEP with either phosphoramidon or SCH32615 increases thymidine incorporation by 166-182% (P < 0.025). The specific BLP receptor antagonist, [Leu13-psi(CH2NH)Leu14]bombesin abolishes these effects on fetal lung growth, suggesting that CD10/NEP modulates BLP-mediated proliferation. CD10/NEP expression in the growing front of airway epithelium and the effects of CD10/NEP inhibitors in lung explants implicate the enzyme in the regulation of peptide-mediated fetal lung growth.

Anti-CD22/CD20 Bispecific antibody with enhanced trogocytosis for treatment of Lupus.

PubMed

Rossi, Edmund A; Chang, Chien-Hsing; Goldenberg, David M

2014-01-01

The humanized anti-CD22 antibody, epratuzumab, has demonstrated therapeutic activity in clinical trials of lymphoma, leukemia and autoimmune diseases, treating currently over 1500 cases of non-Hodgkin lymphoma, acute lymphoblastic leukemias, Waldenström's macroglobulinemia, Sjögren's syndrome, and systemic lupus erythematosus. Because epratuzumab reduces on average only 35% of circulating B cells in patients, and has minimal antibody-dependent cellular cytotoxicity and negligible complement-dependent cytotoxicity when evaluated in vitro, its therapeutic activity may not result completely from B-cell depletion. We reported recently that epratuzumab mediates Fc/FcR-dependent membrane transfer from B cells to effector cells via trogocytosis, resulting in a substantial reduction of multiple BCR modulators, including CD22, CD19, CD21, and CD79b, as well as key cell adhesion molecules, including CD44, CD62L, and β7 integrin, on the surface of B cells in peripheral blood mononuclear cells obtained from normal donors or SLE patients. Rituximab has clinical activity in lupus, but failed to achieve primary endpoints in a Phase III trial. This is the first study of trogocytosis mediated by bispecific antibodies targeting neighboring cell-surface proteins, CD22, CD20, and CD19, as demonstrated by flow cytometry and immunofluorescence microscopy. We show that, compared to epratuzumab, a bispecific hexavalent antibody comprising epratuzumab and veltuzumab (humanized anti-CD20 mAb) exhibits enhanced trogocytosis resulting in major reductions in B-cell surface levels of CD19, CD20, CD21, CD22, CD79b, CD44, CD62L and β7-integrin, and with considerably less immunocompromising B-cell depletion that would result with anti-CD20 mAbs such as veltuzumab or rituximab, given either alone or in combination with epratuzumab. A CD22/CD19 bispecific hexavalent antibody, which exhibited enhanced trogocytosis of some antigens and minimal B-cell depletion, may also be therapeutically useful. The bispecific antibody is a candidate for improved treatment of lupus and other autoimmune diseases, offering advantages over administration of the two parental antibodies in combination.

Comparative Gene Expression Profiling of Primary and Metastatic Renal Cell Carcinoma Stem Cell-Like Cancer Cells

PubMed Central

Czarnecka, Anna M.; Lewicki, Sławomir; Helbrecht, Igor; Brodaczewska, Klaudia; Koch, Irena; Zdanowski, Robert; Król, Magdalena; Szczylik, Cezary

2016-01-01

Background Recent advancement in cancer research has shown that tumors are highly heterogeneous, and multiple phenotypically different cell populations are found in a single tumor. Cancer development and tumor growth are driven by specific types of cells—stem cell-like cancer cells (SCLCCs)—which are also responsible for metastatic spread and drug resistance. This research was designed to verify the presence of SCLCCs in renal cell cancer cell lines. Subsequently, we aimed to characterize phenotype and cell biology of CD105+ cells, defined previously as renal cell carcinoma tumor-initiating cells. The main goal of the project was to describe the gene-expression profile of stem cell-like cancer cells of primary tumor and metastatic origin. Materials and Methods Real-time PCR analysis of stemness genes (Oct-4, Nanog and Ncam) and soft agar colony formation assay were conducted to check the stemness properties of renal cell carcinoma (RCC) cell lines. FACS analysis of CD105+ and CD133+ cells was performed on RCC cells. Isolated CD105+ cells were verified for expression of mesenchymal markers—CD24, CD146, CD90, CD73, CD44, CD11b, CD19, CD34, CD45, HLA-DR and alkaline phosphatase. Hanging drop assay was used to investigate CD105+ cell-cell cohesion. Analysis of free-floating 3D spheres formed by isolated CD105+ was verified, as spheres have been hypothesized to contain undifferentiated multipotent progenitor cells. Finally, CD105+ cells were sorted from primary (Caki-2) and metastatic (ACHN) renal cell cancer cell lines. Gene-expression profiling of sorted CD105+ cells was performed with Agilent’s human GE 4x44K v2 microarrays. Differentially expressed genes were further categorized into canonical pathways. Network analysis and downstream analysis were performed with Ingenuity Pathway Analysis. Results Metastatic RCC cell lines (ACHN and Caki-1) demonstrated higher colony-forming ability in comparison to primary RCC cell lines. Metastatic RCC cell lines harbor numerous CD105+ cell subpopulations and have higher expression of stemness genes (Oct-4 and Nanog). CD105+ cells adopt 3D grape-like floating structures under handing drop conditions. Sorted CD105+ cells are positive for human mesenchymal stem cell (MSC) markers CD90, CD73, CD44, CD146, and alkaline phosphatase activity, but not for CD24 and hematopoietic lineage markers CD34, CD11b, CD19, CD45, and HLA-DR. 1411 genes are commonly differentially expressed in CD105+ cells (both from primary [Caki-2] and metastatic RCC [ACHN] cells) in comparison to a healthy kidney epithelial cell line (ASE-5063). TGF-β, Wnt/β-catenine, epithelial-mesenchymal transition (EMT), Rap1 signaling, PI3K-Akt signaling, and Hippo signaling pathway are deregulated in CD105+ cells. TGFB1, ERBB2, and TNF are the most significant transcriptional regulators activated in these cells. Conclusions All together, RCC-CD105+ cells present stemlike properties. These stem cell-like cancer cells may represent a novel target for therapy. A unique gene-expression profile of CD105+ cells could be used as initial data for subsequent functional studies and drug design. PMID:27812180

The Prognostic Role of Cancer Stem Cell Markers for Long-term Outcome After Resection of Colonic Liver Metastases.

PubMed

Spelt, Lidewij; Sasor, Agata; Ansari, Daniel; Hilmersson, Katarzyna Said; Andersson, Roland

2018-01-01

To assess the expression of cancer stem cell (CSC) markers CD44, CD133 and CD24 in colon cancer liver metastases and analyse their predictive value for overall survival (OS) and disease-free survival (DFS) after liver resection. Patients operated on for colon cancer liver metastases were included. CSC marker expression was determined through immunohistochemistry analysis. OS and DFS were compared between marker-positive and marker-negative patients. Multivariate analysis was performed to select predictive variables for OS and DFS. CD133-positive patients had a worse DFS than CD133-negative patients, with a median DFS of 12 and 25 months (p=0.051). Multivariate analysis selected CD133 expression as a significant predictor for DFS. CD44 and CD24 were not found to predict OS or DFS. CD133 expression in colonic liver metastases is a negative prognostic factor for DFS after liver resection. In the future, CD133 could be used as a biomarker for risk stratification, and possibly for developing novel targeted therapy. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Restricted L1 Estimators and Their Covariances,

DTIC Science & Technology

1980-06-01

CD22 w =e 1-.< l’ -- I -M Ce -=, -2 C 22 CD -. 00 CD LJ L":v, wD .-C >-C CD U., -D -a 22J CD: CD. Vt. U.1 . CD* 31- a UOC CD I" -J zI == CŖ - 1- InCC.J...C 22 CC:.: 2 15Cb In0’C C 4 t’ 3 CD.2-cm I- I.- iC2 CD-3SJ U. cc CDam CC 22 0.-J~ I- iD C Me CC .JJ . A.2 Dt-2C. 2. D ~ 2 CD, Z-i- C -J 2Cn LI CD22 ...22 "I.- bII CD 0.-..CW I -- k- -o. CDCD-c =DDCCC-ce CDI- 22eCCDC.C I-L- 5 2wI-CDUct. I4.CDgCD Ci L., 2=9 E Mm t,2 cm CD22 ,,- D.-.2RCC Ce:CD .Cc n 9

Novel strategies targeting cancer stem cells through phytochemicals and their analogs

PubMed Central

Dandawate, Prasad; Padhye, Subhash; Ahmad, Aamir

2013-01-01

Cancer stem cells (CSCs) are cells that exist within a tumor with a capacity of self-renewal and an ability to differentiate, giving rise to heterogeneous populations of cancer cells. These cells are increasingly being implicated in resistance to conventional therapeutics and have also been implicated in tumor recurrence. Several cellular signaling pathways including Notch, Wnt, phosphoinositide-3-kinase–Akt–mammalian target of rapamycin pathways, and known markers such as CD44, CD133, CD166, ALDH, etc. have been associated with CSCs. Here, we have reviewed our current understanding of self-renewal pathways and factors that help in the survival of CSCs with special emphasis on those that have been documented to be modulated by well characterized natural agents such as curcumin, sulforaphane, resveratrol, genistein, and epigallocatechin gallate. With the inclusion of a novel derivative of curcumin, CDF, we showcase how natural agents can be effectively modified to increase their efficacy, particularly against CSCs. We hope that this article will generate interest among researchers for further mechanistic and clinical studies exploiting the cancer preventive and therapeutic role of nutraceuticals by targeted elimination of CSCs. PMID:24076568

A tissue-engineered subcutaneous pancreatic cancer model for antitumor drug evaluation.

PubMed

He, Qingyi; Wang, Xiaohui; Zhang, Xing; Han, Huifang; Han, Baosan; Xu, Jianzhong; Tang, Kanglai; Fu, Zhiren; Yin, Hao

2013-01-01

The traditional xenograft subcutaneous pancreatic cancer model is notorious for its low incidence of tumor formation, inconsistent results for the chemotherapeutic effects of drug molecules of interest, and a poor predictive capability for the clinical efficacy of novel drugs. These drawbacks are attributed to a variety of factors, including inoculation of heterogeneous tumor cells from patients with different pathological histories, and use of poorly defined Matrigel(®). In this study, we aimed to tissue-engineer a pancreatic cancer model that could readily cultivate a pancreatic tumor derived from highly homogenous CD24(+)CD44(+) pancreatic cancer stem cells delivered by a well defined electrospun scaffold of poly(glycolide-co-trimethylene carbonate) and gelatin. The scaffold supported in vitro tumorigenesis from CD24(+)CD44(+) cancer stem cells for up to 7 days without inducing apoptosis. Moreover, CD24(+)CD44(+) cancer stem cells delivered by the scaffold grew into a native-like mature pancreatic tumor within 8 weeks in vivo and exhibited accelerated tumorigenesis as well as a higher incidence of tumor formation than the traditional model. In the scaffold model, we discovered that oxaliplatin-gemcitabine (OXA-GEM), a chemotherapeutic regimen, induced tumor regression whereas gemcitabine alone only capped tumor growth. The mechanistic study attributed the superior antitumorigenic performance of OXA-GEM to its ability to induce apoptosis of CD24(+)CD44(+) cancer stem cells. Compared with the traditional model, the scaffold model demonstrated a higher incidence of tumor formation and accelerated tumor growth. Use of a tiny population of highly homogenous CD24(+)CD44(+) cancer stem cells delivered by a well defined scaffold greatly reduces the variability associated with the traditional model, which uses a heterogeneous tumor cell population and poorly defined Matrigel. The scaffold model is a robust platform for investigating the antitumorigenesis mechanism of novel chemotherapeutic drugs with a special focus on cancer stem cells.

Production of Pigs by Hand-Made Cloning Using Mesenchymal Stem Cells and Fibroblasts.

PubMed

Yang, Zhenzhen; Vajta, Gábor; Xu, Ying; Luan, Jing; Lin, Mufei; Liu, Cong; Tian, Jianing; Dou, Hongwei; Li, Yong; Liu, Tianbin; Zhang, Yijie; Li, Lin; Yang, Wenxian; Bolund, Lars; Yang, Huanming; Du, Yutao

2016-08-01

Mesenchymal stem cells (MSCs) exhibited self-renewal and less differentiation, making the MSCs promising candidates for adult somatic cell nuclear transfer (SCNT). In this article, we tried to produce genome identical pigs through hand-made cloning (HMC), with MSCs and adult skin fibroblasts as donor cells. MSCs were derived from either adipose tissue or peripheral blood (aMSCs and bMSCs, respectively). MSCs usually showed the expression pattern of CD29, CD73, CD90, and CD105 together with lack of expression of the hematopoietic markers CD34and CD45. Flow cytometry results demonstrated high expression of CD29 and CD90 in both MSC lines, while CD73, CD34, and CD45 expression were not detected. In contrary, in reverse transcription-polymerase chain reaction (RT-PCR) analysis, CD73 and CD34 were detected indicating that human antibodies CD73 and CD34 were not suitable to identify porcine cell surface markers and porcine MSC cellular surface markers of CD34 might be different from other species. MSCs also had potential to differentiate successfully into chondrocytes, osteoblasts, and adipocytes. After HMC, embryos reconstructed with aMSCs had higher blastocyst rate on day 5 and 6 than those reconstructed with bMSCs and fibroblasts (29.6% ± 1.3% and 41.1% ± 1.4% for aMSCs vs. 23.9% ± 1.2% and 35.5% ± 1.6% for bMSCs and 22.1% ± 0.9% and 33.3% ± 1.1% for fibroblasts, respectively). Live birth rate per transferred blastocyst achieved with bMSCs (1.59%) was the highest among the three groups. This article was the first report to compare the efficiency among bMSCs, aMSCs, and fibroblasts for boar cloning, which offered a realistic perspective to use the HMC technology for commercial breeding.

Downregulation of CD147 expression by RNA interference inhibits HT29 cell proliferation, invasion and tumorigenicity in vitro and in vivo.

PubMed

Li, Rui; Pan, Yuqin; He, Bangshun; Xu, Yeqiong; Gao, Tianyi; Song, Guoqi; Sun, Huiling; Deng, Qiwen; Wang, Shukui

2013-12-01

We investigated the effect of CD147 silencing on HT29 cell proliferation and invasion. We constructed a novel short hairpin RNA (shRNA) expression vector pYr-mir30-shRNA. The plasmid was transferred to HT29 cells. The expression of CD147, MCT1 (lactate transporters monocarboxylate transporter 1) and MCT4 (lactate transporters monocarboxylate transporter 4) were monitored by quantitative PCR and western blotting, respectively. The MMP-2 (matrix metalloproteinase-2) and MMP-9 (matrix metalloproteinase-9) activities were determined by gelatin zymography assay, while the intracellular lactate concentration was determined by the lactic acid assay kit. WST-8 assay was used to determine the HT29 cell proliferation and the chemosensitivity. Invasion assay was used to determine the invasion of HT29 cells. In addition, we established a colorectal cancer model, and detected CD147 expression in vivo. The results showed that the expression of CD147 and MCT1 was significantly reduced at both mRNA and protein levels, and also the activity of MMP-2 and MMP-9 was reduced. The proliferation and invasion were decreased, but chemosensitivity to cisplatin was increased. In vivo, the CD147 expression was also significantly decreased, and reduced the tumor growth after CD147 gene silencing. The results demonstrated that silencing of CD147 expression inhibited the proliferation and invasion, suggesting CD147 silencing might be an adjuvant gene therapy strategy to chemotherapy.

CD4 Lymphocyte Count: MedlinePlus Lab Test Information

MedlinePlus

... Clinics Authority; c2017. CD4+ Count Results [updated 2017 Mar 3; cited 2017 Nov 29]; [about 7 screens]. ... Authority; c2017. CD4+ Count Test Overview [updated 2017 Mar 3; cited 2017 Nov 29]; [about 2 screens]. ...

Characterization of kidney CD45intCD11bintF4/80+MHCII+CX3CR1+Ly6C- "intermediate mononuclear phagocytic cells".

PubMed

Lee, Sul A; Noel, Sanjeev; Sadasivam, Mohanraj; Allaf, Mohamad E; Pierorazio, Phillip M; Hamad, Abdel R A; Rabb, Hamid

2018-01-01

Kidney immune cells play important roles in pathogenesis of many diseases, including ischemia-reperfusion injury (IRI) and transplant rejection. While studying murine kidney T cells, we serendipitously identified a kidney mononuclear phagocytic cell (MPC) subset characterized by intermediate surface expression of CD45 and CD11b. These CD45intCD11bint MPCs were further identified as F4/80+MHCII+CX3CR1+Ly6C- cells, comprising ~17% of total CD45+ cells in normal mouse kidney (P < 0.01) and virtually absent from all other organs examined except the heart. Systemic clodronate treatment had more significant depletive effect on the CD45intCD11bint population (77.3%±5.9%, P = 0.03) than on CD45highCD11b+ population (14.8%±16.6%, P = 0.49). In addition, CD45intCD11bint MPCs had higher phagocytic function in the normal kidney (35.6%±3.3% vs. 24.1%±2.2%, P = 0.04), but lower phagocytic capacity in post-ischemic kidney (54.9%±1.0% vs. 67.8%±1.9%, P < 0.01) compared to the CD45highCD11b+ population. Moreover, the CD45intCD11bint population had higher intracellular production of the pro-inflammatory tumor necrosis factor (TNF)-α (58.4%±5.2% vs. 27.3%±0.9%, P < 0.001) after lipopolysaccharide (LPS) stimulation and lower production of the anti-inflammatory interleukin (IL)-10 (7.2%±1.3% vs. 14.9%±2.2%, P = 0.02) following kidney IRI, suggesting a functional role under inflammatory conditions. The CD45intCD11bint cells increased early after IRI, and then abruptly decreased 48h later, whereas CD45highCD11b+ cells steadily increased after IRI before declining at 72h (P = 0.03). We also identified the CD45intCD11bint MPC subtype in human kidney. We conclude that CD45intCD11bint F4/80+MHCII+CX3CR1+Ly6C-population represent a unique subset of MPCs found in both mouse and human kidneys. Future studies will further characterize their role in kidney health and disease.

Immortalized porcine mesenchymal cells derived from nasal mucosa, lungs, lymph nodes, spleen and bone marrow retain their stemness properties and trigger the expression of siglec-1 in co-cultured blood monocytic cells

PubMed Central

Garba, Abubakar; Desmarets, Lowiese M. B.; Acar, Delphine D.; Devriendt, Bert; Nauwynck, Hans J.

2017-01-01

Mesenchymal stromal cells have been isolated from different sources. They are multipotent cells capable of differentiating into many different cell types, including osteocytes, chondrocytes and adipocytes. They possess a therapeutic potential in the management of immune disorders and the repair of damaged tissues. Previous work in our laboratory showed an increase of the percentages of CD172a+, CD14+, CD163+, Siglec-1+, CD4+ and CD8+ hematopoietic cells, when co-cultured with immortalized mesenchymal cells derived from bone marrow. The present work aimed to demonstrate the stemness properties of SV40-immortalized mesenchymal cells derived from nasal mucosa, lungs, spleen, lymph nodes and red bone marrow and their immunomodulatory effect on blood monocytes. Mesenchymal cells from nasal mucosa, lungs, spleen, lymph nodes and red bone marrow were isolated and successfully immortalized using simian virus 40 large T antigen (SV40LT) and later, co-cultured with blood monocytes, in order to examine their differentiation stage (expression of Siglec-1). Flow cytometric analysis revealed that the five mesenchymal cell lines were positive for mesenchymal cell markers CD105, CD44, CD90 and CD29, but lacked the expression of myeloid cell markers CD16 and CD11b. Growth analysis of the cells demonstrated that bone marrow derived-mesenchymal cells proliferated faster compared with those derived from the other tissues. All five mesenchymal cell lines co-cultured with blood monocytes for 1, 2 and 7 days triggered the expression of siglec-1 in the monocytes. In contrast, no siglec-1+ cells were observed in monocyte cultures without mesenchymal cell lines. Mesenchymal cells isolated from nasal mucosa, lungs, spleen, lymph nodes and bone marrow were successfully immortalized and these cell lines retained their stemness properties and displayed immunomodulatory effects on blood monocytes. PMID:29036224

Immortalized porcine mesenchymal cells derived from nasal mucosa, lungs, lymph nodes, spleen and bone marrow retain their stemness properties and trigger the expression of siglec-1 in co-cultured blood monocytic cells.

PubMed

Garba, Abubakar; Desmarets, Lowiese M B; Acar, Delphine D; Devriendt, Bert; Nauwynck, Hans J

2017-01-01

Mesenchymal stromal cells have been isolated from different sources. They are multipotent cells capable of differentiating into many different cell types, including osteocytes, chondrocytes and adipocytes. They possess a therapeutic potential in the management of immune disorders and the repair of damaged tissues. Previous work in our laboratory showed an increase of the percentages of CD172a+, CD14+, CD163+, Siglec-1+, CD4+ and CD8+ hematopoietic cells, when co-cultured with immortalized mesenchymal cells derived from bone marrow. The present work aimed to demonstrate the stemness properties of SV40-immortalized mesenchymal cells derived from nasal mucosa, lungs, spleen, lymph nodes and red bone marrow and their immunomodulatory effect on blood monocytes. Mesenchymal cells from nasal mucosa, lungs, spleen, lymph nodes and red bone marrow were isolated and successfully immortalized using simian virus 40 large T antigen (SV40LT) and later, co-cultured with blood monocytes, in order to examine their differentiation stage (expression of Siglec-1). Flow cytometric analysis revealed that the five mesenchymal cell lines were positive for mesenchymal cell markers CD105, CD44, CD90 and CD29, but lacked the expression of myeloid cell markers CD16 and CD11b. Growth analysis of the cells demonstrated that bone marrow derived-mesenchymal cells proliferated faster compared with those derived from the other tissues. All five mesenchymal cell lines co-cultured with blood monocytes for 1, 2 and 7 days triggered the expression of siglec-1 in the monocytes. In contrast, no siglec-1+ cells were observed in monocyte cultures without mesenchymal cell lines. Mesenchymal cells isolated from nasal mucosa, lungs, spleen, lymph nodes and bone marrow were successfully immortalized and these cell lines retained their stemness properties and displayed immunomodulatory effects on blood monocytes.

Hyaluronan Tumor Cell Interactions in Prostate Cancer Growth and Survival

DTIC Science & Technology

2006-12-01

prognostic value of CD44 standard and variant v3 and v6 isoforms in prostate cancer. Eur Urol, 2001. 39(2): p. 138-44. 32. De Marzo , A.M., et al., CD44...subcutaneous injection model [ 24 ]and in orthotopic or intrafemoral bone injection models (see progress report below). Importantly, the addition of...expression from these cells, completely reverses growth inhibition[ 24 ]. CD44 and Rhamm – Two Hyaladherins with Overlapping Function: The two most

Cancer stem cell markers in patterning differentiation and in prognosis of oral squamous cell carcinoma.

PubMed

Mohanta, Simple; Siddappa, Gangotri; Valiyaveedan, Sindhu Govindan; Dodda Thimmasandra Ramanjanappa, Ravindra; Das, Debashish; Pandian, Ramanan; Khora, Samanta Sekhar; Kuriakose, Moni Abraham; Suresh, Amritha

2017-06-01

Differentiation is a major histological parameter determining tumor aggressiveness and prognosis of the patient; cancer stem cells with their slow dividing and undifferentiated nature might be one of the factors determining the same. This study aims to correlate cancer stem cell markers (CD44 and CD147) with tumor differentiation and evaluate their subsequent effect on prognosis. Immunohistochemical analysis in treatment naïve oral cancer patients (n = 53) indicated that the expression of CD147 was associated with poorly differentiated squamous cell carcinoma and moderately differentiated squamous cell carcinoma (p < 0.01). Furthermore, co-expression analysis showed that 45% each of moderately differentiated squamous cell carcinoma and poorly differentiated squamous cell carcinoma patients were CD44 high /CD147 high as compared to only 10% of patients with well-differentiated squamous cell carcinoma. A three-way analysis indicated that differentiation correlated with recurrence and survival (p < 0.05) in only the patients with CD44 high /CD147 high cohort. Subsequently, relevance of these cancer stem cell markers in patterning the differentiation characteristics was evaluated in oral squamous cell carcinoma cell lines originating from different grades of oral cancer. Flowcytometry-based analysis indicated an increase in CD44 + /CD147 + cells in cell lines of poorly differentiated squamous cell carcinoma (94.35 ± 1.14%, p < 0.001) and moderately differentiated squamous cell carcinoma origin (93.49 ± 0.47%, p < 0.001) as compared to cell line of well-differentiated squamous cell carcinoma origin (23.12% ± 0.49%). Expression profiling indicated higher expression of cancer stem cell and epithelial-mesenchymal transition markers in SCC029B (poorly differentiated squamous cell carcinoma originated; p ≤ 0.001), which was further translated into increased spheroid formation, migration, and invasion (p < 0.001) as compared to cell line of well-differentiated squamous cell carcinoma origin. This study suggests that CD44 and CD147 together improve the prognostic efficacy of tumor differentiation; in vitro results further point out that these markers might be determinant of differentiation characteristics, imparting properties of increased self-renewal, migration, and invasion.

Construction of Hyaluronic Tetrasaccharide Clusters Modified Polyamidoamine siRNA Delivery System.

PubMed

Ma, Yingcong; Sha, Meng; Cheng, Shixuan; Yao, Wang; Li, Zhongjun; Qi, Xian-Rong

2018-06-14

The CD44 protein, as a predominant receptor for hyaluronan (HA), is highly expressed on the surface of multiple tumor cells. HA, as a targeting molecule for a CD44-contained delivery system, increases intracellular drug concentration in tumor tissue. However, due to the weak binding ability of hyaluronan oligosaccharide to CD44, targeting for tumor drug delivery has been restricted. In this study, we first use a HA tetrasaccharide cluster as the target ligand to enhance the binding ability to CD44. A polyamidoamine (PAMAM) dendrimer was modified by a HA tetrasaccharide cluster as a nonviral vector for small interfering RNA (siRNA) delivery. The dendrimer/siRNA nanocomplexes increased the cellular uptake capacity of siRNA through the CD44 receptor-mediated endocytosis pathway, allowing the siRNA to successfully escape the endosome/lysosome. Compared with the control group, nanocomplexes effectively reduced the expression of GFP protein and mRNA in MDA-MB-231-GFP cells. This delivery system provides a foundation to increase the clinical applications of PAMAM nanomaterials.

Selective Resistance of CD44hi T Cells to p53 Dependent Cell Death Results in Persistence of Immunologic Memory after Total Body Irradiation1,2,3

PubMed Central

Yao, Zhenyu; Jones, Jennifer; Kohrt, Holbrook; Strober, Samuel

2011-01-01

Our previous studies showed that treatment of mice with total body irradiation (TBI) or total lymphoid tissue irradiation (TLI) markedly changes the balance of residual T cell subsets to favor CD4+CD44hi natural killer T (NKT) cells due to differential resistance of the latter subset to cell death. The object of the current study was to further elucidate the changed balance and mechanisms of differential radioresistance of T cell subsets after graded doses of TBI. The experimental results show that CD4+ T cells were markedly more resistant than CD8+ T cells, and CD44hi T cells including NKT cells and memory T cells were markedly more resistant than CD44lo (naïve) T cells. The memory T cells immunized to alloantigens persisted even after myeloabloative (1,000cGy) TBI, and were able to prevent engraftment of bone marrow transplants. Although T cell death after 1,000cGy was prevented in p53−/− mice, there was progressive T cell death in p53−/− mice at higher doses. Whereas, p53 dependent T cell death changed the balance of subsets, the p53 independent T cell death did not. In conclusion, resistance of CD44hi T cells to p53 dependent cell death results in the persistence of immunological memory after TBI, and can explain the immune mediated rejection of marrow transplants in sensitized recipients. PMID:21930972

NKG2D and CD94 bind to heparin and sulfate-containing polysaccharides.

PubMed

Higai, Koji; Imaizumi, Yuzo; Suzuki, Chiho; Azuma, Yutaro; Matsumoto, Kojiro

2009-09-04

Killer lectin-like receptors NKG2D and CD94 on natural killer cells trigger cytotoxicity through binding of glycans on target cells including sialyl Lewis X antigen. We previously reported that NKG2D and CD94 recognize alpha2,3-linked NeuAc on multi-antennary N-glycans. Here we further investigated polysaccharide binding by these receptors, using glutathione-S-transferase-fused extracellular domains of NKG2D AA 73-216 (rNKG2Dlec) and CD94 AA 68-179 (rCD94lec). We found that rNKG2Dlec and rCD94lec bind in a dose-dependent manner to plates coated with heparin-conjugated bovine serum albumin (heparin-BSA). Binding to heparin-BSA was suppressed by soluble sulfate-containing polysaccharides, but minimally impacted by 2-O-, 6-O-, and 2-N-desulfated heparin. Mutagenesis revealed that (152)Y and (199)Y of NKG2D and (144)F, (160)N, and (166)C of CD94 were critical for binding to heparin-BSA. The present manuscript provides the first evidence that NKG2D and CD94 bind to heparin and sulfate-containing polysaccharides.

Effects of baicalin in CD4 + CD29 + T cell subsets of ulcerative colitis patients

PubMed Central

Yu, Feng-Yan; Huang, Shao-Gang; Zhang, Hai-Yan; Ye, Hua; Chi, Hong-Gang; Zou, Ying; Lv, Ru-Xi; Zheng, Xue-Bao

2014-01-01

AIM: To evaluate the role of baicalin in ulcerative colitis (UC) with regard to the CD4+CD29+ T helper cell, its surface markers and serum inflammatory cytokines. METHODS: Flow cytometry was used to detect the percentage of CD4+CD29+ cells in patients with UC. Real time polymerase chain reaction was used to detect expression of GATA-3, forkhead box P3, T-box expressed in T cells (T-bet), and retinoic acid-related orphan nuclear hormone receptor C (RORC). Western blotting was used to analyze expression of nuclear factor-κB (NF-κB) p65, phosphorylation of NF-κB (p-NF-κB) p65, STAT4, p-STAT4, STAT6 and p-STAT6. The concentrations of interferon-γ (IFN-γ), interleukin (IL)-4, IL-5, IL-6, IL-10 and TGF-β in serum were determined by ELISA assay. RESULTS: The percentages of CD4+CD29+ T cells were lower in treatment with 40 and 20 μmol/L baicalin than in the treatment of no baicalin. Treatment with 40 or 20 μmol/L baicalin significantly upregulated expression of IL-4, TGF-β1 and IL-10, increased p-STAT6/STAT6 ratio, but downregulated expression of IFN-γ, IL-5, IL-6, RORC, Foxp3 and T-bet, and decreased ratios of T-bet/GATA-3, p-STAT4/STAT4 and p-NF-κB/NF-κB compared to the treatment of no baicalin. CONCLUSION: The results indicate that baicalin regulates immune balance and relieves the ulcerative colitis-induced inflammation reaction by promoting proliferation of CD4+CD29+ cells and modulating immunosuppressive pathways. PMID:25386078

Preparation of Cytokine-activated NK Cells for Use in Adoptive Cell Therapy in Cancer Patients: Protocol Optimization and Therapeutic Potential.

PubMed

van Ostaijen-ten Dam, Monique M; Prins, Henk-Jan; Boerman, Gerharda H; Vervat, Carly; Pende, Daniela; Putter, Hein; Lankester, Arjan; van Tol, Maarten J D; Zwaginga, Jaap J; Schilham, Marco W

2016-01-01

Cell-based immunotherapy using donor-derived natural killer (NK) cells after allogeneic hematopoietic stem cell transplantation may be an attractive treatment of residual leukemia. This study aimed to optimize clinical grade production of a cytokine-activated NK-cell product. NK cells were isolated either by double depletion (CD3(-), CD19(-)) or by sequential depletion and enrichment (CD3(-,) CD56(+)) via CliniMACS from leukapheresis material and cultured in vitro with interleukin (IL)-2 or IL-15. Both NK cell isolation procedures yielded comparable recovery of NK cells and levels of T-cell contamination. After culture with cytokines, the CD3(-)CD56(+) procedure resulted in NK cells of higher purity, that is, less T cells and monocytes, higher viability, and a slightly higher yield than the CD3(-)CD19- procedure. CD69, NKp44, and NKG2A expression were higher on CD3(-)CD56(+) products, whereas lysis of Daudi cells was comparable. Five days of culture led to higher expression of CD69, NKp44, and NKp30 and lysis of K562 and Daudi cell lines. Although CD69 expression and lysis of Daudi cells were slightly higher in cultures with IL-2, T-cell contamination was lower with IL-15. Therefore, further experiments were performed with CD3(-)CD56(+) products cultured with IL-15. Cryopreservation of IL-15-activated NK cells resulted in a loss of cytotoxicity (>92%), whereas thawing of isolated, uncultured NK cells followed by culture with IL-15 yielded cells with about 43% of the original lytic activity. Five-day IL-15-activated NK cells lysed tumor target cell lines and primary leukemic blasts, providing the basis for NK cell–based immunotherapeutic strategies in a clinical setting.

Caveolin-mediated endocytosis of the Chlamydia M278 outer membrane peptide encapsulated in poly(lactic acid)-Poly(ethylene glycol) nanoparticles by mouse primary dendritic cells enhances specific immune effectors mediated by MHC class II and CD4+ T cells.

PubMed

Dixit, Saurabh; Sahu, Rajnish; Verma, Richa; Duncan, Skyla; Giambartolomei, Guillermo H; Singh, Shree R; Dennis, Vida A

2018-03-01

We previously developed a Chlamydia trachomatis nanovaccine (PPM) by encapsulating a chlamydial M278 peptide within poly(lactic acid)-poly(ethylene glycol) biodegradable nanoparticles that immunopotentiated Chlamydia-specific immune effector responses in mice. Herein, we investigated the mechanistic interactions of PPM with mouse bone marrow-derived dendritic cells (DCs) for its uptake, trafficking, and T cell activation. Our results reveal that PPM triggered enhanced expression of effector cytokines and chemokines, surface activation markers (Cd1d2, Fcgr1), pathogen-sensing receptors (TLR2, Nod1), co-stimulatory (CD40, CD80, CD86) and MHC class I and II molecules. Co-culturing of PPM-primed DCs with T cells from C. muridarum vaccinated mice yielded an increase in Chlamydia-specific immune effector responses including CD3 + lymphoproliferation, CD3 + CD4 + IFN-γ-secreting cells along with CD3 + CD4 + memory (CD44 high and CD62L high ) and effector (CD44 high and CD62L low ) phenotypes. Intracellular trafficking analyses revealed an intense expression and colocalization of PPM predominantly in endosomes. PPM also upregulated the transcriptional and protein expression of the endocytic mediator, caveolin-1 in DCs. More importantly, the specific inhibition of caveolin-1 led to decreased expression of PPM-induced cytokines and co-stimulatory molecules. Our investigation shows that PPM provided enhancement of uptake, probably by exploiting the caveolin-mediated endocytosis pathway, endosomal processing, and MHC II presentation to immunopotentiate Chlamydia-specific immune effector responses mediated by CD4 + T cells. Copyright © 2017 Elsevier Ltd. All rights reserved.

Yield and proliferation rate of adipose-derived stromal cells as a function of age, body mass index and harvest site-increasing the yield by use of adherent and supernatant fractions?

PubMed

Buschmann, Johanna; Gao, Shuping; Härter, Luc; Hemmi, Sonja; Welti, Manfred; Werner, Clement M L; Calcagni, Maurizio; Cinelli, Paolo; Wanner, Guido A

2013-09-01

Adipose-derived stem cells are easily accessed and have a relatively high density compared with other mesenchymal stromal cells. Isolation protocols of adipose-derived stem cells (ASC) rely on the cell's ability to adhere to tissue culture plastic overnight. It was evaluated whether the floating ASC fractions are also of interest for cell-based therapies. In addition, the impact of age, body mass index (BMI) and harvest site was assessed. The surface protein profile with the use of flow cytometry, the cell yield and the doubling time of passages 4, 5 and 6 of ASC from 30 donors were determined. Adherent and supernatant fractions were compared. The impact of age, BMI and harvest site on cell yield and doubling times was determined. Both adherent and supernatant fractions showed high mean fluorescence intensities for CD13, CD29, CD44, CD73, CD90 and CD105 and comparatively low mean fluorescence intensities for CD11b, CD62L, intracellular adhesion molecule-1 and CD34. Doubling times of adherent and supernatant fractions did not differ significantly. Whereas the old age group had a significantly lower cell yield compared with the middle aged group, BMI and harvest site had no impact on cell yield. Finally, doubling times for passages 4, 5 and 6 were not influenced by the age and BMI of the donors, nor the tissue-harvesting site. The floating ASC fraction is an equivalent second cell source just like the adherent ASC fraction. Donor age, BMI and harvest site do not influence cell yield and proliferation rate. Copyright © 2013 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Primary Endoscopic Transnasal Transsphenoidal Surgery for Magnetic Resonance Image-Positive Cushing Disease: Outcomes of a Series over 14 Years.

PubMed

Kuo, Chao-Hung; Yen, Yu-Shu; Wu, Jau-Ching; Chen, Yu-Chun; Huang, Wen-Cheng; Cheng, Henrich

2015-09-01

There are scant data of endoscopic transsphenoidal surgery (ETS) with adjuvant therapies of Cushing disease (CD). To report the remission rate, secondary management, and outcomes of a series of CD patients. Patients with CD with magnetic resonance imaging (MRI)-positive adenoma who underwent ETS as the first and primary treatment were included. The diagnostic criteria were a combination of 24-hour urine-free cortisol, elevated serum cortisol levels, or other tests (e.g., inferior petrosal sinus sampling). All clinical and laboratory evaluations and radiological examinations were reviewed. Forty consecutive CD patients, with an average age of 41.0 years, were analyzed with a mean follow-up of 40.2 ± 29.6 months. These included 22 patients with microadenoma and 18 with macroadenoma, including 9 cavernous invasions. The overall remission rate of CD after ETS was 72.5% throughout the entire follow-up. Patients with microadenoma or noninvasive macroadenoma had a higher remission rate than those who had macroadenoma with cavernous sinus invasion (81.8% or 77.8% vs. 44.4%, P = 0.02). After ETS, the patients who had adrenocorticotropic hormone-positive adenoma had a higher remission rate than those who had not (76.5% vs. 50%, P = 0.03). In the 11 patients who had persistent/recurrent CD after the first ETS, 1 underwent secondary ETS, 8 received gamma-knife radiosurgery (GKRS), and 2 underwent both. At the study end point, two (5%) of these CD patients had persistent CD and were under the medication of ketoconazole. For MRI-positive CD patients, primary (i.e., the first) ETS yielded an overall remission rate of 72.5%. Adjuvant therapies, including secondary ETS, GKRS, or both, yielded an ultimate remission rate of 95%. Copyright © 2015 Elsevier Inc. All rights reserved.

Differentiation "in vitro" of primary and immortalized porcine mesenchymal stem cells into cardiomyocytes for cell transplantation.

PubMed

Moscoso, I; Centeno, A; López, E; Rodriguez-Barbosa, J I; Santamarina, I; Filgueira, P; Sánchez, M J; Domínguez-Perles, R; Peñuelas-Rivas, G; Domenech, N

2005-01-01

Cell transplantation to regenerate injured tissues is a promising new treatment for patients suffering several diseases. Bone marrow contains a population of progenitor cells known as mesenchymal stem cells (MSCs), which have the capability to colonize different tissues, replicate, and differentiate into multilineage cells. Our goal was the isolation, characterization, and immortalization of porcine MSCs (pMSCs) to study their potential differentiation "in vitro" into cardiomyocytes. pMSCs were obtained from the aspirated bone marrow of Large-White pigs. After 4 weeks in culture, adherent cells were phenotypically characterized by flow cytometry and immunochemistry by using monoclonal antibodies. Primary pMSCs were transfected with the plasmid pRNS-1 to obtain continuous growing cloned cell lines. Fresh pMSCs and immortalized cells were treated with 5-azacytidine to differentiate them into cardiomyocytes. Flow cytometry analysis of isolated pMSCs demonstrated the following phenotype, CD90(pos), CD29(pos), CD44(pos), SLA-I(pos), CD106(pos), CD46(pos) and CD45(neg), CD14(neg), CD31(neg), and CD11b(neg), similar to that described for human MSC. We derived several stable immortalized MSC cell lines. One of these, called pBMC-2, was chosen for further characterization. After "in vitro" stimulation of both primary or immortalized cells with 5-azacytidine, we obtained different percentages (30%-50%) of cells with cardiomyocyte characteristics, namely, positive for alpha-Actin and T-Troponin. Thus, primary or immortalized pMSCs derived from bone marrow and cultured were able to differentiate "ex vivo" into cardiac-like muscle cells. These elements may be potentials tools to improve cardiac function in a swine myocardial infarct model.

Targeted delivery of CD44s-siRNA by ScFv overcomes de novo resistance to cetuximab in triple negative breast cancer.

PubMed

Fu, Wenyan; Sun, Hefen; Zhao, Yang; Chen, Mengting; Yang, Lipeng; Yang, Xueli; Jin, Wei

2018-05-16

The overexpression of EGFR often occurs in TNBC, and the anti-EGFR receptor antibody cetuximab is used widely to treat metastatic cancer in the clinic. However, EGFR-targeted therapies have been developed for TNBC without clinical success. In this study, we show that impaired EGFR degradation is crucial for resistance to cetuximab, which depends on the cell surface molecule CD44. To further investigate the role of CD44 in EGFR signaling and its treatment potential, we developed a targeting fusion protein composed of an anti-EGFR scFv generated from cetuximab and truncated protamine, called Ce-tP. CD44 siRNA can be specifically delivered into EGFR-positive TNBC cells by Ce-tP. Efficient knockdown of CD44 and suppression of both EGFR and downstream signaling by the Ce-tP/siRNA complex were observed in EGFR-positive TNBC cells. More importantly, our results also showed that targeted delivery of siRNA specific for CD44 can efficiently overcome resistance to EGFR targeting in TNBC cells both in vitro and in vivo. Overall, our results establish a new principle to achieve EGFR inhibition in TNBC and limit drug resistance. Copyright © 2018 Elsevier Ltd. All rights reserved.

Blocking the Adhesion Cascade at the Premetastatic Niche for Prevention of Breast Cancer Metastasis

PubMed Central

Kang, Shin-Ae; Hasan, Nafis; Mann, Aman P; Zheng, Wei; Zhao, Lichao; Morris, Lynsie; Zhu, Weizhu; Zhao, Yan D; Suh, K Stephen; Dooley, William C; Volk, David; Gorenstein, David G; Cristofanilli, Massimo; Rui, Hallgeir; Tanaka, Takemi

2015-01-01

Shear-resistant adhesion and extravasation of disseminated cancer cells at the target organ is a crucial step in hematogenous metastasis. We found that the vascular adhesion molecule E-selectin preferentially promoted the shear-resistant adhesion and transendothelial migration of the estrogen receptor (ER)–/CD44+ hormone-independent breast cancer cells, but not of the ER+/CD44-/low hormone-dependent breast cancer cells. Coincidentally, CD44+ breast cancer cells were abundant in metastatic lung and brain lesions in ER– breast cancer, suggesting that E-selectin supports hematogenous metastasis of ER–/CD44+ breast cancer. In an attempt to prevent hematogenous metastasis through the inhibition of a shear-resistant adhesion of CD44+ cancer cells to E-selectin-expressing blood vessels on the premetastatic niche, an E-selectin targeted aptamer (ESTA) was developed. We demonstrated that a single intravenous injection of ESTA reduced metastases to a baseline level in both syngeneic and xenogeneic forced breast cancer metastasis models without relocating the site of metastasis. The effect of ESTA was absent in E-selectin knockout mice, suggesting that E-selectin is a molecular target of ESTA. Our data highlight the potential application of an E-selectin antagonist for the prevention of hematogenous metastasis of ER–/CD44+ breast cancer. PMID:25815697

Blocking the adhesion cascade at the premetastatic niche for prevention of breast cancer metastasis.

PubMed

Kang, Shin-Ae; Hasan, Nafis; Mann, Aman P; Zheng, Wei; Zhao, Lichao; Morris, Lynsie; Zhu, Weizhu; Zhao, Yan D; Suh, K Stephen; Dooley, William C; Volk, David; Gorenstein, David G; Cristofanilli, Massimo; Rui, Hallgeir; Tanaka, Takemi

2015-06-01

Shear-resistant adhesion and extravasation of disseminated cancer cells at the target organ is a crucial step in hematogenous metastasis. We found that the vascular adhesion molecule E-selectin preferentially promoted the shear-resistant adhesion and transendothelial migration of the estrogen receptor (ER)(-)/CD44(+) hormone-independent breast cancer cells, but not of the ER(+)/CD44(-/low) hormone-dependent breast cancer cells. Coincidentally, CD44(+) breast cancer cells were abundant in metastatic lung and brain lesions in ER(-) breast cancer, suggesting that E-selectin supports hematogenous metastasis of ER(-)/CD44(+) breast cancer. In an attempt to prevent hematogenous metastasis through the inhibition of a shear-resistant adhesion of CD44(+) cancer cells to E-selectin-expressing blood vessels on the premetastatic niche, an E-selectin targeted aptamer (ESTA) was developed. We demonstrated that a single intravenous injection of ESTA reduced metastases to a baseline level in both syngeneic and xenogeneic forced breast cancer metastasis models without relocating the site of metastasis. The effect of ESTA was absent in E-selectin knockout mice, suggesting that E-selectin is a molecular target of ESTA. Our data highlight the potential application of an E-selectin antagonist for the prevention of hematogenous metastasis of ER(-)/CD44(+) breast cancer.

In vitro characterization of CD133lo cancer stem cells in Retinoblastoma Y79 cell line.

PubMed

Nair, Rohini M; Balla, Murali Ms; Khan, Imran; Kalathur, Ravi Kiran Reddy; Kondaiah, Paturu; Vemuganti, Geeta K

2017-11-21

Retinoblastoma (Rb), the most common childhood intraocular malignant tumor, is reported to have cancer stem cells (CSCs) similar to other tumors. Our previous investigation in primary tumors identified the small sized cells with low CD133 (Prominin-1) and high CD44 (Hyaluronic acid receptor) expression to be putative Rb CSCs using flow cytometry (FSC lo /SSC lo /CD133 lo /CD44 hi ). With this preliminary data, we have now utilized a comprehensive approach of in vitro characterization of Y79 Rb cell line following CSC enrichment using CD133 surface marker and subsequent validation to confirm the functional properties of CSCs. The cultured Rb Y79 cells were evaluated for surface markers by flow cytometry and CD133 sorted cells (CD133 lo /CD133 hi ) were compared for CSC characteristics by size/percentage, cell cycle assay, colony formation assay, differentiation, Matrigel transwell invasion assay, cytotoxicity assay, gene expression using microarray and validation by semi-quantitative PCR. Rb Y79 cell line shared the profile (CD133, CD90, CXCR4 and ABCB1) of primary tumors except for CD44 expression. The CD133 lo cells (16.1 ± 0.2%) were FSC lo /SSC lo , predominantly within the G0/G1 phase, formed larger and higher number of colonies with ability to differentiate to CD133 hi cells, exhibited increased invasive potential in a matrigel transwell assay (p < 0.05) and were resistant to Carboplatin treatment (p < 0.001) as compared to CD133 hi cells. The CD133 lo cells showed higher expression of several embryonic stem cell genes (HOXB2, HOXA9, SALL1, NANOG, OCT4, LEFTY), stem cells/progenitor genes (MSI2, BMI1, PROX1, ABCB1, ABCB5, ABCG2), and metastasis related gene- MACC1, when compared to the CD133 hi cells. This study validates the observation from our earlier primary tumor study that CSC properties in Rb Y79 cell line are endowed within the CD133 lo population, evident by their characteristics- i.e. small sized, dormant in nature, increased colony forming ability, differentiation to CD133 hi cells, higher invasiveness potential, drug resistance and primitive gene expression pattern. These findings provide a proof of concept for methodological characterization of the retinoblastoma CSCs with future implications for improved diagnostic and treatment strategies.

Myosin 1g Contributes to CD44 Adhesion Protein and Lipid Rafts Recycling and Controls CD44 Capping and Cell Migration in B Lymphocytes

PubMed Central

López-Ortega, Orestes; Santos-Argumedo, Leopoldo

2017-01-01

Cell migration and adhesion are critical for immune system function and involve many proteins, which must be continuously transported and recycled in the cell. Recycling of adhesion molecules requires the participation of several proteins, including actin, tubulin, and GTPases, and of membrane components such as sphingolipids and cholesterol. However, roles of actin motor proteins in adhesion molecule recycling are poorly understood. In this study, we identified myosin 1g as one of the important motor proteins that drives recycling of the adhesion protein CD44 in B lymphocytes. We demonstrate that the lack of Myo1g decreases the cell-surface levels of CD44 and of the lipid raft surrogate GM1. In cells depleted of Myo1g, the recycling of CD44 was delayed, the delay seems to be caused at the level of formation of recycling complex and entry into recycling endosomes. Moreover, a defective lipid raft recycling in Myo1g-deficient cells had an impact both on the capping of CD44 and on cell migration. Both processes required the transportation of lipid rafts to the cell surface to deliver signaling components. Furthermore, the extramembrane was essential for cell expansion and remodeling of the plasma membrane topology. Therefore, Myo1g is important during the recycling of lipid rafts to the membrane and to the accompanied proteins that regulate plasma membrane plasticity. Thus, Myosin 1g contributes to cell adhesion and cell migration through CD44 recycling in B lymphocytes. PMID:29321775

CD44-positive cells are candidates for astrocyte precursor cells in developing mouse cerebellum.

PubMed

Cai, Na; Kurachi, Masashi; Shibasaki, Koji; Okano-Uchida, Takayuki; Ishizaki, Yasuki

2012-03-01

Neural stem cells are generally considered to be committed to becoming precursor cells before terminally differentiating into either neurons or glial cells during neural development. Neuronal and oligodendrocyte precursor cells have been identified in several areas in the murine central nervous system. The presence of astrocyte precursor cells (APCs) is not so well understood. The present study provides several lines of evidence that CD44-positive cells are APCs in the early postnatal mouse cerebellum. In developing mouse cerebellum, CD44-positive cells, mostly located in the white matter, were positive for the markers of the astrocyte lineage, but negative for the markers of mature astrocytes. CD44-positive cells were purified from postnatal cerebellum by fluorescence-activated cell sorting and characterized in vitro. In the absence of any signaling molecule, many cells died by apoptosis. The surviving cells gradually expressed glial fibrillary acidic protein, a marker for mature astrocytes, indicating that differentiation into mature astrocytes is the default program for these cells. The cells produced no neurospheres nor neurons nor oligodendrocytes under any condition examined, indicating these cells are not neural stem cells. Leukemia inhibitory factor greatly promoted astrocytic differentiation of CD44-positive cells, whereas bone morphogenetic protein 4 (BMP4) did not. Fibroblast growth factor-2 was a potent mitogen for these cells, but was insufficient for survival. BMP4 inhibited activation of caspase-3 and greatly promoted survival, suggesting a novel role for BMP4 in the control of development of astrocytes in cerebellum. We isolated and characterized only CD44 strongly positive large cells and discarded small and/or CD44 weakly positive cells in this study. Further studies are necessary to characterize these cells to help determine whether CD44 is a selective and specific marker for APCs in the developing mouse cerebellum. In conclusion, we succeeded in preparing APC candidates from developing mouse cerebellum, characterized them in vitro, and found that BMPs are survival factors for these cells.

The Pluripotent Stem-Cell Marker Alkaline Phosphatase is Highly Expressed in Refractory Glioblastoma with DNA Hypomethylation.

PubMed

Iwadate, Yasuo; Suganami, Akiko; Tamura, Yutaka; Matsutani, Tomoo; Hirono, Seiichiro; Shinozaki, Natsuki; Hiwasa, Takaki; Takiguchi, Masaki; Saeki, Naokatsu

2017-02-01

Hypomethylation of genomic DNA induces stem-cell properties in cancer cells and contributes to the treatment resistance of various malignancies. To examine the correlation between the methylation status of stem-cell-related genes and the treatment outcomes in patients with glioblastoma (GBM). The genome-wide DNA methylation status was determined using HumanMethylation450 BeadChips, and the methylation status was compared between a group of patients with good prognosis (survival > 4 yr) and a group with poor prognosis (survival < 1 yr). Immunohistochemistry for proteins translated from hypomethylated genes, including alkaline phosphatase (ALPL), CD133, and CD44, was performed in 70 GBMs and 60 oligodendroglial tumors. The genomic DNA in refractory GBM was more hypomethylated than in GBM from patients with relatively long survival (P = .0111). Stem-cell-related genes including ALPL, CD133, and CD44 were also significantly hypomethylated. A validation study using immunohistochemistry showed that DNA hypomethylation was strongly correlated with high protein expression of ALPL, CD133, and CD44. GBM patients with short survival showed high expression of these stem-cell markers. Multivariate analysis confirmed that co-expression of ALPL + CD133 or ALPL + CD44 was a strong predictor of short survival. Anaplastic oligodendroglial tumors without isocitrate dehydrogenase 1 mutation were significantly correlated with high ALPL expression and poor survival. Accumulation of stem-cell properties due to aberrant DNA hypomethylation is associated with the refractory nature of GBM. Copyright © 2017 by the Congress of Neurological Surgeons

Systematic review: treatment pattern and clinical effectiveness and safety of pharmaceutical therapies for Crohn’s disease in Europe

PubMed Central

Lelli, Filippo; Nuhoho, Solomon; Lee, Xin Ying; Xu, Weiwei

2016-01-01

Background Although many clinical trials have been conducted in treatments of Crohn’s disease (CD), whether the trial results were representative of daily practice needs to be supported by studies conducted in real-world settings. Aim This study aims to identify how CD is treated and what are the clinical effectiveness and safety of the pharmaceutical therapies of CD in real-world settings. Methods A systematic literature review was conducted based on Medline®, Embase®, and Cochrane. All publications were assessed for title/abstract and full-text according to a predefined study protocol. Data were extracted and reported. Results A total of 1,998 publications were identified. Fifty studies including six publications reporting treatment pattern and 44 studies reporting clinical effectiveness and safety of pharmaceutical therapies in CD management in Europe were included. 5-Aminosalicylic acid and corticosteroids were reported to be used among 14%–74% of CD patients. Immunomodulators were used by 14%–25% and 29%–31% of CD patients as an initial and follow-up treatment, respectively. Biological therapies were used by 25%–33% of CD patients. A trend toward an increasing use of immunomodulators and biological therapies in Europe has been reported in recent years. Approximately 50% of patients achieved remission on immunomodulator or biologic treatment, although a relapse rate of up to 23% has been reported. Conclusion There is a trend of treatment shift to immunomodulators and biologics in CD management. Clinical effectiveness of immunomodulators and biologics has been demonstrated, though with a lack of sustainability of the effectiveness. PMID:27785086

Systematic review: treatment pattern and clinical effectiveness and safety of pharmaceutical therapies for Crohn's disease in Europe.

PubMed

Lelli, Filippo; Nuhoho, Solomon; Lee, Xin Ying; Xu, Weiwei

2016-01-01

Although many clinical trials have been conducted in treatments of Crohn's disease (CD), whether the trial results were representative of daily practice needs to be supported by studies conducted in real-world settings. This study aims to identify how CD is treated and what are the clinical effectiveness and safety of the pharmaceutical therapies of CD in real-world settings. A systematic literature review was conducted based on Medline ® , Embase ® , and Cochrane. All publications were assessed for title/abstract and full-text according to a predefined study protocol. Data were extracted and reported. A total of 1,998 publications were identified. Fifty studies including six publications reporting treatment pattern and 44 studies reporting clinical effectiveness and safety of pharmaceutical therapies in CD management in Europe were included. 5-Aminosalicylic acid and corticosteroids were reported to be used among 14%-74% of CD patients. Immunomodulators were used by 14%-25% and 29%-31% of CD patients as an initial and follow-up treatment, respectively. Biological therapies were used by 25%-33% of CD patients. A trend toward an increasing use of immunomodulators and biological therapies in Europe has been reported in recent years. Approximately 50% of patients achieved remission on immunomodulator or biologic treatment, although a relapse rate of up to 23% has been reported. There is a trend of treatment shift to immunomodulators and biologics in CD management. Clinical effectiveness of immunomodulators and biologics has been demonstrated, though with a lack of sustainability of the effectiveness.

Cadmium in the Coastal Upwelling Area Adjacent to the California Mexico Border

NASA Astrophysics Data System (ADS)

Segovia-Zavala, J. A.; Delgadillo-Hinojosa, F.; Alvarez-Borrego, S.

1998-04-01

Cadmium concentrations ([Cd]) were measured in samples from the water column of the coastal upwelling zone adjacent to the California - Mexico border. Temperature and nutrient distributions showed an intense upwelling event during our sampling. Lowest [Cd] were found at locations offshore (50 km) (0·03-0·058 nM), whereas the maximum concentrations were found inshore (0·14-0·166 nM). Both nutrients and [Cd] were enriched in coastal waters. Our inshore [Cd] values are about 25% of those reported for waters off central California. This is possibly due to the intrusion of oligotrophic waters from the eastern edge of the North Pacific Central Gyre to the Southern California Bight. Multivariate analysis indicates that high [Cd]s were associated with high phytoplankton biomass, nutrients and low temperature. Our data present no evidence of a [Cd] gradient due to the San Diego and Tijuana sewage discharges, which indicates that they maintain a very local effect.

A reproducible method for the isolation and expansion of ovine mesenchymal stromal cells from bone marrow for use in regenerative medicine preclinical studies.

PubMed

Caminal, Marta; Vélez, Roberto; Rabanal, Rosa Maria; Vivas, Daniel; Batlle-Morera, Laura; Aguirre, Màrius; Barquinero, Jordi; García, Joan; Vives, Joaquim

2017-12-01

The use of multipotent mesenchymal stromal cells (MSCs) as candidate medicines for treating a variety of pathologies is based on their qualities as either progenitors for the regeneration of damaged tissue or producers of a number of molecules with pharmacological properties. Preclinical product development programmes include the use of well characterized cell populations for proof of efficacy and safety studies before testing in humans. In the field of orthopaedics, an increasing number of translational studies use sheep as an in vivo test system because of the similarities with humans in size and musculoskeletal architecture. However, robust and reproducible methods for the isolation, expansion, manipulation and characterization of ovine MSCs have not yet been standardised. The present study describes a method for isolation and expansion of fibroblastic-like, adherent ovine MSCs that express CD44, CD90, CD140a, CD105 and CD166, and display trilineage differentiation potential. The 3-week bioprocess proposed here typically yielded cell densities of 1.4 × 10 4 MSCs/cm 2 at passage 2, with an expansion factor of 37.8 and approximately eight cumulative population doublings. The osteogenic potential of MSCs derived following this methodology was further evaluated in vivo in a translational model of osteonecrosis of the femoral head, in which the persistence of grafted cells in the host tissue and their lineage commitment into osteoblasts and osteocytes was demonstrated by tracking enhanced green fluorescent protein-labelled cells. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

In situ targeting of dendritic cells sets tolerogenic environment and ameliorates CD4+ T-cell response in the postischemic liver.

PubMed

Funken, Dominik; Ishikawa-Ankerhold, Hellen; Uhl, Bernd; Lerchenberger, Maximilian; Rentsch, Markus; Mayr, Doris; Massberg, Steffen; Werner, Jens; Khandoga, Andrej

2017-11-01

CD4 + T cells recruited to the liver play a key role in the pathogenesis of ischemia/reperfusion (I/R) injury. The mechanism of their activation during alloantigen-independent I/R is not completely understood. We hypothesized that liver-resident dendritic cells (DCs) interact with CD4 + T cells in the postischemic liver and that modulation of DCs or T-cell-DC interactions attenuates liver inflammation. In mice, warm hepatic I/R (90/120-240 min) was induced. Tolerogenic DCs were generated in situ by pretreatment of animals with the vitamin D analog paricalcitol. A mAb-CD44 was used for blockade of CD4 + T-cell-DC interactions. As shown by 2-photon in vivo microscopy as well as confocal microscopy, CD4 + T cells were closely colocalized with DCs in the postischemic liver. Pretreatment with paricalcitol attenuated I/R-induced maturation of DCs (flow cytometry), CD4 + T-cell recruitment into the liver (intravital microscopy), and hepatocellular/microvascular damage (intravital microscopy, alanine aminotransferase/aspartate aminotransferase, histology). However, interruption of T-cell-DC interaction increased proinflammatory DC maturation and even enhanced tissue damage. Simultaneous treatment with an anti-CD44mAb completely abolished the beneficial effect of paricalcitol on T-cell migration and tissue injury. Our study demonstrates for the first time that hepatic DCs interact with CD4 + T cells in the postischemic liver in vivo ; modulation of DCs and/or generation of tolerogenic DCs attenuates intrahepatic CD4 + T-cell recruitment and reduces I/R injury; and interruption of CD44-dependent CD4 + T-cell-DC interactions enhances tissue injury by preventing the modulatory effect of hepatic DCs on T cells, especially type 1 T helper effector cells. Thus, hepatic DCs are strongly involved in the promotion of CD4 + T-cell-dependent postischemic liver inflammation.-Funken, D., Ishikawa-Ankerhold, H., Uhl, B., Lerchenberger, M., Rentsch, M., Mayr, D., Massberg, S., Werner, J., Khandoga, A. In situ targeting of dendritic cells sets tolerogenic environment and ameliorates CD4 + T-cell response in the postischemic liver. © FASEB.

Three-Dimensional Cellular Arrangement in Epithelial Ovarian Cancer Cell Lines TOV-21G and SKOV-3 is Associated with Apoptosis-Related miRNA Expression Modulation.

PubMed

de Lima, Aline Brito; Silva, Luciana Maria; Gonçales, Nikole Gontijo; Carvalho, Maria Raquel Santos; da Silva Filho, Agnaldo Lopes; da Conceição Braga, Letícia

2018-01-06

Epithelial ovarian cancer (EOC) is the most lethal gynecological malignancy, and the lack of chemoresistance biomarkers contributes to the poor prognosis. Cancer stem cells (CSC) have been investigated in EOC to understand its relationship with chemoresistance and recurrence. In this context, in vitro cultivation-models are important tools for CSC studies. MicroRNAs (miRNAs) play key roles in cancer, CSC regulation and apoptosis. Thus, this study aims to evaluate the tumorsphere model as CSC-enrichment method in EOC studies and investigate apoptosis-related miRNAs in tumorspheres-derived EOC cell lines. TOV-21G and SKOV-3 were cultured in monolayer and tumorspheres. Genetic profiles of cell lines were obtained using COSMIC database. CD24/CD44/CD146/CD177 and ALDH1 markers were evaluated in cell lines and tumorspheres-derived by flow cytometry. Eleven miRNAs were selected by in silico analysis for qPCR analysis. According to COSMIC, TOV-21G and SKOV-3 have eight and nine cancer-related mutations, respectively. TOV-21G showed a CD44 +/high /CD24 -/low /CD117 -/low /CD146 -/low /ALDH1 low profile in both culture models; thus, no significant difference between cultivation models was identified. SKOV-3 showed a CD44 +/high /CD24 +/high / CD117 -/low /CD146 -/low /ALDH1 low profile in both culture models, although the tumorsphere model showed a significant increase in CD24 +/high subpopulation (ovarian CSC-like). Among eleven miRNAs, we observed differences in miRNA expression between culture models. MiR-26a was overexpressed in TOV-21G tumorspheres, albeit downregulated in SKOV-3 tumorspheres. MiR-125b-5p, miR-17-5p and miR-221 was downregulated in tumorsphere model in both cell lines. Given that tumorsphere-derived SKOV-3 had a higher ratio of CD24 +/high cells, we suggest that miR-26a, miR-125b-5p, miR-17-5p and miR-221 downregulation could be related to poor EOC prognosis.

Platelets Play Differential Role During the Initiation and Progression of Autoimmune Neuroinflammation

PubMed Central

Starossom, Sarah C.; Veremeyko, Tatyana; Yung, Amanda W. Y.; Dukhinova, Marina; Au, Cheryl; Lau, Alexander Y.; Weiner, Howard L.; Ponomarev, Eugene D.

2015-01-01

Rationale Platelets are known to participate in vascular pathologies; however, their role in neuroinflammatory diseases such as multiples sclerosis (MS) is unknown. Autoimmune CD4 T cells have been the main focus of studies of MS, although the factors that regulate T cell differentiation towards pathogenic Th1/Th17 phenotypes are not completely understood. Objectives We investigated the role of platelets in the modulation of CD4 T cell functions in MS patients and in mice with experimental autoimmune encephalitis (EAE), an animal model for MS. Methods and Results We found that early in MS and EAE platelets degranulated and produced a number of soluble factors serotonin (5HT), PF4 and PAF, which specifically stimulated differentiation of T cells towards pathogenic Th1, Th17 and IFN-γ/IL-17-producing CD4 T cells. At the later stages of MS and EAE platelets became exhausted in their ability to produce proinflammatory factors and stimulate CD4 T cells, but substantially increased their ability to form aggregates with CD4 T cells. Formation of platelet-CD4 T cell aggregates involved interaction of CD62P on activated platelets with adhesion molecule CD166 on activated CD4 T cells, contributing to downmodulation of CD4 T cell activation, proliferation and production of IFN-γ. Blocking of formation of platelet-CD4 T cell aggregates during progression of EAE substantially enhanced proliferation of CD4 T cell in the CNS and the periphery leading to exacerbation of the disease. Conclusion Our study indicates differential roles for platelets in the regulation of functions of pathogenic CD4 T cells during initiation and progression of CNS autoimmune inflammation. PMID:26294656

Viremic HIV Infected Individuals with High CD4 T Cells and Functional Envelope Proteins Show Anti-gp41 Antibodies with Unique Specificity and Function

PubMed Central

Curriu, Marta; Fausther-Bovendo, Hughes; Pernas, María; Massanella, Marta; Carrillo, Jorge; Cabrera, Cecilia; López-Galíndez, Cecilio; Clotet, Bonaventura; Debré, Patrice; Vieillard, Vincent; Blanco, Julià

2012-01-01

Background CD4 T-cell decay is variable among HIV-infected individuals. In exceptional cases, CD4 T-cell counts remain stable despite high plasma viremia. HIV envelope glycoprotein (Env) properties, namely tropism, fusion or the ability to induce the NK ligand NKp44L, or host factors that modulate Env cytopathic mechanisms may be modified in such situation. Methods We identified untreated HIV-infected individuals showing non-cytopathic replication (VL>10,000 copies/mL and CD4 T-cell decay<50 cells/µL/year, Viremic Non Progressors, VNP) or rapid progression (CD4 T-cells<350 cells/µL within three years post-infection, RP). We isolated full-length Env clones and analyzed their functions (tropism, fusion activity and capacity to induce NKp44L expression on CD4 cells). Anti-Env humoral responses were also analyzed. Results Env clones isolated from VNP or RP individuals showed no major phenotypic differences. The percentage of functional clones was similar in both groups. All clones tested were CCR5-tropic and showed comparable expression and fusogenic activity. Moreover, no differences were observed in their capacity to induce NKp44L expression on CD4 T cells from healthy donors through the 3S epitope of gp41. In contrast, anti- Env antibodies showed clear functional differences: plasma from VNPs had significantly higher capacity than RPs to block NKp44L induction by autologous viruses. Consistently, CD4 T-cells isolated from VNPs showed undetectable NKp44L expression and specific antibodies against a variable region flanking the highly conserved 3S epitope were identified in plasma samples from these patients. Conversely, despite continuous antigen stimulation, VNPs were unable to mount a broad neutralizing response against HIV. Conclusions Env functions (fusion and induction of NKp44L) were similar in viremic patients with slow or rapid progression to AIDS. However, differences in humoral responses against gp41 epitopes nearby 3S sequence may contribute to the lack of CD4 T cell decay in VNPs by blocking the induction of NKp44L by gp41. PMID:22312424

Human umbilical cord mesenchymal stromal cells suppress MHC class II expression on rat vascular endothelium and prolong survival time of cardiac allograft

PubMed Central

Qiu, Ying; Yun, Mark M; Han, Xia; Zhao, Ruidong; Zhou, Erxia; Yun, Sheng

2014-01-01

Background: Human umbilical cord mesenchymal stromal cells (UC-MSCs) have low immunogenicity and immune regulation. To investigate immunomodulatory effects of human UC-MSCs on MHC class II expression and allograft, we transplanted heart of transgenic rats with MHC class II expression on vascular endothelium. Methods: UC-MSCs were obtained from human umbilical cords and confirmed with flow cytometry analysis. Transgenic rat line was established using the construct of human MHC class II transactivator gene (CIITA) under mouse ICAM-2 promoter control. The induced MHC class II expression on transgenic rat vascular endothelial cells (VECs) was assessed with immunohistological staining. And the survival time of cardiac allograft was compared between the recipients with and without UC-MSC transfusion. Results: Flow cytometry confirmed that the human UC-MSCs were positive for CD29, CD44, CD73, CD90, CD105, CD271, and negative for CD34 and HLA-DR. Repeated infusion of human UC-MSCs reduced MHC class II expression on vascular endothelia of transplanted hearts, and increased survival time of allograft. The UC-MSCs increased regulatory cytokines IL10, transforming growth factor (TGF)-β1 and suppressed proinflammatory cytokines IL2 and IFN-γ in vivo. The UC-MSC culture supernatant had similar effects on cytokine expression, and decreased lymphocyte proliferation in vitro. Conclusions: Repeated transfusion of the human UC-MSCs reduced MHC class II expression on vascular endothelia and prolonged the survival time of rat cardiac allograft. PMID:25126177

Investigation of celiac disease followed by immune thrombocytopenic purpura diagnosis in patients and comparison with literature

PubMed Central

Sarbay, Hakan; Kocamaz, Halil; Akin, Mehmet; Ozhan, Bayram

2017-01-01

OBJECTIVE: Celiac disease (CD) and Immune thrombocytopenic purpura (ITP) may occur together as a result of similar autoimmune mechanisms. The aim of this study was to assess the frequency of CD in a group of ITP patients and in the literature. METHODS: A total of 29 patients in Pamukkale University Faculty of Medicine Hospital Pediatric Hematology and Oncology Department with ITP were included in the study. Test was performed for the antibodies related to CD. Positive result for celiac antibodies was confirmed with biopsy. The results were compared with the literature. RESULTS: Of the study group, 13 patients (44.8%) were female and 16 (55.2%) were male. The mean age was 7.2±4.7 years and mean platelet count at the time of admission was 13,440±11,110/mm3 (range: 2000-41,000/mm3). Twelve patients (41.4%) were diagnosed as acute ITP, 6 patients (20.7%) as persistent ITP, and 11 patients (37.9%) as chronic ITP, according to the duration of thrombocytopenia. Antibody positivity was detected in 1 patient. Histological evaluation was compatible with CD. Results were compared with studies regarding the prevalence of CD in the population. No significant difference was found. CONCLUSION: Although it is not necessary to perform CD test in every case of ITP, the presence of differential diagnosis of CD is important to prevent unnecessary treatment, especially in ITP patients with growth retardation or malabsorption findings. PMID:28971174

Installation Restoration Program, Phase 2. Confirmation/Quantification, Stage 2, Hancock Air National Guard Base, Syracuse, New York. Volume 2. Appendices

DTIC Science & Technology

1989-06-01

4) 0) C)-D C 0401 C) 0 -c eL4 CD CD r, r- *,-A (mz61L 00 rC- 4ic 0Cm q) CD) C) -DC DC )C wl ~ ~ ’ %0 0 0 0 koko%04 4 J 4 J I+ C-M 44. 0 4J 44) -) 4...SAC) and 5,6-Dihydro-5-Azacytidine (H5AC) in L1210 Cell Culture Samples by Gas Chromatography (GC) and Mass Spectrometry (MS), C.J. Nielson, S.W

HgCdTe Surface and Defect Study Program.

DTIC Science & Technology

1986-03-01

different potential for Hg and Cd and hence be reflected in the electronic structure. The techniques of PES and ARPES available to our research group ...D-A166 795 HOME SURFCE ND DEFECT STUDY PROQRN(U) SATA / BARBRA RESEARCH CENTER GOLETA CALXF J A WILSON ET AL. USI FE MAR 86 SBRC-60411 ND93-63-C...0168 FO2/2 N L6 ILO 1.5 1. 11111 .6 .ICnrnp CHR HgCdTo SURFACE AND DEFECT STUDY PROGRAM J. A. Wilson and V. A. Cotton Santa Barbara Research Center

Peripheral Blood Mononuclear Cells Enhance Cartilage Repair in in vivo Osteochondral Defect Model.

PubMed

Hopper, Niina; Wardale, John; Brooks, Roger; Power, Jonathan; Rushton, Neil; Henson, Frances

2015-01-01

This study characterized peripheral blood mononuclear cells (PBMC) in terms of their potential in cartilage repair and investigated their ability to improve the healing in a pre-clinical large animal model. Human PBMCs were isolated with gradient centrifugation and adherent PBMC's were evaluated for their ability to differentiate into adipogenic, chondrogenic and osteogenic lineages and also for their expression of musculoskeletal genes. The phenotype of the PBMCs was evaluated using Stro-1, CD34, CD44, CD45, CD90, CD106, CD105, CD146 and CD166 cell surface markers. Osteochondral defects were created in the medial femoral condyle (MFC) of 24 Welsh mountain sheep and evaluated at a six month time point. Four cell treatment groups were evaluated in combination with collagen-GAG-scaffold: (1) MSC alone; (2) MSCs and PBMCs at a ratio of 20:1; (3) MSCs and PBMC at a ratio of 2:1 and (4) PBMCs alone. Samples from the surgical site were evaluated for mechanical properties, ICRS score and histological repair. Fresh PBMC samples were 90% positive for hematopoietic cell surface markers and negative for the MSC antibody panel (<1%, p = 0.006). However, the adherent PBMC population expressed mesenchymal stem cell markers in hypoxic culture and lacked CD34/45 positive cells (<0.2%). This finding demonstrated that the adherent cells had acquired an MSC-like phenotype and transformed in hypoxia from their original hematopoietic lineage. Four key genes in muskuloskeletal biology were significantly upregulated in adherent PBMCs by hypoxia: BMP2 4.2-fold (p = 0.0007), BMP6 10.7-fold (p = 0.0004), GDF5 2.0-fold (p = 0.002) and COL1 5.0-fold (p = 0.046). The monolayer multilineage analysis confirmed the trilineage mesenchymal potential of the adherent PBMCs. PBMC cell therapy was equally good as bone marrow MSC therapy for defects in the ovine large animal model. Our results show that PBMCs support cartilage healing and oxygen tension of the environment was found to have a key effect on the derivation of a novel adherent cell population with an MSC-like phenotype. This study presents a novel and easily attainable point-of-care cell therapy with PBMCs to treat osteochondral defects in the knee avoiding any cell manipulations outside the surgical room.

Spatiotemporal expression dynamics of selectins govern the sequential extravasation of neutrophils and monocytes in the acute inflammatory response.

PubMed

Zuchtriegel, Gabriele; Uhl, Bernd; Hessenauer, Maximilian E T; Kurz, Angela R M; Rehberg, Markus; Lauber, Kirsten; Krombach, Fritz; Reichel, Christoph A

2015-04-01

Leukocyte recruitment to the site of inflammation is a key event in a variety of cardiovascular pathologies. Infiltrating neutrophils constitute the first line of defense that precedes a second wave of emigrating monocytes reinforcing the inflammatory reaction. The mechanisms initiating this sequential process remained largely obscure. Using advanced in vivo microscopy and in vitro/ex vivo techniques, we identified individual spatiotemporal expression patterns of selectins and their principal interaction partners on neutrophils, resident/inflammatory monocytes, and endothelial cells. Coordinating the intraluminal trafficking of neutrophils and inflammatory monocytes to common sites of extravasation, selectins assign different sites to these immune cells for their initial interactions with the microvascular endothelium. Whereas constitutively expressed leukocyte L-selectin/CD62L and endothelial P-selectin/CD62P together with CD44 and P-selectin glycoprotein ligand-1/CD162 initiate the emigration of neutrophils, de novo synthesis of endothelial E-selectin/CD62E launches the delayed secondary recruitment of inflammatory monocytes. In this context, P-selectin/CD62P and L-selectin/CD62L together with P-selectin glycoprotein ligand-1/CD162 and CD44 were found to regulate the flux of rolling neutrophils and inflammatory monocytes, whereas E-selectin/CD62E selectively adjusts the rolling velocity of inflammatory monocytes. Moreover, selectins and their interaction partners P-selectin glycoprotein ligand-1/CD162 and CD44 differentially control the intraluminal crawling behavior of neutrophils and inflammatory monocytes collectively enabling the sequential extravasation of these immune cells to inflamed tissue. Our findings provide novel insights into the mechanisms initiating the sequential infiltration of the perivascular tissue by neutrophils and monocytes in the acute inflammatory response and might thereby contribute to the development of targeted therapeutic strategies for prevention and treatment of cardiovascular diseases. © 2015 American Heart Association, Inc.

Analysis of Etched CdZnTe Substrates

NASA Astrophysics Data System (ADS)

Benson, J. D.; Bubulac, L. O.; Jaime-Vasquez, M.; Lennon, C. M.; Arias, J. M.; Smith, P. J.; Jacobs, R. N.; Markunas, J. K.; Almeida, L. A.; Stoltz, A.; Wijewarnasuriya, P. S.; Peterson, J.; Reddy, M.; Jones, K.; Johnson, S. M.; Lofgreen, D. D.

2016-09-01

State-of-the-art as-received (112)B CdZnTe substrates have been examined for surface impurity contamination and polishing residue. Two 4 cm × 4 cm and one 6 cm × 6 cm (112)B state-of-the-art as-received CdZnTe wafers were analyzed. A maximum surface impurity concentration of Al = 1.7 × 1015 atoms cm-2, Si = 3.7 × 1013 atoms cm-2, Cl = 3.12 × 1015 atoms cm-2, S = 1.7 × 1014 atoms cm-2, P = 1.1 × 1014 atoms cm-2, Fe = 1.0 × 1013 atoms cm-2, Br = 1.2 × 1014 atoms cm-2, and Cu = 4 × 1012 atoms cm-2 was observed on the as-received CdZnTe wafers. CdZnTe particulates and residual SiO2 polishing grit were observed on the surface of the as-received (112)B CdZnTe substrates. The polishing grit/CdZnTe particulate density on CdZnTe wafers was observed to vary across a 6 cm × 6 cm wafer from ˜4 × 107 cm-2 to 2.5 × 108 cm-2. The surface impurity and damage layer of the (112)B CdZnTe wafers dictate that a molecular beam epitaxy (MBE) preparation etch is required. The contamination for one 4 cm × 4 cm and one 6 cm × 6 cm CdZnTe wafer after a standard MBE Br:methanol preparation etch procedure was also analyzed. A maximum surface impurity concentration of Al = 2.4 × 1015 atoms cm-2, Si = 4.0 × 1013 atoms cm-2, Cl = 7.5 × 1013 atoms cm-2, S = 4.4 × 1013 atoms cm-2, P = 9.8 × 1013 atoms cm-2, Fe = 1.0 × 1013 atoms cm-2, Br = 2.9 × 1014 atoms cm-2, and Cu = 5.2 × 1012 atoms cm-2 was observed on the MBE preparation-etched CdZnTe wafers. The MBE preparation-etched surface contamination consists of Cd(Zn)Te particles/flakes. No residual SiO2 polishing grit was observed on the (112)B surface.

Hyaluronan, CD44, and Emmprin Regulate Lactate Efflux and Membrane Localization of Monocarboxylate Transporters in Human Breast Carcinoma Cells

PubMed Central

Slomiany, Mark G.; Grass, G. Daniel; Robertson, Angela D.; Yang, Xiao Y.; Maria, Bernard L.; Beeson, Craig; Toole, Bryan P.

2013-01-01

Interactions of hyaluronan with CD44 in tumor cells play important cooperative roles in various aspects of malignancy and drug resistance. Emmprin (CD147; basigin)is a cell surface glycoprotein of the immunoglobulin superfamily that is highly up-regulated in malignant cancer cells and stimulates hyaluronan production, as well as several downstream signaling pathways. Emmprin also interacts with various monocarboxylate transporters (MCT). Malignant cancer cells use the glycolytic pathway and require MCTs to efflux lactate that results from glycolysis. Glycolysis and lactate secretion contribute to malignant cell behaviors and drug resistance in tumor cells. In the present study, we find that perturbation of endogenous hyaluronan, using small hyaluronan oligosaccharides, rapidly inhibits lactate efflux from breast carcinoma cells; down-regulation of emmprin, using emmprin small interfering RNA, also results in decreased efflux. In addition, we find that CD44 coimmunoprecipitates with MCT1, MCT4, and emmprin and colocalizes with these proteins at the plasma membrane. Moreover, after treatment of the cells with hyaluronan oligosaccharides, CD44, MCT1, and MCT4 become localized intracellularly whereas emmprin remains at the cell membrane. Together, these data indicate that constitutive interactions among hyaluronan, CD44, and emmprin contribute to regulation of MCT localization and function in the plasma membrane of breast carcinoma cells. PMID:19176383

Alanine-170 and proline-172 are critical determinants for extracellular CD20 epitopes; heterogeneity in the fine specificity of CD20 monoclonal antibodies is defined by additional requirements imposed by both amino acid sequence and quaternary structure.

PubMed

Polyak, Maria J; Deans, Julie P

2002-05-01

In vivo ablation of malignant B cells can be achieved using antibodies directed against the CD20 antigen. Fine specificity differences among CD20 monoclonal antibodies (mAbs) are assumed not to be a factor in determining their efficacy because evidence from antibody-blocking studies indicates limited epitope diversity with only 2 overlapping extracellular CD20 epitopes. However, in this report a high degree of heterogeneity among antihuman CD20 mAbs is demonstrated. Mutation of alanine and proline at positions 170 and 172 (AxP) (single-letter amino acid codes; x indicates the identical amino acid at the same position in the murine and human CD20 sequences) in human CD20 abrogated the binding of all CD20 mAbs tested. Introduction of AxP into the equivalent positions in the murine sequence, which is not otherwise recognized by antihuman CD20 mAbs, fully reconstituted the epitope recognized by B1, the prototypic anti-CD20 mAb. 2H7, a mAb previously thought to recognize the same epitope as B1, did not recognize the murine AxP mutant. Reconstitution of the 2H7 epitope was achieved with additional mutations replacing VDxxD in the murine sequence for INxxN (positions 162-166 in the human sequence). The integrity of the 2H7 epitope, unlike that of B1, further depends on the maintenance of CD20 in an oligomeric complex. The majority of 16 antihuman CD20 mAbs tested, including rituximab, bound to murine CD20 containing the AxP mutations. Heterogeneity in the fine specificity of these antibodies was indicated by marked differences in their ability to induce homotypic cellular aggregation and translocation of CD20 to a detergent-insoluble membrane compartment previously identified as lipid rafts.

Standardization of the CFU-GM assay: Advantages of plating a fixed number of CD34+ cells in collagen gels.

PubMed

Dobo, Irène; Pineau, Danielle; Robillard, Nelly; Geneviève, Frank; Piard, Nicole; Zandecki, Marc; Hermouet, Sylvie

2003-10-01

We investigated whether plating a stable amount of CD34(+) cells improves the CFU-GM assay. Data of CFU-GM assays performed with leukaphereses products in two transplant centers using a commercial collagen-based medium and unified CFU-GM scoring criteria were pooled and analyzed according to the numbers of CD34(+) cells plated. A first series of 113 CFU-GM assays was performed with a fixed number of mononuclear cells (i.e., a variable number of CD34(+) cells). In these cultures the CFU-GM/CD34 ratio varied according to the number of CD34(+) cells plated: median CFUGM/CD34 ratios were 1/6.2 to 1/6.6 for grafts containing <2% CD34(+) cells, vs. 1/10.2 for grafts containing > or =2% CD34(+) cells. The median CFU-GM/CD34 ratio also varied depending on pathology: 1/9.3 for multiple myeloma (MM), 1/6.8 for Hodgkin's disease (HD), 1/6.5 for non-Hodgkin lymphoma (NHL), and 1/4.5 for solid tumors (ST). A second series of 95 CFU-GM assays was performed with a fixed number of CD34(+) cells (220/ml). The range of median CFU-GM/CD34 ratios was narrowed to 1/7.0 to 1/5.2, and coefficients of variation for CFU-GM counts decreased by half to 38.1% (NHL), 36.1% (MM), 49.9% (HD), and 22.4% (ST). In addition, CFU-GM scoring was facilitated as the percentages of cultures with >50 CFU/GM/ml decreased from 6.7% to 43.8% when a variable number of CD34(+) cells was plated, to 4.5% to 16.7% when 220 CD34(+) cells/ml were plated. Hence, plating a fixed number of CD34(+) cells in collagen gels improves the CFU-GM assay by eliminating cell number-related variability and reducing pathology-related variability in colony growth.

Characterization of polyethylene glycol-grafted polyethylenimine and superparamagnetic iron oxide nanoparticles (PEG-g-PEI-SPION) as an MRI-visible vector for siRNA delivery in gastric cancer in vitro and in vivo.

PubMed

Chen, Yinting; Lian, Guoda; Liao, Chengde; Wang, Weiwei; Zeng, Linjuan; Qian, Chenchen; Huang, Kaihong; Shuai, Xintao

2013-07-01

Gene therapy is a promising therapeutic method but is severely hampered due to its lack of an ideal delivery system. Therefore, in this study, a nonviral and magnetic resonance imaging (MRI) visible vector, polyethylene glycol-grafted polyethylenimine and superparamagnetic iron oxide nanoparticles (PEG-g-PEI-SPION) was used as a nanocarrier for small interfering RNA (siRNA) delivery in gastric cancer. Biophysical characterization of PEG-g-PEI-SPION was systematically analyzed, including size, zeta potential, siRNA condensation capacity, cell viability, transfection efficiency, cellular uptake, and MRI-visible function in vivo. Besides, CD44 variant isoform 6 (CD44v6), a protein marker for metastatic behavior in gastric cancer, and was chose as the target gene to further analyze the siRNA delivery function of PEG-g-PEI-SPION. Under comprehensive analysis, the appropriate N/P ratio of PEG-g-PEI-SPION/siRNA was 10, and siRNA targeting at human CD44v6 (siCD44v6) transferred by PEG-g-PEI-SPION was effective at downregulating the CD44v6 expression of gastric carcinoma cell line SGC-7901 in vitro. Moreover, knockdown of CD44v6 impaired migrating and invasive abilities of SGC-7901 cells. Furthermore, PEG-g-PEI-SPION was a highly efficient contrast agent for MRI scan in vivo. PEG-g-PEI-SPION was a promising nonviral vector with molecular image tracing capacity for cancer gene therapy. And CD44v6 was a potential target gene for the prevention and detection of metastatic behavior in gastric cancer.

Hyaluronic acid-modified polyamidoamine dendrimer G5-entrapped gold nanoparticles delivering METase gene inhibits gastric tumor growth via targeting CD44+ gastric cancer cells.

PubMed

Li, Yi-Fan; Zhang, Hou-Ting; Xin, Lin

2018-06-01

Gastric cancer (GC) is the second most common leading cause of cancer-related death. Cancer stem cell (CSC) with the mark of CD44 played an important role in GC. rMETase was wildly exploited as chemotherapeutic option for GC. Polymers synthetic nanoparticle drug delivery systems have been commonly used for cancer therapy. With the decorating of Hyaluronic acid (HA), a receptor of CD44, nanoparticles exhibit with good biocompatibility and aqueous solubility. The characteristic of nanoparticles (NPs) was analyzed by TEM and DLS. The viability and proliferation of GC cells were examined by MTT assays. The levels of CD44, Cyt C, and c-caspase 3 were examined by Western blot. The level of ROS was measured by DCFH-DA assays. The morphology of tissues was detected using hematoxylin-eosin (H&E) stain. Nude mice xenograft models were used to evaluate the effect of HA-PAMAM-Au-METase on GC. The transfection of rMETase carried by HA-G5 PAMAM-Au visibly inhibited the proliferation and tumorsphere formation of GC cells through obviously enhancing METase activity. Elevation of METase activity suppressed the proliferation of CD44(+) GC cells through down-regulating MET in cellular supernatant that resulted in the increase of Cyc C and ROS levels. The number of CD44(+) GC cells in nude mice injected with G5 PAMAM-Au-METase decorated by HA was markly declined resulting in the inhibition of tumor growth. HA-G5 PAMAM-Au-METase significantly suppressed tumor growth of GC by targeted damaging the mitochondrial function of CD44(+) gastric CSCs.

Unique CD44 intronic SNP is associated with tumor grade in breast cancer: a case control study and in silico analysis.

PubMed

Esmaeili, Rezvan; Abdoli, Nasrin; Yadegari, Fatemeh; Neishaboury, Mohamadreza; Farahmand, Leila; Kaviani, Ahmad; Majidzadeh-A, Keivan

2018-01-01

CD44 encoded by a single gene is a cell surface transmembrane glycoprotein. Exon 2 is one of the important exons to bind CD44 protein to hyaluronan. Experimental evidences show that hyaluronan-CD44 interaction intensifies the proliferation, migration, and invasion of breast cancer cells. Therefore, the current study aimed at investigating the association between specific polymorphisms in exon 2 and its flanking region of CD44 with predisposition to breast cancer. In the current study, 175 Iranian female patients with breast cancer and 175 age-matched healthy controls were recruited in biobank, Breast Cancer Research Center, Tehran, Iran. Single nucleotide polymorphisms of CD44 exon 2 and its flanking were analyzed via polymerase chain reaction and gene sequencing techniques. Association between the observed variation with breast cancer risk and clinico-pathological characteristics were studied. Subsequently, bioinformatics analysis was conducted to predict potential exonic splicing enhancer (ESE) motifs changed as the result of a mutation. A unique polymorphism of the gene encoding CD44 was identified at position 14 nucleotide upstream of exon 2 (A37692→G) by the sequencing method. The A > G polymorphism exhibited a significant association with higher-grades of breast cancer, although no significant relation was found between this polymorphism and breast cancer risk. Finally, computational analysis revealed that the intronic mutation generated a new consensus-binding motif for the splicing factor, SC35, within intron 1. The current study results indicated that A > G polymorphism was associated with breast cancer development; in addition, in silico analysis with ESE finder prediction software showed that the change created a new SC35 binding site.

Immunohistochemistry Analysis of CD44, EGFR, and p16 in Oral Cavity and Oropharyngeal Squamous Cell Carcinoma.

PubMed

Cohen, Erin R; Reis, Isildinha M; Gomez, Carmen; Pereira, Lutecia; Freiser, Monika E; Hoosien, Gia; Franzmann, Elizabeth J

2017-08-01

Objectives We analyze the relationship between CD44, epidermal growth factor receptor (EGFR), and p16 expression in oral cavity and oropharyngeal cancers in a diverse population. We also describe whether particular patterns of staining are associated with progression-free survival and overall survival. Study Design Prospective study, single-blind to pathologist and laboratory technologist. Setting Hospital based. Subjects and Methods Immunohistochemistry, comprising gross staining and cellular expression, was performed and interpreted in a blinded fashion on 24 lip/oral cavity and 40 oropharyngeal cancer specimens collected between 2007 and 2012 from participants of a larger study. Information on overall survival and progression-free survival was obtained from medical records. Results Nineteen cases were clinically p16 positive, 16 of which were oropharyngeal. Oral cavity lesions were more likely to exhibit strong CD44 membrane staining ( P = .0002). Strong CD44 membrane and strong EGFR membrane and/or cytoplasmic staining were more common in p16-negative cancers ( P = .006). Peripheral/mixed gross p16 staining pattern was associated with worse survival than the universal staining on univariate and multivariate analyses ( P = .006, P = .030). This held true when combining gross and cellular localization for p16. For CD44, universal gross staining demonstrated poorer overall survival compared with the peripheral/mixed group ( P = .039). CD44 peripheral/mixed group alone and when combined with universal p16 demonstrated the best survival on multivariate analysis ( P = .010). Conclusion In a diverse population, systematic analysis applying p16, CD44, and EGFR gross staining and cellular localization on immunohistochemistry demonstrates distinct patterns that may have prognostic potential exceeding current methods. Larger studies are warranted to investigate these findings further.

Prognostic implications of adhesion molecule expression in colorectal cancer.

PubMed

Seo, Kyung-Jin; Kim, Maru; Kim, Jeana

2015-01-01

Research on the expression of adhesion molecules, E-cadherin (ECAD), CD24, CD44 and osteopontin (OPN) in colorectal cancer (CRC) has been limited, even though CRC is one of the leading causes of cancer-related deaths. This study was conducted to evaluate the expression of adhesion molecules in CRC and to determine their relationships with clinicopathologic variables, and the prognostic significance. The expression of ECAD, CD24, CD44 and OPN was examined in 174 stage II and III CRC specimens by immunohistochemistry of TMA. Negative ECAD expression was significantly correlated with advanced nodal stage and poor tumor differentiation. Multivariate analysis showed that both negative expression of ECAD and positive expression of CD24 were independent prognostic factors for disease-free survival (DFS) in CRC patients (P<0.001, relative risk [RR] = 5.596, 95% CI = 2.712-11.549; P = 0.038, RR = 3.768, 95% CI = 1.077-13.185, respectively). However, for overall survival (OS), only ECAD negativity showed statistically significant results in multivariate analysis (P<0.001, RR = 4.819, 95% CI = 2.515-9.234). Positive expression of CD24 was associated with poor OS in univariate analysis but was of no prognostic value in multivariate analysis. In conclusion, our study suggests that among these four adhesion molecules, ECAD and CD24 expression can be considered independent prognostic factors. The role of CD44 and OPN may need further evaluation.

Prognostic implications of adhesion molecule expression in colorectal cancer

PubMed Central

Seo, Kyung-Jin; Kim, Maru; Kim, Jeana

2015-01-01

Research on the expression of adhesion molecules, E-cadherin (ECAD), CD24, CD44 and osteopontin (OPN) in colorectal cancer (CRC) has been limited, even though CRC is one of the leading causes of cancer-related deaths. This study was conducted to evaluate the expression of adhesion molecules in CRC and to determine their relationships with clinicopathologic variables, and the prognostic significance. The expression of ECAD, CD24, CD44 and OPN was examined in 174 stage II and III CRC specimens by immunohistochemistry of TMA. Negative ECAD expression was significantly correlated with advanced nodal stage and poor tumor differentiation. Multivariate analysis showed that both negative expression of ECAD and positive expression of CD24 were independent prognostic factors for disease-free survival (DFS) in CRC patients (P<0.001, relative risk [RR] = 5.596, 95% CI = 2.712-11.549; P = 0.038, RR = 3.768, 95% CI = 1.077-13.185, respectively). However, for overall survival (OS), only ECAD negativity showed statistically significant results in multivariate analysis (P<0.001, RR = 4.819, 95% CI = 2.515-9.234). Positive expression of CD24 was associated with poor OS in univariate analysis but was of no prognostic value in multivariate analysis. In conclusion, our study suggests that among these four adhesion molecules, ECAD and CD24 expression can be considered independent prognostic factors. The role of CD44 and OPN may need further evaluation. PMID:26097606

Isolation and characterization of porcine adipose tissue-derived adult stem cells.

PubMed

Williams, Kellie J; Picou, Alicia A; Kish, Sharon L; Giraldo, Angelica M; Godke, Robert A; Bondioli, Kenneth R

2008-01-01

Stem cell characteristics such as self-renewal, differentiation and expression of CD34 and CD44 stem cell markers have not been identified in porcine adipose tissue-derived adult stem (ADAS) cells. The objective of this study was to develop a protocol for the isolation and culture of porcine adipose tissue-derived cells and to determine stem cell-like characteristics. Primary cultures were established and cell cultures were maintained. Cloning capacity was determined using a ring cloning procedure. Primary cultures and clones were differentiated and stained for multiple differentiated phenotypes. CD34 and CD44 messenger ribonucleic acid (mRNA) was isolated and reverse transcriptase polymerase chain reaction was used to compare expression profiles. An average of 2,700,000 nucleated cells/ml was isolated; 26% were adherent, and cells completed a cell cycle approximately every 3.3 days. Ring cloning identified 19 colonies. Primary cultures and clones were determined to differentiate along osteogenic, adipogenic and chondrogenic tissue lineages. The mRNA expression profiles showed CD34 expression was higher for undifferentiated ADAS cells versus differentiated cell types and the CD34 expression level was lower than that of CD44 among differentiated cells. Improved culture conditions and defined cellular characteristics of these porcine ADAS cells have been identified. Porcine ADAS can self-renew, can differentiate into multiple tissue lineages and they express CD34. Copyright 2008 S. Karger AG, Basel.

Optimum Aeroelastic Characteristics for Composite Supermaneuverable Aircraft.

DTIC Science & Technology

1986-07-31

1󈨌D22 1 k 16 ’ cD 22YI Y6 WL k 17 ’ cD 22 Y, Y7 k 18 ’ -2cD 26YI1 Y6 gi 0 k19 : -2cD26Y I y 7 k1,10 12 2c Bo 10 1 󈧐D22 1 10 22 cD22 (y2 )2 k23 -2cD...2 6y1Y2 k24 -2cD 2 6 Y2Y 2 3 ,, 11 10 k -S-D + 2cD a 25 12 D22 5 2 12 5 2 k26 cD22 Y2 Y6 k27 cD22 y2 Y7 28 -2cD 2 6 Y2 Y6 k29 -2cD 26Y2 Y7 k’ "" 0y

Comparative study of β-catenin and CD44 immunoexpression in oral lichen planus and squamous cell carcinoma.

PubMed

Zargaran, Massoumeh; Baghaei, Fahimeh; Moghimbeigi, Abbas

2018-04-24

Dysfunction of adhesion molecules is believed to play an early and important role in developing cancer. Accordingly, this study aims to compare beta-catenin (β-catenin) and CD44 expression in oral lichen planus (OLP) as a condition with malignant potential and oral squamous cell carcinoma (OSCC). β-Catenin and CD44 expression were evaluated in 15 patients with epithelial hyperplasia (group A), 20 OLP (group B), and 20 OSCC (group C) by immunohistochemistry. Quantitative and semi-quantitative evaluations revealed β-catenin, and CD44 membranous expression had significant differences among the three groups. Expression of these markers in the OSCC group decreased significantly compared to that of the OLP. Also, nuclear/cytoplasmic expression of β-catenin was significantly different among the three groups, considering that nuclear expression was not observed in any of the epithelial hyperplasia and OLP samples. According to the findings of this study, β-catenin and CD44 can differentiate between behavior of OLP and OSCC, while the precancerous nature of OLP and malignant transformation potential of it are not suggested. © 2018 The International Society of Dermatology.

Clinical relevance of TRKA expression on neuroblastoma: comparison with N-MYC amplification and CD44 expression.

PubMed Central

Combaret, V.; Gross, N.; Lasset, C.; Balmas, K.; Bouvier, R.; Frappaz, D.; Beretta-Brognara, C.; Philip, T.; Favrot, M. C.; Coll, J. L.

1997-01-01

TRKA expression was evaluated on 122 untreated neuroblastomas by immunohistochemistry using an antibody with predetermined specificity. This procedure is simple and reliable for protein detection at cellular level in a routine clinical setting. Fourteen tumours were classified as benign ganglioneuroma with a restricted expression of TRKA on ganglion cells; these patients were excluded from the following analysis. A total of 108 tumours were classified as neuroblastoma or ganglioneuroblastoma; 74 expressed TRKA protein, which strongly correlated with low stage, absence of N-MYC amplification, age (<1 year), CD44 expression and favourable clinical outcome. In a univariate analysis including tumour stage, age, histology, N-MYC amplification, CD44 and TRKA expression, all parameters had significant prognostic value. The absence of TRKA expression on CD44-positive or N-MYC non-amplified tumours permits the characterization of a subgroup of patients with intermediate prognosis. However, in a multivariate analysis taking into consideration the prognostic factors mentioned above, CD44 and tumour stage were the only independent prognostic factors for the prediction of patients' event-free survival. PMID:9099963

Shear Stress Regulates Adhesion and Rolling of CD44+ Leukemic and Hematopoietic Progenitor Cells on Hyaluronan

PubMed Central

Christophis, Christof; Taubert, Isabel; Meseck, Georg R.; Schubert, Mario; Grunze, Michael; Ho, Anthony D.; Rosenhahn, Axel

2011-01-01

Leukemic cells and human hematopoietic progenitor cells expressing CD44 receptors have the ability to attach and roll on hyaluronan. We investigated quantitatively the adhesion behavior of leukemic cell lines and hematopoietic progenitor cells on thin films of the polysaccharides hyaluronan and alginate in a microfluidic system. An applied flow enhances the interaction between CD44-positive cells and hyaluronan if a threshold shear stress of 0.2 dyn/cm2 is exceeded. At shear stress ∼1 dyn/cm2, the cell rolling speed reaches a maximum of 15 μm/s. Leukemic Jurkat and Kasumi-1 cells lacking CD44-expression showed no adhesion or rolling on the polysaccharides whereas the CD44-expressing leukemic cells KG-1a, HL-60, K-562, and hematopoietic progenitor cells attached and rolled on hyaluronan. Interestingly, the observations of flow-induced cell rolling are related to those found in the recruitment of leukocytes to inflammatory sites and the mechanisms of stem-cell homing into the bone marrow. PMID:21806926

Immobilization remediation of Cd-polluted soil with different water condition.

PubMed

Li, Jianrui; Xu, Yingming

2017-05-15

To demonstrate effects of water management on soil Cd immobilization using palygorskite, the investigation evaluated impacts of palygorskite on uptake of Cd present in soils with different water condition by rice plant. Pot experiment results showed that, pH, available Fe and P in untreated soils were higher in continuous flooding than in traditional irrigation and wetting irrigation, which were reasons for lower soil exchangeable Cd and plant Cd in continuous flooding. In control group (untreated soils), compared to traditional irrigation, continuous flooding reduced brown rice Cd by 37.9%, that in wetting irrigation increased by 31.0%. At palygorskite concentrations of 5 g kg -1 , 10 g kg -1 and 15 g kg -1 , brown rice Cd reduced by 16.7%, 44.4% and 55.6% under continuous flooding, 13.8%, 34.5% and 44.8% under traditional irrigation, 13.1%, 36.8% and 47.3% under wetting irrigation (p < 0.05). At the same palygorskite addition, decreasing amplitude of brown rice Cd was higher in continuous flooding than in traditional irrigation and wetting irrigation. Competition for adsorption sites in root coating between Cd 2+ and Fe 2+ was another factor governing plant Cd. In control group, compared to traditional irrigation, root coating Fe(II) increased by 124.5% and root coating Cd reduced by 17.6% upon continuous flooding (p < 0.05). In conclusion, palygorskite addition combined with continuous flooding was an efficacious technique to stabilize Cd in paddy soils. Copyright © 2017 Elsevier Ltd. All rights reserved.

Dexamethasone Suppresses Oxysterol-Induced Differentiation of Monocytic Cells

PubMed Central

Son, Yonghae; Kim, Bo-Young; Eo, Seong-Kug; Park, Young Chul; Kim, Koanhoi

2016-01-01

Oxysterol like 27-hydroxycholesterol (27OHChol) has been reported to induce differentiation of monocytic cells into a mature dendritic cell phenotype. We examined whether dexamethasone (Dx) affects 27OHChol-induced differentiation using THP-1 cells. Treatment of monocytic cells with Dx resulted in almost complete inhibition of transcription and surface expression of CD80, CD83, and CD88 induced by 27OHChol. Elevated surface levels of MHC class I and II molecules induced by 27OHChol were reduced to basal levels by treatment with Dx. A decreased endocytosis ability caused by 27OHChol was recovered by Dx. We also examined effects of Dx on expression of CD molecules involved in atherosclerosis. Increased levels of surface protein and transcription of CD105, CD137, and CD166 by treatment with 27OHChol were significantly inhibited by cotreatment with Dx. These results indicate that Dx inhibits 27OHChol-induced differentiation of monocytic cells into a mature dendritic cell phenotype and expression of CD molecules whose levels are associated with atherosclerosis. In addition, we examined phosphorylation of AKT induced by 27OHChol and effect of Dx, where cotreatment with Dx inhibited the phosphorylation of AKT. The current study reports that Dx regulates oxysterol-mediated dendritic cell differentiation of monocytic cells. PMID:27340507

Dexamethasone Suppresses Oxysterol-Induced Differentiation of Monocytic Cells.

PubMed

Son, Yonghae; Kim, Bo-Young; Eo, Seong-Kug; Park, Young Chul; Kim, Koanhoi

2016-01-01

Oxysterol like 27-hydroxycholesterol (27OHChol) has been reported to induce differentiation of monocytic cells into a mature dendritic cell phenotype. We examined whether dexamethasone (Dx) affects 27OHChol-induced differentiation using THP-1 cells. Treatment of monocytic cells with Dx resulted in almost complete inhibition of transcription and surface expression of CD80, CD83, and CD88 induced by 27OHChol. Elevated surface levels of MHC class I and II molecules induced by 27OHChol were reduced to basal levels by treatment with Dx. A decreased endocytosis ability caused by 27OHChol was recovered by Dx. We also examined effects of Dx on expression of CD molecules involved in atherosclerosis. Increased levels of surface protein and transcription of CD105, CD137, and CD166 by treatment with 27OHChol were significantly inhibited by cotreatment with Dx. These results indicate that Dx inhibits 27OHChol-induced differentiation of monocytic cells into a mature dendritic cell phenotype and expression of CD molecules whose levels are associated with atherosclerosis. In addition, we examined phosphorylation of AKT induced by 27OHChol and effect of Dx, where cotreatment with Dx inhibited the phosphorylation of AKT. The current study reports that Dx regulates oxysterol-mediated dendritic cell differentiation of monocytic cells.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Naqvi, S.M.; Howell, R.D.; Sholas, M.

The whole-body residues of Cd and Pb in the tissues of Louisiana swamp crayfish (Procambarus clarkii) were determined by flame AAS technique. Test animals were collected from roadside ditches alongside major highways. The water and soil samples were also collected from the same sites. The mean Cd and Pb concentrations in crayfish tissues were 0.46 and 0.07, respectively. The levels of Cd and Pb in the water were 0.09 and 0.04; and in soil were 2.85 and 0.87 mg/1, respectively. The concentration of cadmium was 32 and Pb 12 times more than in the water. The bioaccumulation factors (BF) formore » Cd and Pb in crayfish tissues were 5.1 and 1.7, respectively. Alligator weed (Alternanthera philoxiroides) plants were exposed to 0.5 mg/1 Cd-chloride or Pb-nitrate solutions for 3 wk period, thrice. The mean Pb accumulation in roots was 1.31 mg/1, followed by stem (0.078 mg/1), but Cd only accumulated in root (0.83 mg/1). The BF for Pb and Cd in plant tissues were 14.8 and 16.6, respectively. The uptake of metals was time-dependent. These data suggest that although there is no biomagnification of Cd and Pb from alligator weed to crayfish, both metals readily accumulate in field-collected crayfish and laboratory-exposed alligator weed.« less

Umbilical cord-derived mesenchymal stem cells inhibit growth and promote apoptosis of HepG2 cells.

PubMed

Tang, Ying-Mei; Bao, Wei-Min; Yang, Jin-Hui; Ma, Lin-Kun; Yang, Jing; Xu, Ying; Yang, Li-Hong; Sha, Feng; Xu, Zhi-Yuan; Wu, Hua-Mei; Zhou, Wei; Li, Yan; Li, Yu-Hua

2016-09-01

Hepatocellular carcinoma is the fifth most common type of cancer worldwide and remains difficult to treat. The aim of this study was to investigate the effects of mesenchymal stem cells (MSCs) derived from the umbilical cord (UC‑MSCs) on HepG2 hepatocellular carcinoma cells. UC‑MSCs were co‑cultured with HepG2 cells and biomarkers of UC‑MSCs were analyzed by flow cytometry. mRNA and protein expression of genes were determined by reverse transcription‑polymerase chain reaction and flow cytometry, respectively. Passage three and seven UC‑MSCs expressed CD29, CD44, CD90 and CD105, whereas CD34 and CD45 were absent on these cells. Co‑culture with UC‑MSCs inhibited proliferation and promoted apoptosis of HepG2 cells in a time‑dependent manner. The initial seeding density of UC‑MSCs also influenced the proliferation and apoptosis of HepG2 cells, with an increased number of UC‑MSCs causing enhanced proliferation inhibition and cell apoptosis. Co‑culture with UC‑MSCs downregulated mRNA and protein expression of α‑fetoprotein (AFP), Bcl‑2 and Survivin in HepG2 cells. Thus, UC‑MSCs may inhibit growth and promote apoptosis of HepG2 cells through downregulation of AFP, Bcl‑2 and Survivin. US-MSCs may be used as a novel therapy for treating hepatocellular carcinoma in the future.

[Proportion and significance of CD1d(hi)CD5⁺CD19⁺ regulatory B cell in peripheral blood of patients with neuromyelitis optica].

PubMed

Yang, Fen; Huang, Dehui; Cheng, Chen; Wu, Weiping

2015-03-01

To detect the proportion of CD1d(hi)CD5⁺CD19⁺ regulatory B cells (Bregs) in peripheral blood of the patients with neuromyelitis optica (NMO), and explore whether CD1d(hi)CD5⁺CD19⁺ Bregs can play a role as a biomarker in the diagnosis of NMO versus multiple sclerosis (MS). Flow cytometry was performed to detect the proportion of CD1d(hi)CD5⁺CD19⁺ Bregs in peripheral blood from 44 cases of NMO, 38 cases of MS, and 30 healthy controls. The serum level of aquaporin-4 antibody (AQP4-Ab) of patients with NMO was detected by indirect immunofluorescence assay. The proportion of CD1d(hi)CD5⁺CD19⁺ Bregs in CD19⁺ B cells and lymphocytes was significantly lower in NMO group than in MS and control groups; however, there was no significant difference between MS group and control group. The proportion of CD1d(hi)CD5⁺CD19⁺ Bregs in CD19⁺ B cells and lymphocytes was lower in AQP4-Ab-positive NMO patients than in AQP4-Ab-negative NMO patients, and the difference was statistically significant. CD1d(hi)CD5⁺CD19⁺ Bregs may be a biomarker in the differential diagnosis of NMO versus MS.

Mycobacterium tuberculosis infection modulates adipose tissue biology

PubMed Central

Kühl, Anja A.; Kupz, Andreas; Vogelzang, Alexis; Mollenkopf, Hans-Joachim; Löwe, Delia; Bandermann, Silke; Dorhoi, Anca; Brinkmann, Volker

2017-01-01

Mycobacterium tuberculosis (Mtb) primarily resides in the lung but can also persist in extrapulmonary sites. Macrophages are considered the prime cellular habitat in all tissues. Here we demonstrate that Mtb resides inside adipocytes of fat tissue where it expresses stress-related genes. Moreover, perigonadal fat of Mtb-infected mice disseminated the infection when transferred to uninfected animals. Adipose tissue harbors leukocytes in addition to adipocytes and other cell types and we observed that Mtb infection induces changes in adipose tissue biology depending on stage of infection. Mice infected via aerosol showed infiltration of inducible nitric oxide synthase (iNOS) or arginase 1 (Arg1)-negative F4/80+ cells, despite recruitment of CD3+, CD4+ and CD8+ T cells. Gene expression analysis of adipose tissue of aerosol Mtb-infected mice provided evidence for upregulated expression of genes associated with T cells and NK cells at 28 days post-infection. Strikingly, IFN-γ-producing NK cells and Mtb-specific CD8+ T cells were identified in perigonadal fat, specifically CD8+CD44-CD69+ and CD8+CD44-CD103+ subpopulations. Gene expression analysis of these cells revealed that they expressed IFN-γ and the lectin-like receptor Klrg1 and down-regulated CD27 and CD62L, consistent with an effector phenotype of Mtb-specific CD8+ T cells. Sorted NK cells expressed higher abundance of Klrg1 upon infection, as well. Our results reveal the ability of Mtb to persist in adipose tissue in a stressed state, and that NK cells and Mtb-specific CD8+ T cells infiltrate infected adipose tissue where they produce IFN-γ and assume an effector phenotype. We conclude that adipose tissue is a potential niche for Mtb and that due to infection CD8+ T cells and NK cells are attracted to this tissue. PMID:29040326

Cyclodextrin Complexes of Reduced Bromonoscapine in Guar Gum Microspheres Enhance Colonic Drug Delivery

PubMed Central

2015-01-01

Here, we report improved solubility and enhanced colonic delivery of reduced bromonoscapine (Red-Br-Nos), a cyclic ether brominated analogue of noscapine, upon encapsulation of its cyclodextrin (CD) complexes in bioresponsive guar gum microspheres (GGM). Phase–solubility analysis suggested that Red-Br-Nos complexed with β-CD and methyl-β-CD in a 1:1 stoichiometry, with a stability constant (Kc) of 2.29 × 103 M–1 and 4.27 × 103 M–1. Fourier transforms infrared spectroscopy indicated entrance of an O–CH2 or OCH3–C6H4–OCH3 moiety of Red-Br-Nos in the β-CD or methyl-β-CD cavity. Furthermore, the cage complex of Red-Br-Nos with β-CD and methyl-β-CD was validated by several spectral techniques. Rotating frame Overhauser enhancement spectroscopy revealed that the Ha proton of the OCH3–C6H4–OCH3 moiety was closer to the H5 proton of β-CD and the H3 proton of the methyl-β-CD cavity. The solubility of Red-Br-Nos in phosphate buffer saline (PBS, pH ∼ 7.4) was improved by ∼10.7-fold and ∼21.2-fold when mixed with β-CD and methyl-β-CD, respectively. This increase in solubility led to a favorable decline in the IC50 by ∼2-fold and ∼3-fold for Red-Br-Nos−β-CD-GGM and Red-Br-Nos–methyl-β-CD-GGM formulations respectively, compared to free Red-Br-Nos−β-CD and Red-Br-Nos–methyl-β-CD in human colon HT-29 cells. GGM-bearing drug complex formulations were found to be highly cytotoxic to the HT-29 cell line and further effective with simultaneous continuous release of Red-Br-Nos from microspheres. This is the first study to showing the preparation of drug-complex loaded GGMS for colon delivery of Red-Br-Nos that warrants preclinical assessment for the effective management of colon cancer. PMID:25350222

Prenatal cadmium exposure produces persistent changes to thymus and spleen cell phenotypic repertoire as well as the acquired immune response

DOE Office of Scientific and Technical Information (OSTI.GOV)

Holásková, Ida; Elliott, Meenal; Hanson, Miranda L.

2012-12-01

Cadmium (Cd) is a common environmental contaminant. Adult exposure to Cd alters the immune system, however, there are limited studies on the effects of prenatal exposure to Cd. Pregnant C57Bl/6 mice were exposed to an environmentally relevant dose of CdCl{sub 2} (10 ppm) and the effects on the immune system of the offspring were assessed at 20 weeks of age. Prenatal Cd exposure caused an increase in the percent of CD4{sup −}CD8{sup −}CD44{sup +}CD25{sup −} (DN1) thymocytes in both sexes and a decrease in the percent of CD4{sup −}CD8{sup −}CD44{sup −}CD25{sup +} (DN3) thymocytes in females. Females had an increasemore » in the percent of splenic CD4{sup +} T cells, CD8{sup +} T cells, and CD45R/B220{sup +} B cells and a decrease in the percent of NK cells and granulocytes (Gr-1{sup +}). Males had an increase in the percent of splenic CD4{sup +} T cells and CD45R/B220{sup +} B cells and a decrease in the percent of CD8{sup +} T cells, NK cells, and granulocytes. The percentage of neutrophils and myeloid-derived suppressor cells were reduced in both sexes. The percent of splenic nTreg cells was decreased in all Cd-exposed offspring. Cd-exposed offspring were immunized with a streptococcal vaccine and the antibody response was determined. PC-specific serum antibody titers were decreased in Cd exposed female offspring but increased in the males. PspA-specific serum IgG titers were increased in both females and males compared to control animals. Females had a decrease in PspA-specific serum IgM antibody titers. Females and males had a decrease in the number of splenic anti-PspA antibody-secreting cells when standardized to the number of B cells. These findings demonstrate that very low levels of Cd exposure during gestation can result in long term sex-specific alterations on the immune system of the offspring. -- Highlights: ► Prenatal exposure to cadmium alters the immune system of 20 week old offspring. ► The percentage of DN1 and DN3 thymocytes was changed. ► Males and females had changed percentages of numerous splenic cell populations. ► The antibody response of a streptococcal vaccine showed numerous changes.« less

Human mesenchymal stem cells - current trends and future prospective

PubMed Central

Ullah, Imran; Subbarao, Raghavendra Baregundi; Rho, Gyu Jin

2015-01-01

Stem cells are cells specialized cell, capable of renewing themselves through cell division and can differentiate into multi-lineage cells. These cells are categorized as embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs) and adult stem cells. Mesenchymal stem cells (MSCs) are adult stem cells which can be isolated from human and animal sources. Human MSCs (hMSCs) are the non-haematopoietic, multipotent stem cells with the capacity to differentiate into mesodermal lineage such as osteocytes, adipocytes and chondrocytes as well ectodermal (neurocytes) and endodermal lineages (hepatocytes). MSCs express cell surface markers like cluster of differentiation (CD)29, CD44, CD73, CD90, CD105 and lack the expression of CD14, CD34, CD45 and HLA (human leucocyte antigen)-DR. hMSCs for the first time were reported in the bone marrow and till now they have been isolated from various tissues, including adipose tissue, amniotic fluid, endometrium, dental tissues, umbilical cord and Wharton's jelly which harbours potential MSCs. hMSCs have been cultured long-term in specific media without any severe abnormalities. Furthermore, MSCs have immunomodulatory features, secrete cytokines and immune-receptors which regulate the microenvironment in the host tissue. Multilineage potential, immunomodulation and secretion of anti-inflammatory molecules makes MSCs an effective tool in the treatment of chronic diseases. In the present review, we have highlighted recent research findings in the area of hMSCs sources, expression of cell surface markers, long-term in vitro culturing, in vitro differentiation potential, immunomodulatory features, its homing capacity, banking and cryopreservation, its application in the treatment of chronic diseases and its use in clinical trials. PMID:25797907

Fabrication of human hair keratin/jellyfish collagen/eggshell-derived hydroxyapatite osteoinductive biocomposite scaffolds for bone tissue engineering: From waste to regenerative medicine products.

PubMed

Arslan, Yavuz Emre; Sezgin Arslan, Tugba; Derkus, Burak; Emregul, Emel; Emregul, Kaan C

2017-06-01

In the present study, we aimed at fabricating an osteoinductive biocomposite scaffold using keratin obtained from human hair, jellyfish collagen and eggshell-derived nano-sized spherical hydroxyapatite (nHA) for bone tissue engineering applications. Keratin, collagen and nHA were characterized with the modified Lowry method, free-sulfhydryl groups and hydroxyproline content analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), attenuated total reflectance-fourier transform infrared spectroscopy (ATR-FTIR) and thermal gravimetric analysis (TGA) which confirmed the success of the extraction and/or isolation processes. Human adipose mesenchymal stem cells (hAMSCs) were isolated and the cell surface markers were characterized via flow cytometry analysis in addition to multilineage differentiation capacity. The undifferentiated hAMSCs were highly positive for CD29, CD44, CD73, CD90 and CD105, but were not seen to express hematopoietic cell surface markers such as CD14, CD34 and CD45. The cells were successfully directed towards osteogenic, chondrogenic and adipogenic lineages in vitro. The microarchitecture of the scaffolds and cell attachment were evaluated using scanning electron microscopy (SEM). The cell viability on the scaffolds was assessed by the MTT assay which revealed no evidence of cytotoxicity. The osteogenic differentiation of hAMSCs on the scaffolds was determined histologically using alizarin red S, osteopontin and osteonectin stainings. Early osteogenic differentiation markers of hAMSCs were significantly expressed on the collagen-keratin-nHA scaffolds. In conclusion, it is believed that collagen-keratin-nHA osteoinductive biocomposite scaffolds have the potential of being used in bone tissue engineering. Copyright © 2017 Elsevier B.V. All rights reserved.

Association Between Cd Exposure and Risk of Prostate Cancer: A PRISMA-Compliant Systematic Review and Meta-Analysis.

PubMed

Ju-Kun, Song; Yuan, Dong-Bo; Rao, Hao-Fu; Chen, Tian-Fei; Luan, Bo-Shi; Xu, Xiao-Ming; Jiang, Fu-Neng; Zhong, Wei-De; Zhu, Jian-Guo

2016-02-01

Several observational studies on the association between Cd exposure and risk of prostate cancer have yielded inconsistent results. To address this issue, we conducted a meta-analysis to evaluate the correlation between Cd exposure and risk of prostate cancer.Relevant studies in PubMed and Embase databases were retrieved until October 2015. We compared the highest and lowest meta-analyses to quantitatively evaluate the relationship between Cd exposure and risk of prostate cancer. Summary estimates were obtained using a random-effects model.In the general population, high Cd exposure was not associated with increased prostate cancer (OR 1.21; 95% CI 0.91-1.64), whereas the combined standardized mortality ratio of the association between Cd exposure and risk of prostate cancer was 1.66 (95% CI 1.10-2.50) in populations exposed to occupational Cd. In addition, high D-Cd intake (OR 1.07; 95% CI 0.96-1.20) and U-Cd concentration (OR 0.86; 95% CI 0.48-1.55) among the general population was not related to the increased risk of prostate cancer. In the dose analysis, the summary relative risk was 1.07 (95% CI 0.73-1.57) for each 0.5 μg/g creatinine increase in U-Cd and 1.02 (95% CI 0.99-1.06) for each 10 μg/day increase of dietary Cd intake. However, compared with nonoccupational exposure, high occupational Cd exposure may be associated with the increased risk of prostate cancer.This meta-analysis suggests high Cd exposure as a risk factor for prostate cancer in occupational rather than nonoccupational populations. However, these results should be carefully interpreted because of the significant heterogeneity among studies. Additional large-scale and high-quality prospective studies are needed to confirm the association between Cd exposure and risk of prostate cancer.

Association Between Cd Exposure and Risk of Prostate Cancer

PubMed Central

Ju-Kun, Song; Yuan, Dong-Bo; Rao, Hao-Fu; Chen, Tian-Fei; Luan, Bo-Shi; Xu, Xiao-Ming; Jiang, Fu-Neng; Zhong, Wei-De; Zhu, Jian-Guo

2016-01-01

Abstract Several observational studies on the association between Cd exposure and risk of prostate cancer have yielded inconsistent results. To address this issue, we conducted a meta-analysis to evaluate the correlation between Cd exposure and risk of prostate cancer. Relevant studies in PubMed and Embase databases were retrieved until October 2015. We compared the highest and lowest meta-analyses to quantitatively evaluate the relationship between Cd exposure and risk of prostate cancer. Summary estimates were obtained using a random-effects model. In the general population, high Cd exposure was not associated with increased prostate cancer (OR 1.21; 95% CI 0.91–1.64), whereas the combined standardized mortality ratio of the association between Cd exposure and risk of prostate cancer was 1.66 (95% CI 1.10–2.50) in populations exposed to occupational Cd. In addition, high D-Cd intake (OR 1.07; 95% CI 0.96–1.20) and U-Cd concentration (OR 0.86; 95% CI 0.48–1.55) among the general population was not related to the increased risk of prostate cancer. In the dose analysis, the summary relative risk was 1.07 (95% CI 0.73–1.57) for each 0.5 μg/g creatinine increase in U-Cd and 1.02 (95% CI 0.99–1.06) for each 10 μg/day increase of dietary Cd intake. However, compared with nonoccupational exposure, high occupational Cd exposure may be associated with the increased risk of prostate cancer. This meta-analysis suggests high Cd exposure as a risk factor for prostate cancer in occupational rather than nonoccupational populations. However, these results should be carefully interpreted because of the significant heterogeneity among studies. Additional large-scale and high-quality prospective studies are needed to confirm the association between Cd exposure and risk of prostate cancer. PMID:26871808

Mouse and human HSPC immobilization in liquid culture by CD43- or CD44-antibody coating.

PubMed

Loeffler, Dirk; Wang, Weijia; Hopf, Alois; Hilsenbeck, Oliver; Bourgine, Paul E; Rudolf, Fabian; Martin, Ivan; Schroeder, Timm

2018-03-29

Keeping track of individual cell identifications is imperative to the study of dynamic single-cell behavior over time. Highly motile hematopoietic stem and progenitor cells (HSPCs) migrate quickly and do not adhere, and thus must be imaged very frequently to keep cell identifications. Even worse, they are also flushed away during medium exchange. To overcome these limitations, we tested antibody coating for reducing HSPC motility in vitro. Anti-CD43- and anti-CD44-antibody coating reduced the cell motility of mouse and human HSPCs in a concentration-dependent manner. This enables 2-dimensional (2D) colony formation without cell mixing in liquid cultures, massively increases time-lapse imaging throughput, and also maintains cell positions during media exchange. Anti-CD43 but not anti-CD44 coating reduces mouse HSPC proliferation with increasing concentrations. No relevant effects on cell survival or myeloid and megakaryocyte differentiation of hematopoietic stem cells and multipotent progenitors 1-5 were detected. Human umbilical cord hematopoietic CD34 + cell survival, proliferation, and differentiation were not affected by either coating. This approach both massively simplifies and accelerates continuous analysis of suspension cells, and enables the study of their behavior in dynamic rather than static culture conditions over time. © 2018 by The American Society of Hematology.

Analysis of circulating CD14+/CD16+ monocyte-derived macrophages (MDMs) in the peripheral blood of patients with oral squamous cell carcinoma.

PubMed

Grimm, Martin; Feyen, Oliver; Coy, Johannes F; Hofmann, Heiko; Teriete, Peter; Reinert, Siegmar

2016-03-01

Monocytes/macrophages are regarded as the first line of defense in tumors. Therefore, analyzing monocyte subtypes in oral squamous cell carcinoma (OSCC) may be of value in disease monitoring and to explore immunotherapeutic strategies for cancer patients. Circulating peripheral blood CD14+/CD16+ monocyte-derived macrophages (MDMs) were evaluated in OSCC patients with oral squamous cell carcinoma (n = 44) compared with controls (n = 85). Moreover, epitope detection in monocytes (EDIM) technology was used to detect biomarkers Apo10 and transketolase-like-1 in CD14+/CD16+ MDMs. Compared with controls, no significant (P = .3646) difference (control group 9.8%, OSCC group 8.8%) in CD14+/CD16+ MDM were noted in OSCC. However, EDIM-Apo10 and EDIM-TKTL1 scores detected in the CD14+/CD16+ MDMs were increased in OSCC compared with controls (P < .0001). Analyzing CD14+/CD16+ MDMs represents a stable cell population for detecting biomarkers in cancer disease monitoring. Copyright © 2016 Elsevier Inc. All rights reserved.

CD24 negative lung cancer cells, possessing partial cancer stem cell properties, cannot be considered as cancer stem cells.

PubMed

Xu, Haineng; Mu, Jiasheng; Xiao, Jing; Wu, Xiangsong; Li, Maolan; Liu, Tianrun; Liu, Xinyuan

2016-01-01

Cancer stem cells (CSCs) play vital role in lung cancer progression, resistance, metastasis and relapse. Identifying lung CSCs makers for lung CSCs targeting researches are critical for lung cancer therapy. In this study, utilizing previous identified lung CSCs as model, we compared the expression of CD24, CD133 and CD44 between CSCs and non-stem cancer cells. Increased ratio of CD24- cells were found in CSCs. CD24- cells were then sorted by flow cytometry and their proliferative ability, chemo-resistance property and in vivo tumor formation abilities were detected. A549 CD24- cells formed smaller colonies, slower proliferated in comparison to A549 CD24+ cells. Besides, A549 CD24- exhibited stronger resistance to chemotherapy drug. However, A549 CD24- didn't exert any stronger tumor formation ability in vivo, which is the gold standard of CSCs. These results showed that CD24- A549 cells showed some properties of CSCs but not actually CSCs. This study provides evidence that CD24 cannot be considered as lung CSCs marker.

The role of CD133 in the identification and characterisation of tumour-initiating cells in non-small-cell lung cancer.

PubMed

Tirino, Virginia; Camerlingo, Rosa; Franco, Renato; Malanga, Donatella; La Rocca, Antonello; Viglietto, Giuseppe; Rocco, Gaetano; Pirozzi, Giuseppe

2009-09-01

Emerging evidence suggests that specific sub-populations of cancer cells with stem cell characteristics within the bulk of tumours are implicated in the pathogenesis of heterogeneous malignant tumours. The cells that drive tumour growth have been denoted cancer-initiating cells or cancer stem cells (hereafter CSCs). CSCs have been isolated initially from leukaemias and subsequently from several solid tumours including brain, breast, prostate, colon and lung cancer. This study aimed at isolating and characterising the population of tumour-initiating cells in non-small-cell lung cancer (NSCLC). Specimens of NSCLC obtained from 89 patients undergoing tumour resection at the Cancer National Institute of Naples were analysed. Three methods to isolate the tumour-initiating cells were used: (1) flow cytometry analysis for identification of positive cells for surface markers such as CD24, CD29, CD31, CD34, CD44, CD133 and CD326; (2) Hoechst 33342 dye exclusion test for the identification of a side-population characteristic for the presence of stem cells; (3) non-adherent culture condition able to form spheres with stem cell-like characteristics. Definition of the tumourigenic potential of the cells through soft agar assay and injection into NOD/SCID mice were used to functionally define (in vitro and in vivo) putative CSCs isolated from NSCLC samples. Upon flow cytometry analysis of NSCLC samples, CD133-positive cells were found in 72% of 89 fresh specimens analysed and, on average, represented 6% of the total cells. Moreover, the number of CD133-positive cells increased markedly when the cells, isolated from NSCLC specimens, were grown as spheres in non-adherent culture conditions. Cells from NSCLC, grown as spheres, when assayed in soft agar, give rise to a 3.8-fold larger number of colonies in culture and are more tumourigenic in non-obese diabetic (NOD)/severe combined immunodeficiency (SCID) mice compared with the corresponding adherent cells. We have isolated and characterised a population of CD133-positive cells from NSCLC that is able to give rise to spheres and can act as tumour-initiating cells.

Does haemochromatosis predispose to coeliac disease? A study of 29,096 coeliac disease patients

PubMed Central

Ludvigsson, Jonas F; Murray, Joseph A; Adams, Paul C; Elmberg, Maria

2013-01-01

Background and aim Case reports suggest an association between hereditary haemochromatosis (HH) and coeliac disease (CD), but estimates of association are lacking. We estimated the association between HH and CD in a population-based study. Materials and methods Case-control study. We identified 29,096 individuals with biopsy-verified CD (equal to villous atrophy, Marsh stage III) through biopsy reports from all 28 pathology departments in Sweden. We then investigated the risk of a clinical diagnosis of HH in CD and in 144,522 controls matched for age, sex, county and calendar year. Conditional logistic regression was used to calculate odds ratios (ORs) for CD in patients with HH. Results HH was seen in 30 patients with CD and in 60 matched controls. HH was hence associated with an increased risk of CD (OR=2.30; 95%CI=1.53–3.25). Restricting HH to individuals with at least 2 records of HH, the OR for CD was 2.54 (95%CI=1.57–4.11), with a similar risk estimate when we only looked at HH diagnosed before CD (and matched date in controls) (OR=2.64; 95%CI=1.24–5.60). Conclusion HH seems to be associated with an increased risk of CD. PMID:23256862

Cysteine-rich Domain 1 of CD40 Mediates Receptor Self-assembly*

PubMed Central

Smulski, Cristian R.; Beyrath, Julien; Decossas, Marion; Chekkat, Neila; Wolff, Philippe; Estieu-Gionnet, Karine; Guichard, Gilles; Speiser, Daniel; Schneider, Pascal; Fournel, Sylvie

2013-01-01

The activation of CD40 on B cells, macrophages, and dendritic cells by its ligand CD154 (CD40L) is essential for the development of humoral and cellular immune responses. CD40L and other TNF superfamily ligands are noncovalent homotrimers, but the form under which CD40 exists in the absence of ligand remains to be elucidated. Here, we show that both cell surface-expressed and soluble CD40 self-assemble, most probably as noncovalent dimers. The cysteine-rich domain 1 (CRD1) of CD40 participated to dimerization and was also required for efficient receptor expression. Modelization of a CD40 dimer allowed the identification of lysine 29 in CRD1, whose mutation decreased CD40 self-interaction without affecting expression or response to ligand. When expressed alone, recombinant CD40-CRD1 bound CD40 with a KD of 0.6 μm. This molecule triggered expression of maturation markers on human dendritic cells and potentiated CD40L activity. These results suggest that CD40 self-assembly modulates signaling, possibly by maintaining the receptor in a quiescent state. PMID:23463508

Immunohistochemical expression of CD44s in human neuroblastic tumors: Moroccan experience and highlights on current data

PubMed Central

2013-01-01

Background Peripheral neuroblastic tumors (pNTs), including neuroblastoma (NB), ganglioneuroblastoma (GNB) and ganglioneuroma (GN), are extremely heterogeneous pediatric tumors responsible for 15 % of childhood cancer death. The aim of the study was to evaluate the expression of CD44s (‘s’: standard form) cell adhesion molecule by comparison with other specific prognostic markers. Methods An immunohistochemical profile of 32 formalin-fixed paraffin-embedded pNTs tissues, diagnosed between January 2007 and December 2010, was carried out. Results Our results have demonstrated the association of CD44s negative pNTs cells to lack of differentiation and tumour progression. A significant association between absence of CD44s expression and metastasis in human pNTs has been reported. We also found that expression of CD44s defines subgroups of patients without MYCN amplification as evidenced by its association with low INSS stages, absence of metastasis and favorable Shimada histology. Discussion These findings support the thesis of the role of CD44s glycoprotein in the invasive growth potential of neoplastic cells and suggest that its expression could be taken into consideration in the therapeutic approaches targeting metastases. Virtual Slides The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1034403150888863 Résumé Introduction les tumeurs neuroblastiques périphériques (TNPs), comprenant le neuroblastome (NB), le ganglioneuroblastome (GNB) et le ganglioneurome (GN), sont des tumeurs pédiatriques extrêmement hétérogènes responsables de 15% des décès par cancer chez les enfants. Le but de cette étude était d’évaluer l’expression de la molécule d’adhésion cellulaire CD44s (‘s’: pour standard) par rapport à d’autres facteurs pronostiques spécifiques. Méthodes Un profil immunohistochimique de 32 TNPs fixées au formol et incluses en paraffine, diagnostiquées entre Janvier 2007 et Décembre 2010, a été réalisé. Résultats Nos résultats ont mis en évidence l’association des TNPs n’exprimant pas le CD44s avec une perte de différenciation et une progression tumorale et nous avons rapporté une association significative entre l’absence d’expression du CD44s et la présence de métastases. Nous avons également constaté que l’expression du CD44s définit des sous-groupes de patients dans les tumeurs n’amplifiant pas le MYCN, comme en témoigne son association avec les stades INSS bas, l’absence de métastases et l’histologie favorable de Shimada. Discussion Ces résultats appuient l’hypothèse du rôle de la glycoprotéine CD44s dans le potentiel de croissance invasive des cellules néoplasiques et suggèrent que son expression pourrait être prise en considération dans des voies thérapeutiques ciblant les métastases. PMID:23445749

A Case-Cohort Study of Cadmium Body Burden and Gestational Diabetes Mellitus in American Women.

PubMed

Romano, Megan E; Enquobahrie, Daniel A; Simpson, Christopher D; Checkoway, Harvey; Williams, Michelle A

2015-10-01

Environmental cadmium (Cd) exposure is associated with type 2 diabetes. However, the association of Cd and gestational diabetes mellitus (GDM) is unknown. We examined the association between body burden of Cd and GDM risk. We used 140 GDM cases and 481 randomly selected noncase subcohort members from the Omega Study to conduct a case-cohort study. Creatinine (Cr)-corrected Cd in early pregnancy urine (U-Cd) was measured by inductively coupled plasma mass spectrometry. Tertiles (< 0.29; 0.29-0.42; ≥ 0.43 μg/g Cr) were defined using the subcohort's U-Cd distribution. GDM was diagnosed using the 2004 American Diabetes Association guidelines. Adjusted odds ratios (ORs) and 95% confidence intervals (CIs) were estimated using logistic regression. GDM cases had higher geometric mean U-Cd (0.39 μg/g Cr; 95% CI: 0.37, 0.41) than noncases (0.31 μg/g Cr; 95% CI: 0.29, 0.33). Odds ratios for GDM increased with increasing U-Cd tertile (OR = 1.64; 95% CI: 0.88, 3.05 for middle vs. low tertile; OR = 2.07; 95% CI: 1.15, 3.73 for high vs. low tertile; p-trend = 0.015). Overweight/obesity (body mass index ≥ 25 kg/m(2)) did not modify the association between U-Cd and GDM (p = 0.26). Our findings suggest that body burden of Cd increases risk of GDM in a dose-dependent manner. Improved understanding of environmental factors influencing GDM may facilitate early identification of women at high risk of GDM.

A placebo controlled observer blind immunocytochemical and histologic study of epithelium adjacent to anogenital warts in patients treated with systemic interferon alpha in combination with cryotherapy or cryotherapy alone.

PubMed Central

Handley, J M; Maw, R D; Horner, T; Lawther, H; Walsh, M; Dinsmore, W W

1992-01-01

OBJECTIVE--To examine biopsy specimens of tissue immediately adjacent to anogenital (AG) warts which had been treated with either cryotherapy plus subcutaneous interferon (IFN) alpha 2a or cryotherapy alone, for histological features of (a) human papilloma virus (HPV) infection (b) localised cellular immune responses, to further characterise any cellular immune infiltrates with tissue immunocytochemistry, and to relate any histological, immunocytochemical findings to the treatment response of nearby AG warts. DESIGN--A randomised placebo controlled observer blind study. SETTING--Genitourinary Medicine clinic, Department of Immunopathology, Royal Victoria Hospital, Belfast, N. Ireland. SUBJECTS--Thirty patients with AG warts; 16 treated with IFN alpha 2a plus cryotherapy, and 14 treated with cryotherapy alone. OUTCOME MEASURES--(1) Light microscopic features associated with HPV infection and local cellular immune responses. (2) Indirect immunofluorescence detection of the following cell surface markers: HLA DR, alpha one antitrypsin, CD1, CD3, CD4, CD8, CD22. (3) Clinical response of AG warts to treatment. RESULTS--In pre-treatment biopsies only non specific indicators of HPV infection (acanthosis, 29/30 biopsies, and hyperkeratosis, 7/30 biopsies) were seen on light microscopy. Mononuclear cells were seen both throughout the upper dermis and centred around dermal blood vessels in 19/30 (63.3%) biopsies, and infiltrating into the epidermis in 12/30 (40%) biopsies. On indirect immunofluorescence CD3, CD8, CD4 antigen was detected on the surface of cells throughout the upper dermis in 24/29 (82.7%), 15/29 (51.7%), and 3/29 (10.3%), of biopsy specimens respectively. CD3 antigen, CD8 antigen and CD4 antigen was detected on the surface of cells infiltrating into the epidermis in 18/29 (62%), 7/29 (24.1%), and 6/29 (20.7%) of biopsy specimens respectively. CD1 antigen was seen on the surface of dendritic cells throughout the epidermis in all specimens; CD1 positive cells infiltrated into the upper dermis in 5/29 (17.2%). HLA DR was detected on the surface of dendritic cells throughout the epidermis in 22/29 (75.9%) of specimens, and on the surface of cells scattered both diffusely throughout the upper dermis and centred around dermal blood vessels in all specimens. Alpha one antitrypsin (A1AT) antigen was seen on the surface of cells in the upper dermis in 6/29 (20.7%) of biopsy specimens; no cells expressing CD22 surface antigen were seen. The nature of this local cellular immune response was not altered by treatment of nearby warts with either cryotherapy alone or cryotherapy plus systemic IFN alpha 2a, or related to the therapeutic outcome of these warts. CONCLUSIONS--(1) No convincing histological evidence of HPV infection was seen in epithelium surrounding AG warts. (2) A predominantly T cell-mediated immune response (the target of which is uncertain) was seen in this perilesional epithelium. (3) In the dosage regimens used in this study, treatment of AG warts with either systemic IFN alpha 2a plus cryotherapy or cryotherapy alone did not appear to augment localised cellular immune responses (against any presumed subclinical HPV infection) in epithelium surrounding AG warts. Images PMID:1316307

Semisynthesis of SY-1 for investigation of breast cancer stem cell selectivity of C-ring-modified salinomycin analogues.

PubMed

Huang, Xiaoli; Borgström, Björn; Månsson, Linda; Persson, Lo; Oredsson, Stina; Hegardt, Cecilia; Strand, Daniel

2014-07-18

Salinomycin, a naturally occurring polyether ionophore was recently found to selectively reduce the proportion of CD44(+)/CD24(-) cells, a phenotype associated with breast cancer stem cells. Subsequent studies from our group showed that chemical modification of the allylic C20 hydroxyl of salinomycin, located at the C-ring, can enhance the activity of derivatives against breast cancer cells over 5-fold compared to the native structure. Access to C-ring-modified salinomycin analogues is thus of interest from both a mechanistic and a synthetic perspective. Here, we report efficient strategies for gram scale synthesis of the natural product SY-1 (20-deoxy salinomycin), and a saturated analogue, 18,19-dihydro SY-1, for a comparative in vitro investigation of the biological profiles of these compounds with that of salinomycin. Across several assays, the deoxygenated structures required higher concentrations to elicit similar cellular responses to that of salinomycin. Similarly to salinomycin, SY-1 or 18,19-dihydro SY-1 treatment was found to reduce the proportion of CD44(+)/CD24(-) cells with essentially complete selectivity up to ∼IC25. Importantly, the proportion of CD44(+)/CD24(-) cells showed a pronounced U-shaped dose response curve for salinomycin and its derivatives, but not for paclitaxel. The concentration for maximum response in this assay followed differences in IC50 for salinomycin and its analogues, which emphasizes the importance of taking concentration dependence into account when comparing effects on the CD44(+)/CD24(-) phenotype. Small differences in the global conformation within the triad of compounds investigated together with differences in activity across assays emphasize the importance of substitution at C20 for the activity of salinomycin and its derivatives.

Clinically relevant morphological structures in breast cancer represent transcriptionally distinct tumor cell populations with varied degrees of epithelial-mesenchymal transition and CD44+CD24- stemness

PubMed Central

Denisov, Evgeny V.; Skryabin, Nikolay A.; Gerashchenko, Tatiana S.; Tashireva, Lubov A.; Wilhelm, Jochen; Buldakov, Mikhail A.; Sleptcov, Aleksei A.; Lebedev, Igor N.; Vtorushin, Sergey V.; Zavyalova, Marina V.; Cherdyntseva, Nadezhda V.; Perelmuter, Vladimir M.

2017-01-01

Intratumor morphological heterogeneity in breast cancer is represented by different morphological structures (tubular, alveolar, solid, trabecular, and discrete) and contributes to poor prognosis; however, the mechanisms involved remain unclear. In this study, we performed 3D imaging, laser microdissection-assisted array comparative genomic hybridization and gene expression microarray analysis of different morphological structures and examined their association with the standard immunohistochemistry scorings and CD44+CD24- cancer stem cells. We found that the intratumor morphological heterogeneity is not associated with chromosomal aberrations. By contrast, morphological structures were characterized by specific gene expression profiles and signaling pathways and significantly differed in progesterone receptor and Ki-67 expression. Most importantly, we observed significant differences between structures in the number of expressed genes of the epithelial and mesenchymal phenotypes and the association with cancer invasion pathways. Tubular (tube-shaped) and alveolar (spheroid-shaped) structures were transcriptionally similar and demonstrated co-expression of epithelial and mesenchymal markers. Solid (large shapeless) structures retained epithelial features but demonstrated an increase in mesenchymal traits and collective cell migration hallmarks. Mesenchymal genes and cancer invasion pathways, as well as Ki-67 expression, were enriched in trabecular (one/two rows of tumor cells) and discrete groups (single cells and/or arrangements of 2-5 cells). Surprisingly, the number of CD44+CD24- cells was found to be the lowest in discrete groups and the highest in alveolar and solid structures. Overall, our findings indicate the association of intratumor morphological heterogeneity in breast cancer with the epithelial-mesenchymal transition and CD44+CD24- stemness and the appeal of this heterogeneity as a model for the study of cancer invasion. PMID:28977854

Coexpression of CD14 and CD326 discriminate hepatic precursors in the human fetal liver.

PubMed

Fomin, Marina E; Tai, Lung-Kuo; Bárcena, Alicia; Muench, Marcus O

2011-07-01

The molecular and cellular profile of liver cells during early human development is incomplete, complicating the isolation and study of hepatocytes, cholangiocytes, and hepatic stem cells from the complex amalgam of hepatic and hematopoietic cells, that is, the fetal liver. Epithelial cell adhesion molecule, CD326, has emerged as a marker of hepatic stem cells, and lipopolysaccharide receptor CD14 is known to be expressed on adult hepatocytes. Using flow cytometry, we studied the breadth of CD326 and CD14 expression in midgestation liver. Both CD45(+) hematopoietic and CD45(-) nonhematopoietic cells expressed CD326. Moreover, diverse cell types expressing CD326 were revealed among CD45(-) cells by costaining for CD14. Fluorescence-activated cell sorting was used to isolate nonhematopoietic cells distinguished by expression of high levels of CD326 and low CD14 (CD326(++)CD14(lo)), which were characterized for gene expression associated with liver development. CD326(++)CD14(lo) cells expressed the genes albumin, α-fetoprotein, hepatic nuclear factor 3α, prospero-related homeobox 1, cytochrome P450 3A7, α(1)-antitrypsin, and transferrin. Proteins expressed included cell-surface CD24, CD26, CD29, CD34, CD49f, CD243, and CD324 and, in the cytoplasm, cytokeratins-7/8 (CAM 5.2 antigen) and some cytokeratin-19. Cultured CD326(++)CD14(lo) cells yielded albumin(+) hepatocytes, cytokeratin-19(+) cholangiocytes, and hepatoblasts expressing both markers. Using epifluorescence microscopy we observed CD326 and CD14 expression on fetal hepatocytes comprising the liver parenchyma, as well as on cells associated with ductal plates and surrounding large vessels. These findings indicate that expression of CD14 and CD326 can be used to identify functionally distinct subsets of fetal liver cells, including CD326(++)CD14(lo) cells, representing a mixture of parenchymal cells, cholangiocytes, and hepatoblasts.

Mammary stem cell and macrophage markers are enriched in normal tissue adjacent to inflammatory breast cancer.

PubMed

Reddy, Jay P; Atkinson, Rachel L; Larson, Richard; Burks, Jared K; Smith, Daniel; Debeb, Bisrat G; Ruffell, Brian; Creighton, Chad J; Bambhroliya, Arvind; Reuben, James M; Van Laere, Steven J; Krishnamurthy, Savitri; Symmans, William F; Brewster, Abenaa M; Woodward, Wendy A

2018-06-01

We hypothesized that breast tissue not involved by tumor in inflammatory breast cancer (IBC) patients contains intrinsic differences, including increased mammary stem cells and macrophage infiltration, which may promote the IBC phenotype. Normal breast parenchyma ≥ 5 cm away from primary tumors was obtained from mastectomy specimens. This included an initial cohort of 8 IBC patients and 60 non-IBC patients followed by a validation cohort of 19 IBC patients and 25 non-IBC patients. Samples were immunostained for either CD44 + CD49f + CD133/2 + mammary stem cell markers or the CD68 macrophage marker and correlated with IBC status. Quantitation of positive cells was determined using inForm software from PerkinElmer. We also examined the association between IBC status and previously published tumorigenic stem cell and IBC tumor signatures in the validation cohort samples. 8 of 8 IBC samples expressed isolated CD44 + CD49f + CD133/2 + stem cell marked cells in the initial cohort as opposed to 0/60 non-IBC samples (p = 0.001). Similarly, the median number of CD44 + CD49f + CD133/2 + cells was significantly higher in the IBC validation cohort as opposed to the non-IBC validation cohort (25.7 vs. 14.2, p = 0.007). 7 of 8 IBC samples expressed CD68 + histologically confirmed macrophages in initial cohort as opposed to 12/48 non-IBC samples (p = 0.001). In the validation cohort, the median number of CD68 + cells in IBC was 3.7 versus 1.0 in the non-IBC cohort (p = 0.06). IBC normal tissue was positively associated with a tumorigenic stem cell signature (p = 0.02) and with a 79-gene IBC signature (p < 0.001). Normal tissue from IBC patients is enriched for both mammary stem cells and macrophages and has higher association with both a tumorigenic stem cell signature and IBC-specific tumor signature. Collectively, these data suggest that IBC normal tissue differs from non-IBC tissue. Whether these changes occur before the tumor develops or is induced by tumor warrants further investigation.

Combined overexpression of cadherin 6, cadherin 11 and cluster of differentiation 44 is associated with lymph node metastasis and poor prognosis in oral squamous cell carcinoma.

PubMed

Ma, Chao; Zhao, Ji-Zhi; Lin, Run-Tai; Zhou, Lian; Chen, Yong-Ning; Yu, Li-Jiang; Shi, Tian-Yin; Wang, Mu; Liu, Man-Man; Liu, Yao-Ran; Zhang, Tao

2018-06-01

Oral squamous cell carcinoma (OSCC) is a highly invasive lesion that frequently metastasizes to the cervical lymph nodes and is associated with a poor prognosis. Several adhesion factors, including cadherin 6 (CDH6), cadherin 11 (CDH11) and cluster of differentiation 44 (CD44), have been reported to be involved in the invasion and metastasis of multiple types of cancer. Therefore, the aim of the present study was to determine the expression of CDH6, CDH11 and CD44 in tumor tissues from patients with OSCC, and whether this was associated with the metastasis and survival of OSCC. The mRNA expression of the human tumor metastasis-related cytokines was examined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) in OSCC tumors with or without lymph node metastasis (n=10/group). The expression of CDH6, CDH11 and CD44 in 101 OSCC and 10 normal oral mucosa samples was examined by immunohistochemical staining. The association between overall and disease-specific survival times of patients with OSCC and the expression of these three proteins was evaluated using Kaplan-Meier curves and the log-rank test. RT-qPCR results indicated that the mRNA expression of CDH6, CDH11 and CD44 was increased in OSCC patients with lymph node metastasis (2.93-, 2.01- and 1.92-fold; P<0.05). Overexpression of CDH6, CDH11 and CD44 was observed in 31/35 (89%), 25/35 (71%) and 31/35 (89%) patients, respectively. The number of OSCC patients with lymph node metastasis exhibiting CDH6, CDH11 and CD44 overexpression was significantly higher than the number of patients without lymph node metastasis exhibiting overexpression of these proteins (P=0.017, P=0.038 and P=0.007, respectively). OSCC patients with high co-expression of CDH6, CDH11 and CD44 exhibited lower disease-specific survival times (P=0.047; χ 2 =3.933) when compared with OSCC patients with low co-expression of these adhesion factors. CDH6, CDH11 and CD44 serve important roles in OSCC metastasis and the combined use of these factors as biomarkers may improve the accuracy of the prediction of cancer metastases and prognosis.

MIA PaCa-2 and PANC-1 - pancreas ductal adenocarcinoma cell lines with neuroendocrine differentiation and somatostatin receptors.

PubMed

Gradiz, Rui; Silva, Henriqueta C; Carvalho, Lina; Botelho, Maria Filomena; Mota-Pinto, Anabela

2016-02-17

Studies using cell lines should always characterize these cells to ensure that the results are not distorted by unexpected morphological or genetic changes possibly due to culture time or passage number. Thus, the aim of this study was to describe those MIA PaCa-2 and PANC-1 cell line phenotype and genotype characteristics that may play a crucial role in pancreatic cancer therapeutic assays, namely neuroendocrine chemotherapy and peptide receptor radionuclide therapy. Epithelial, mesenchymal, endocrine and stem cell marker characterization was performed by immunohistochemistry and flow cytometry, and genotyping by PCR, gene sequencing and capillary electrophoresis. MIA PaCa-2 (polymorphism) expresses CK5.6, AE1/AE3, E-cadherin, vimentin, chromogranin A, synaptophysin, SSTR2 and NTR1 but not CD56. PANC-1 (pleomorphism) expresses CK5.6, MNF-116, vimentin, chromogranin A, CD56 and SSTR2 but not E-cadherin, synaptophysin or NTR1. MIA PaCA-1 is CD24(-), CD44(+/++), CD326(-/+) and CD133/1(-), while PANC-1 is CD24(-/+), CD44(+), CD326(-/+) and CD133/1(-). Both cell lines have KRAS and TP53 mutations and homozygous deletions including the first 3 exons of CDKN2A/p16(INK4A), but no SMAD4/DPC4 mutations or microsatellite instability. Both have neuroendocrine differentiation and SSTR2 receptors, precisely the features making them suitable for the therapies we propose to assay in future studies.

The ThPOK transcription factor differentially affects the development and function of self-specific CD8(+) T cells and regulatory CD4(+) T cells.

PubMed

Twu, Yuh-Ching; Teh, Hung-Sia

2014-03-01

The zinc finger transcription factor ThPOK plays a crucial role in CD4 T-cell development and CD4/CD8 lineage decision. In ThPOK-deficient mice, developing T cells expressing MHC class II-restricted T-cell receptors are redirected into the CD8 T-cell lineage. In this study, we investigated whether the ThPOK transgene affected the development and function of two additional types of T cells, namely self-specific CD8 T cells and CD4(+) FoxP3(+) T regulatory cells. Self-specific CD8 T cells are characterized by high expression of CD44, CD122, Ly6C, 1B11 and proliferation in response to either IL-2 or IL-15. The ThPOK transgene converted these self-specific CD8 T cells into CD4 T cells. The converted CD4(+) T cells are no longer self-reactive, lose the characteristics of self-specific CD8 T cells, acquire the properties of conventional CD4 T cells and survive poorly in peripheral lymphoid organs. By contrast, the ThPOK transgene promoted the development of CD4(+) FoxP3(+) regulatory T cells resulting in an increased recovery of CD4(+) FoxP3(+) regulatory T cells that expressed higher transforming growth factor-β-dependent suppressor activity. These studies indicate that the ThPOK transcription factor differentially affects the development and function of self-specific CD8 T cells and CD4(+) FoxP3(+) regulatory T cells. © 2013 John Wiley & Sons Ltd.

Targeting CD44 with nanoparticles in head and neck squamous cell carcinoma: A novel therapeutic strategy against cancer stem cells

NASA Astrophysics Data System (ADS)

Thapa, Ranjeeta

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common type of cancer worldwide and is associated with significant morbidity and mortality. Advances in multi-modality treatments have only minimally improved survival rates in the past several years. Recent attention has been focused on the hypothesis that cancer stem cells (CSCs) may be responsible for the failure of current treatments. In HNSCC, a CSC population is contained within the cell fraction that expresses high levels of CD44. CD44 is a cell surface glycoprotein and was the first CSC marker to be described in solid malignancies. in this study, hyaluronan conjugated, dextran-coated super paramagnetic iron-oxide nanoparticles (HA-DESPIONs) were used to target the CD44 population in CD44-overexpressed HNSCC cell lines for treatment by establishing the interaction of HA-DESPIONs with radiation and hyperthermia therapy. The first part of this dissertation studied the cytotoxic, radiosensitizing, and hyperthermic properties of the HA-DESPIONs using cell proliferation and clonogenic survival assays. Cells were grown, plated, treated with HA-DESPIONs, irradiated/exposed to local hyperthermia, and then analyzed for apoptosis. HA-DESPIONs proved to be relatively non-toxic and nonradiosensitizing. However, temperature-dependent cell survival reduction upon incubation with HA-DESPIONs was observed with evidence of apoptotic cell death. These results supported further development of an alternating magnetic field (AMF) approach to activate the HADESPIONs attached to CSCs. In the second part of the dissertation, an AMF generator was constructed and its heat generating effect was tested via kinetic and dose-dependent bulk heating experiments by exposing magnetic nanoparticles to AMF. For elimination of the CD44 population, cells were treated with HA-DESPIONs/DESPIONs, exposed to AMF, and processed for flow cytometrybased apoptosis analysis. Magnetic nanoparticles caused concentration-dependent bulk heating in response to AMF resulting in a significant temperature rise. Following exposure to AMF, DESPIONs were unable to induce targeted hyperthermia and hence had no effect on CD44 cell death in HNSCC cells. However, there was significant cell death in the CD44 population treated with HA-DESPIONs and exposed to AMF. This effect was observed only when the AMF was turned on. These results demonstrated that HA-DESPIONs caused targeted cell-death in CD44overexpressing cells. This may be a promising strategy to specifically target CSCs for the treatment of HNSCC.

Neonatal Desensitization Supports Long-Term Survival and Functional Integration of Human Embryonic Stem Cell-Derived Mesenchymal Stem Cells in Rat Joint Cartilage Without Immunosuppression

PubMed Central

Zhang, Shufang; Jiang, Yang Zi; Zhang, Wei; Chen, Longkun; Tong, Tong; Liu, Wanlu; Mu, Qin; Liu, Hua; Ji, Junfeng; Ouyang, Hong Wei

2013-01-01

Immunological response hampers the investigation of human embryonic stem cells (hESCs) or their derivates for tissue regeneration in vivo. Immunosuppression is often used after surgery, but exhibits side effects of significant weight loss and allows only short-term observation. The purpose of this study was to investigate whether neonatal desensitization supports relative long-term survival of hESC-derived mesenchymal stem cells (hESC-MSCs) and promotes cartilage regeneration. hESC-MSCs were injected on the day of birth in rats. Six weeks after neonatal injection, a full-thickness cylindrical cartilage defect was created and transplanted with a hESC-MSC-seeded collagen bilayer scaffold (group d+s+c) or a collagen bilayer scaffold (group d+s). Rats without neonatal injection were transplanted with the hESC-MSC-seeded collagen bilayer scaffold to serve as controls (group s+c). Cartilage regeneration was evaluated by histological analysis, immunohistochemical staining, and biomechanical test. The role of hESC-MSCs in cartilage regeneration was analyzed by CD4 immunostaining, cell death detection, and visualization of human cells in regenerated tissues. hESC-MSCs expressed CD105, CD73, CD90, CD29, and CD44, but not CD45 and CD34, and possessed trilineage differentiation potential. Group d+s+c exhibited greater International Cartilage Repair Society (ICRS) scores than group d+s or group s+c. Abundant collagen type II and improved mechanical properties were detected in group d+s+c. There were less CD4+ inflammatory cell infiltration and cell death at week 1, and hESC-MSCs were found to survive as long as 8 weeks after transplantation in group d+s+c. Our study suggests that neonatal desensitization before transplantation may be an efficient way to develop a powerful tool for preclinical study of human cell-based therapies in animal models. PMID:22788986

Primary adipose-derived stem cells enriched by growth factor treatment improves cell adaptability toward cardiovascular differentiation in a rodent model of acute myocardial infarction.

PubMed

Chang, Jui-Chih; Lee, Ping-Chun; Lin, Yu-Chun; Lee, Kung-Wei; Hsu, Shan-hui

2011-01-01

The heterogeneous cell population in primary adipose-derived adult stem cells (ADAS) and difficulty in keeping their primitive properties have posed certain limitations on using these cells for cell therapy. Therefore, our objective was to generate a population of cells enriched from the adipose stromal-vascular fraction (SVF) with greater differentiation potential than ADAS and to explore the mechanism behind the repair of the injured myocardium in vivo. The distinct population of adipose stromal cells was enriched by immediate treatment of the growth factor cocktail (EGF and PDGF-BB) to the freshly isolated SVF. These cells (ADAS-GFs) had distinct cell morphology from ADAS and in average had a smaller size. They presented co-expression of CD140a (pericytic markers) and CD34 (hematopoietic marker), more obvious mesenchymal (CD13, CD29, CD44, CD90 and CD117) markers, but rare KDR, and were negative for CD45 and CD31. ADAS-GFs not only spontaneously expressed endothelial cell markers and formed capillary-like tubes on Matrigel but also clearly expressed early cardiomyocyte marker genes when embedded in methylcellulose-based medium. In Sprague-Dawley (SD) rats with left anterior descending artery (LAD)-induced myocardial infarction (MI), the ADAS-GFs transplanted group had the left ventricular function significantly improved compared with the ADAS transplanted group or the control group at 12 weeks post transplantation. The immunofluorescence staining revealed that the transplanted ADAS-GFs expressed GATA4, betamyosin heavy chain and troponin T protein but not vWF. More capillaries were also observed around the infarcted zone in the ADAS-GFs transplanted group. These data suggested that ADAS-GFs with a higher proangiogenic potential may restore the cardiac function of infarcted myocardium via the direct cardiomyocyte differentiation as well as angiogenesis recruitment.

Neonatal desensitization supports long-term survival and functional integration of human embryonic stem cell-derived mesenchymal stem cells in rat joint cartilage without immunosuppression.

PubMed

Zhang, Shufang; Jiang, Yang Zi; Zhang, Wei; Chen, Longkun; Tong, Tong; Liu, Wanlu; Mu, Qin; Liu, Hua; Ji, Junfeng; Ouyang, Hong Wei; Zou, Xiaohui

2013-01-01

Immunological response hampers the investigation of human embryonic stem cells (hESCs) or their derivates for tissue regeneration in vivo. Immunosuppression is often used after surgery, but exhibits side effects of significant weight loss and allows only short-term observation. The purpose of this study was to investigate whether neonatal desensitization supports relative long-term survival of hESC-derived mesenchymal stem cells (hESC-MSCs) and promotes cartilage regeneration. hESC-MSCs were injected on the day of birth in rats. Six weeks after neonatal injection, a full-thickness cylindrical cartilage defect was created and transplanted with a hESC-MSC-seeded collagen bilayer scaffold (group d+s+c) or a collagen bilayer scaffold (group d+s). Rats without neonatal injection were transplanted with the hESC-MSC-seeded collagen bilayer scaffold to serve as controls (group s+c). Cartilage regeneration was evaluated by histological analysis, immunohistochemical staining, and biomechanical test. The role of hESC-MSCs in cartilage regeneration was analyzed by CD4 immunostaining, cell death detection, and visualization of human cells in regenerated tissues. hESC-MSCs expressed CD105, CD73, CD90, CD29, and CD44, but not CD45 and CD34, and possessed trilineage differentiation potential. Group d+s+c exhibited greater International Cartilage Repair Society (ICRS) scores than group d+s or group s+c. Abundant collagen type II and improved mechanical properties were detected in group d+s+c. There were less CD4+ inflammatory cell infiltration and cell death at week 1, and hESC-MSCs were found to survive as long as 8 weeks after transplantation in group d+s+c. Our study suggests that neonatal desensitization before transplantation may be an efficient way to develop a powerful tool for preclinical study of human cell-based therapies in animal models.

Lung cells from neonates show a mesenchymal stem cell phenotype.

PubMed

Hennrick, Kenneth T; Keeton, Angela G; Nanua, Suparna; Kijek, Theresa G; Goldsmith, Adam M; Sajjan, Umadevi S; Bentley, J Kelley; Lama, Vibha N; Moore, Bethany B; Schumacher, Robert E; Thannickal, Victor J; Hershenson, Marc B

2007-06-01

Mesenchymal stem cells have been isolated from adult bone marrow, peripheral blood, adipose tissue, trabecular bone, articular synovium, and bronchial submucosa. We hypothesized that the lungs of premature infants undergoing mechanical ventilation contain fibroblast-like cells with features of mesenchymal stem cells. Tracheal aspirate fluid from mechanically ventilated, premature (< 30 wk gestation) infants 7 days old or younger was obtained from routine suctioning and plated on plastic culture dishes. A total of 11 of 20 patients studied demonstrated fibroblast-like cells, which were identified as early as 6 hours after plating. Cells were found to express the mesenchymal stem cell markers STRO-1, CD73, CD90, CD105, and CD166, as well as CCR2b, CD13, prolyl 4-hydroxylase, and alpha-smooth muscle actin. Cells were negative for the hematopoietic and endothelial cell markers CD11b, CD31, CD34, or CD45. Tracheal aspirate monocyte chemoattractant protein-1/CCL2 levels were ninefold higher in aspirates in which fibroblast-like cells were found, and cells demonstrated chemotaxis in response to monocyte chemoattractant protein. Placement of cells into appropriate media resulted in adipogenic, osteogenic, and myofibroblastic differentiation. Patients from whom mesenchymal stem cells were isolated tended to require more days of mechanical ventilation and supplemental oxygen. Together, these data demonstrate that tracheal aspirate fluid from premature, mechanically ventilated infants contains fibroblasts with cell markers and differentiation potential typically found in mesenchymal stem cells.

Postnatal extra-embryonic tissues as a source of multiple cell types for regenerative medicine applications.

PubMed

Gubar, O S; Rodnichenko, A E; Vasyliev, R G; Zlatska, A V; Zubov, D O

2017-09-01

We aimed to isolate and characterize the cell types which could be obtained from postnatal extra-embryonic tissues. Fresh tissues (no more than 12 h after delivery) were used for enzymatic or explants methods of cell isolation. Obtained cultures were further maintained at 5% oxygen. At P3 cell phenotype was assessed by fluorescence-activated cell sorting, population doubling time was calculated and the multilineage differentiation assay was performed. We have isolated multiple cell types from postnatal tissues. Namely, placental mesenchymal stromal cells from placenta chorionic disc, chorionic membrane mesenchymal stromal cells (ChM-MSC) from free chorionic membrane, umbilical cord MSC (UC-MSC) from whole umbilical cord, human umbilical vein endothelial cells (HUVEC) from umbilical vein, amniotic epithelial cells (AEC) and amniotic MSC (AMSC) from amniotic membrane. All isolated cell types displayed high proliferation rate together with the typical MSC phenotype: CD73 + CD90 + CD105 + CD146 + CD166+CD34 - CD45 - HLA-DR - . HUVEC constitutively expressed key markers CD31 and CD309. Most MSC and AEC were capable of osteogenic and adipogenic differentiation. We have shown that a wide variety of cell types can be easily isolated from extra-embryonic tissues and expanded ex vivo for regenerative medicine applications. These cells possess typical MSC properties and can be considered an alternative for adult MSC obtained from bone marrow or fat, especially for allogeneic use.

Platelet-, monocyte-derived and tissue factor-carrying circulating microparticles are related to acute myocardial infarction severity.

PubMed

Chiva-Blanch, Gemma; Laake, Kristian; Myhre, Peder; Bratseth, Vibeke; Arnesen, Harald; Solheim, Svein; Badimon, Lina; Seljeflot, Ingebjørg

2017-01-01

Circulating microparticles (cMPs) are phospholipid-rich vesicles released from cells when activated or injured, and contribute to the formation of intracoronary thrombi. Tissue factor (TF, CD142) is the main trigger of fibrin formation and TF-carrying cMPs are considered one of the most procoagulant cMPs. Similar types of atherosclerotic lesions may lead to different types of AMI, although the mechanisms behind are unresolved. Therefore, we aimed to investigate the phenotype of cMPs found in plasma of ACS patients and its relation to AMI severity and thrombotic burden. In a cross-sectional study, two hundred patients aged 75±4 years were included in the study 2-8 weeks after suffering an AMI. Annexin V positive (AV+)-cMPs derived from blood and vascular cells were measured by flow cytometry. Plasma procoagulant activity (TF-PCA) was measured through a chromogenic assay. STEMI patients (n = 75) showed higher levels of platelet-derived cMPs [CD61+/AV+, CD31+/AV+, CD42b+/AV+ and CD31+/CD42b+/AV+, P = 0.048, 0.038, 0.009 and 0.006, respectively], compared to NSTEMI patients (n = 125). Patients who suffered a heart failure during AMI (n = 17) had increased levels of platelet (CD61+)-and monocyte (CD14+)-derived cMPs carrying TF (CD142+) (P<0.0001 and 0.004, respectively). Additionally, NYHA class III (n = 23) patients showed higher levels of CD142+/AV+, CD14+/AV+ and CD14+/CD142+/AV+ cMPs than those in class I/II (P = 0.001, 0.015 and 0.014, respectively). The levels of these cMPs positively correlated with TF-PCA (r≥0.166, P≤0.027, all). Platelets and monocytes remain activated in AMI patients treated as per guidelines and release cMPs that discriminate AMI severity. Therefore, TF-MPs, and platelet- and monocyte-MPs may reflect thrombotic burden in AMI patients.

Relationship of PCNA, C-erbB2 and CD44s expression with tumor grade and stage in urothelial carcinomas of the bladder

PubMed Central

Yıldırım, Ayhan; Kösem, Mustafa; Sayar, İlyas; Gelincik, İbrahim; Yavuz, Alparslan; Bozkurt, Aliseydi; Erkorkmaz, Ünal; Bayram, İrfan

2014-01-01

In the present study, the intention was to reveal the relationship of histological grade and stage with c-erbB2, CD44s, and PCNA immunoreactivity in bladder urothelial carcinomas (UC). In our study, we evaluated 46 items of transurethral resection material of patients submitted by YYU Faculty of Medicine, Main Department of Pathology, with a mass revealed in their bladder after clinical and radiological studies at our laboratories and who were diagnosed with urothelial carcinomas. PCNA, c-erbB2, and CD44s were applied in an immunohistochemical manner comprised from nine low-malignant potential papillary urothelial neoplasia, 23 low-grade papillary urothelial carcinoma, and 14 high-grade papillary urothelial carcinoma. Immunostaining was scored according to the percentage of positive cells. The immunohistochemical study demonstrated that the c-erbB2 and PCNA staining ratio increased when an increase occurred in stage and grade. The CD44s staining ratio decreased. C-erbB2, PCNA, and CD44s appear to be a useful marker in the assessment of the prognosis and treatment options in urothelial carcinomas. PMID:25035774

Installation Restoration Program. Site Investigation Report for 166th Tactical Airlift Group, Delaware Air National Guard, Greater Wilmington Airport, New Castle, Delaware

DTIC Science & Technology

1990-05-01

It -ui ’a , CC r~~ ’a ana .- ,3- *. sce’tI eAt ke :-.a 5qsr, r s c na r rs ’ . n: -.- aL . ~ ~ ’’ T’ttC , ~ ~ ."’e.50l... S * 4-7y K 0%,Wj *00t0j 30-Zk + M L ..... 7-f 3y# IkeA ~IVe~A~ ___ __ __ j4IC i ..... 4r0 3SO S -9t~ r~ r.A ieew...I ?- 4’~~~o - IW 4 4 LA -. i a- a SI - pn S 4c ’ 0 Ci .0 616.. It .&CNILjW a 0 U m -l -m Ci CD c o C44 Fnn CDC x C--t Fnn 90 CD x do CC2 a- -% ’A xa.

Design and evaluation of dual CD44 receptor and folate receptor-targeting double-smart pH-response multifunctional nanocarrier

NASA Astrophysics Data System (ADS)

Chen, Daquan; Song, Xiaoyan; Wang, Kaili; Guo, Chunjing; Yu, Yueming; Fan, Huaying; Zhao, Feng

2017-12-01

In this article, in order to enhance the bioavailiability and tumor targeting of curcumin (Cur), the oligosaccharides of hyaluronan conjugates, folic acid-oligosaccharides of hyaluronan-acetal-menthone 1,2-glycerol ketal (FA-oHA-Ace-MGK) carried oHA as a ligand to CD44 receptor, double-pH-sensitive Ace-MGK as hydrophobic moieties, and FA as the target of folate receptor. The structure characteristics of this smart response multifunctional dual-targeting nano-sized carrier was measured by fourier-transform infrared (FT-IR) and nuclear magnetic resonance (1H-NMR). Cur, an anticancer drug, was successfully loaded in FA-oHA-Ace-MGK micelles by self-assembly. The measurement results of transmission electron microscopy (TEM) presented that the Cur-loaded micelles were spherical in shape with the average size of 166.3 ± 2.12 nm and zeta potential - 30.07 mV. Much more encapsulated Cur could be released at mildly acidic environments than at pH 7.4, from the Cur-FA-oHA-Ace-MGK micelles. Cytotoxicity assay indicated that non-Cur loaded micelles mostly had no cytotoxicity to MCF-7 cells and A549 cells, and Cur-loaded micelles had significantly lower survival rate than Cur suspension in the same concentration, which proved that the drug-loaded micelles can effectively inhibit tumor cell growth. The targeting of CD44 receptors and folate receptors was proved in vitro cellular uptake assay. These results showed the promising potential of FA-oHA-Ace-MGK as an effective nano-sized carrier for anti-tumor drug delivery.

Activated T cell subsets in human type 1 diabetes: evidence for expansion of the DR+ CD30+ subpopulation in new-onset disease

PubMed Central

Baker, C; Chang, L; Elsegood, K A; Bishop, A J; Gannon, D H; Narendran, P; Leech, N J; Dayan, C M

2007-01-01

An important limitation in T cell studies of human autoimmune (type 1) diabetes is lack of direct access to cells infiltrating the pancreas. We hypothesized that cells recently released from the pancreas into the blood might express a characteristic combination of markers of activation. We therefore examined the recently activated circulating T cell population [CD3+, human leucocyte antigen D-related (HLA-DR+)] using cytokine production and 10 additional subset markers [CD69, CD25, CD122, CD30, CD44v6, CD57, CD71, CCR3 (CD193), CCR5 (CD195) or CXCR3 (CD183)], comparing newly diagnosed adult (ND) (age 18–40 years) patients (n = 19) to patients with diabetes for > 10 years [long-standing (LS), n = 19] and HLA-matched controls (C, n = 16). CD3+ DR+ cells were enriched by two-step immunomagnetic separation. No differences in basal or stimulated production of interleukin (IL)-4, IL-10, IL-13 or interferon (IFN)-γ by CD3+ DR+ enriched cells were observed between the different groups of subjects. However, among the CD3+ DR+ population, significant expansions appeared to be present in the very small CD30+, CD69+ and CD122+ subpopulations. A confirmatory study was then performed using new subjects (ND = 26, LS = 15), three-colour flow cytometry, unseparated cells and three additional subset markers (CD38, CD134, CD4/CD25). This confirmed the expansion of the CD3+ DR+ CD30+ subpopulation in ND subjects. We conclude that a relative expansion in the T cell subpopulation with the activated phenotype CD3+ DR+ CD30+ is seen in the peripheral blood of subjects with newly diagnosed type 1 diabetes. This subpopulation represents less than 0·7% of circulating T cells and may provide a rich source of disease-specific T cells that can be isolated from blood. PMID:17302896

[Expressions of angiogenesis-related factors: CD105, EphA2 and EphrinA1 in laryngeal squamous cell carcinoma and clinical implication].

PubMed

Su, J; Ji, X B; Xie, J H; Li, W

2016-12-07

Objective: To investigate the expressions of endoglin (CD105), erythropoietin-producing hepatocyte receptor A2 (EphA2) and its ligand ephrinA1 proteins in laryngeal squamous cell carcinoma (LSCC) and the relationship between their expressions and the clinicopathological factors of LSCC. Methods: The expressions of CD105, EphA2 and EphrinA1 proteins were detected with immunohistochemical staining in LSCC in 76 cases and adjacent normal laryngeal tissues (ANLT) (S-P) in 25 cases.SPSS 17.0 software was used to analyze the data. Results: The mean microvessel density (MVD) value marked by CD105 staining in LSCC was 10.33±2.29, which was significantly higher than that in ANLT(1.20±1.04, t =18.732, P <0.05). The CD105-MVD was correlated with T stage, histological grading, clinical stage, lymph node metastasis, recurrence and prognosis in LSCC (F value was 5.34, 4.79, 5.36, t value was -2.70, 2.56, all P <0.05). The positive expression rates of EphA2 and EphrinA1 in LSCC were 78.95% (60/76), and 81.85% (62/76), which were respectively significantly higher than 40% (10/25) for EphA2 expression and 44% (11/25) for EphrinA1, expression in ANLT (χ 2 value was 13.41, 13.26, both P <0.05). EphA2 expression was correlated with histological grading, T stage, clinical stage, lymph node metastasis, recurrence and prognosis in LSCC (χ 2 value was 6.25, 14.60, 15.11, 8.52, 5.54, all P <0.05). EphrinA1 expression was correlated with T stage, clinical stage, lymph node metastasis, recurrence and prognosis in LSCC (χ 2 value was 6.44, 12.28, 16.78, 6.44, all P <0.05). The expressions of CD105, EphA2 and EphrinA1 were positively correlated with each other r value was 0.72, 0.74, 0.64, all P <0.05. Survival analysis indicated that the expressions of CD105 and EphA2, histological grading, lymph node metastasis, clinical stage and recurrence were independent factors for tumor prognosis in LSCC ( P <0.05). Conclusions: The expressions of CD105, EphA2 and EphrinA1 protein were positively correlated with each other in LSCC. They may play important roles in the tumorigenesis, malignant progression and poor prognosis of LSCC.

Expression Profiles of Ligands for Activating Natural Killer Cell Receptors on HIV Infected and Uninfected CD4⁺ T Cells.

PubMed

Tremblay-McLean, Alexandra; Bruneau, Julie; Lebouché, Bertrand; Lisovsky, Irene; Song, Rujun; Bernard, Nicole F

2017-10-12

Natural Killer (NK) cell responses to HIV-infected CD4 T cells (iCD4) depend on the integration of signals received through inhibitory (iNKR) and activating NK receptors (aNKR). iCD4 activate NK cells to inhibit HIV replication. HIV infection-dependent changes in the human leukocyte antigen (HLA) ligands for iNKR on iCD4 are well documented. By contrast, less is known regarding the HIV infection related changes in ligands for aNKR on iCD4. We examined the aNKR ligand profiles HIV p24⁺ HIV iCD4s that maintained cell surface CD4 (iCD4⁺), did not maintain CD4 (iCD4 - ) and uninfected CD4 (unCD4) T cells for expression of unique long (UL)-16 binding proteins-1 (ULBP-1), ULBP-2/5/6, ULBP-3, major histocompatibility complex (MHC) class 1-related (MIC)-A, MIC-B, CD48, CD80, CD86, CD112, CD155, Intercellular adhesion molecule (ICAM)-1, ICAM-2, HLA-E, HLA-F, HLA-A2, HLA-C, and the ligands to NKp30, NKp44, NKp46, and killer immunoglobulin-like receptor 3DS1 (KIR3DS1) by flow cytometry on CD4 T cells from 17 HIV-1 seronegative donors activated and infected with HIV. iCD4⁺ cells had higher expression of aNKR ligands than did unCD4. However, the expression of aNKR ligands on iCD4 where CD4 was downregulated (iCD4 - ) was similar to (ULBP-1, ULBP-2/5/6, ULBP-3, MIC-A, CD48, CD80, CD86 and CD155) or significantly lower than (MIC-B, CD112 and ICAM-2) what was observed on unCD4. Thus, HIV infection can be associated with increased expression of aNKR ligands or either baseline or lower than baseline levels of aNKR ligands, concomitantly with the HIV-mediated downregulation of cell surface CD4 on infected cells.

An enteric pathogen Salmonella enterica serovar Typhimurium suppresses tumor growth by downregulating CD44high and CD4T regulatory (Treg) cell expression in mice: the critical role of lipopolysaccharide and Braun lipoprotein in modulating tumor growth.

PubMed

Liu, T; Chopra, A K

2010-02-01

An antitumor activity associated with several bacterial pathogens, including Salmonella enterica serovar Typhimurium, has been reported; however, the underlying immunological mechanism(s) that lead to an antitumor effect are currently unclear. Furthermore, such pathogens cannot be used to suppress tumor growth because of their potential for causing sepsis. Recently, we reported the characterization of S. Typhimurium isogenic mutants from which Braun lipoprotein genes (lppA and B) and the multicopy repressor of high temperature requirement (msbB) gene were deleted. In a mouse infection model, two mutants, namely, lppB/msbB and lppAB/msbB, minimally induced proinflammatory cytokine production at high doses and were nonlethal to animals. We showed that immunization of mice with these mutants, followed by challenge with the wild-type S. Typhimurium, could significantly suppress tumor growth, as evidenced by an 88% regression in tumor size in lppB/msbB mutant-immunized animals over a 24-day period. However, the lppAB/msbB mutant alone was not effective in modulating tumor growth in mice, although the lppB/msbB mutant alone caused marginal regression in tumor size. Importantly, we showed that CD44(+) cells grew much faster than CD44(-) cells from human liver tumors in mice, leading us to examine the possibility that S. Typhimurium might downregulate CD44 in tumors and splenocytes of mice. Consequently, we found in S. Typhimurium-infected mice that tumor size regression could indeed be related to the downregulation of CD44(high) and CD4(+)CD25(+) T(reg) cells. Importantly, the role of lipopolysaccharide and Braun lipoprotein was critical in S. Typhimurium-induced antitumor immune responses. Taken together, we have defined new immune mechanisms leading to tumor suppression in mice by S. Typhimurium.

Elevated numbers of CD163+ macrophages in hearts of simian immunodeficiency virus-infected monkeys correlate with cardiac pathology and fibrosis.

PubMed

Walker, Joshua A; Sulciner, Megan L; Nowicki, Katherine D; Miller, Andrew D; Burdo, Tricia H; Williams, Kenneth C

2014-07-01

The role of macrophage activation, traffic, and accumulation on cardiac pathology was examined in 23 animals. Seventeen animals were simian immunodeficiency virus (SIV) infected, 12 were CD8 lymphocyte depleted, and the remaining six were uninfected controls (two CD8 lymphocyte depleted, four nondepleted). None of the uninfected controls had cardiac pathology. One of five (20%) SIV-infected, non-CD8 lymphocyte-depleted animals had minor cardiac pathology with increased numbers of macrophages in ventricular tissue compared to controls. Seven of the 12 (58%) SIV-infected, CD8 lymphocyte-depleted animals had cardiac pathology in ventricular tissues, including macrophage infiltration and myocardial degeneration. The extent of fibrosis (measured as the percentage of collagen per tissue area) was increased 41% in SIV-infected, CD8 lymphocyte-depleted animals with cardiac pathology compared to animals without pathological abnormalities. The number of CD163+ macrophages increased significantly in SIV-infected, CD8 lymphocyte-depleted animals with cardiac pathology compared to ones without pathology (1.66-fold) and controls (5.42-fold). The percent of collagen (percentage of collagen per total tissue area) positively correlated with macrophage numbers in ventricular tissue in SIV-infected animals. There was an increase of BrdU+ monocytes in the heart during late SIV infection, regardless of pathology. These data implicate monocyte/macrophage activation and accumulation in the development of cardiac pathology with SIV infection.

Association of high post-transplant soluble CD30 serum levels with chronic allograft nephropathy.

PubMed

Grenzi, Patricia C; Campos, Érika F; Tedesco-Silva, Hélio; Felipe, Claudia R; Franco, Marcello F; Soares, Maria Fernanda; Medina-Pestana, José Osmar; Gerbase-Delima, Maria

2013-12-01

The purpose of this study was to evaluate the association of post-transplant soluble CD30 (sCD30) levels, isolated or in combination with of anti-HLA class II antibodies and of serum creatinine levels, with kidney graft loss due to chronic allograft nephropathy (CAN), and type of lesions in graft biopsies for cause. The study comprised 511 first kidney graft recipients, transplanted at a single center, with a graft functioning for at least 2.8 years. A single blood sample was collected from each patient. sCD30 levels were determined by ELISA, and HLA antibodies by Luminex assay. The minimum follow-up after testing was 9.3 years. High sCD30 levels, set at sCD30 ≥ 34.15 ng/mL, the presence of HLA class II antibodies, and serum creatinine ≥ 1.9 mg/dL were independently associated with CAN-graft loss (P values <0.0001, 0.05, <0.0001, respectively), and the combined hazard ratio for CAN-graft loss was 20.2. Analyses of 166 biopsies for cause showed that high sCD30 levels and creatinine were independently associated with interstitial lesions. Post-transplant sCD30 serum levels, especially in conjunction with information regarding HLA class II antibodies and serum creatinine levels, provide valuable information regarding graft outcome and could be useful for the management of kidney transplant recipients. © 2013.

Multi-color flow cytometry for evaluating age-related changes in memory lymphocyte subsets in dogs.

PubMed

Withers, Sita S; Moore, Peter F; Chang, Hong; Choi, Jin W; McSorley, Stephen J; Kent, Michael S; Monjazeb, Arta M; Canter, Robert J; Murphy, William J; Sparger, Ellen E; Rebhun, Robert B

2018-05-31

While dogs are increasingly being utilized as large-animal models of disease, important features of age-related immunosenescence in the dog have yet to be evaluated due to the lack of defined naïve vs. memory T lymphocyte phenotypes. We therefore performed multi-color flow cytometry on peripheral blood mononuclear cells from young and aged beagles, and determined the differential cytokine production by proposed memory subsets. CD4+ and CD8+ T lymphocytes in aged dogs displayed increased cytokine production, and decreased proliferative capacity. Antibodies targeting CD45RA and CD62L, but less so CD28 or CD44, defined canine cells that consistently exhibited properties of naïve-, central memory-, effector memory-, and terminal effector-like CD4+ and CD8+ T lymphocyte subsets. Older dogs demonstrated decreased frequencies of naïve-like CD4+ and CD8+ T lymphocytes, and an increased frequency of terminal effector-like CD8+ T lymphocytes. Overall findings revealed that aged dogs displayed features of immunosenescence similar to those reported in other species. Copyright © 2018 Elsevier Ltd. All rights reserved.

Cutting edge: high molecular weight hyaluronan promotes the suppressive effects of CD4+CD25+ regulatory T cells.

PubMed

Bollyky, Paul L; Lord, James D; Masewicz, Susan A; Evanko, Stephen P; Buckner, Jane H; Wight, Thomas N; Nepom, Gerald T

2007-07-15

Hyaluronan is a glycosaminoglycan present in the extracellular matrix. When hyaluronan is degraded during infection and injury, low m.w. forms are generated whose interactions influence inflammation and angiogenesis. Intact high m.w. hyaluronan, conversely, conveys anti-inflammatory signals. We demonstrate that high m.w. hyaluronan enhances human CD4(+)CD25(+) regulatory T cell functional suppression of responder cell proliferation, whereas low m.w. hyaluronan does not. High m.w. hyaluronan also up-regulates the transcription factor FOXP3 on CD4(+)CD25(+) regulatory T cells. These effects are only seen with activated CD4(+)CD25(+) regulatory T cells and are associated with the expression of CD44 isomers that more highly bind high m.w. hyaluronan. At higher concentrations, high m.w. hyaluronan also has direct suppressive effects on T cells. We propose that the state of HA in the matrix environment provides contextual cues to CD4(+)CD25(+) regulatory T cells and T cells, thereby providing a link between the innate inflammatory network and the regulation of adaptive immune responses.

Activated leucocyte cell adhesion molecule (ALCAM/CD166) regulates T cell responses in a murine model of food allergy.

PubMed

Kim, Y S; Kim, M N; Lee, K E; Hong, J Y; Oh, M S; Kim, S Y; Kim, K W; Sohn, M H

2018-05-01

Food allergy is a major public health problem. Studies have shown that long-term interactions between activated leucocyte cell adhesion molecule (ALCAM/CD166) on the surface of antigen-presenting cells, and CD6, a co-stimulatory molecule, influence immune responses. However, there are currently no studies on the functions of ALCAM in food allergy. Therefore, we aimed to identify the functions of ALCAM in ovalbumin (OVA)-induced food allergy using ALCAM-deficient mice. Wild-type (WT) and ALCAM-deficient (ALCAM -/- ) mice were sensitized intraperitoneally and with orally fed OVA. The mice were killed, and parameters related to food allergy and T helper type 2 (Th2) immune responses were analysed. ALCAM serum levels increased and mRNA expression decreased in OVA-challenged WT mice. Serum immunoglobulin (Ig)E levels, Th2 cytokine mRNA and histological injuries were higher in OVA-challenged WT mice than in control mice, and these were attenuated in ALCAM -/- mice. T cell proliferation of total cells, CD3 + CD4 + T cells and activated T cells in immune tissues were diminished in OVA-challenged ALCAM -/- mice. Proliferation of co-cultured T cells and dendritic cells (DCs) was decreased by the anti-CD6 antibody. In addition, WT mice sensitized by adoptive transfer of OVA-pulsed ALCAM -/- BM-derived DCs showed reduced immune responses. Lastly, serum ALCAM levels were higher in children with food allergy than in control subjects. In this study, serum levels of ALCAM were elevated in food allergy-induced WT mice and children with food allergy. Moreover, immune responses and T cell activation were attenuated in OVA-challenged ALCAM -/- mice. These results indicate that ALCAM regulates food allergy by affecting T cell activation. © 2018 British Society for Immunology.

The PBX1 lupus susceptibility gene regulates CD44 expression

PubMed Central

Niu, Yuxin; Sengupta, Mayami; Titov, Anton A.; Choi, Seung-Chul; Morel, Laurence

2017-01-01

PBX1-d is novel splice isoform of pre-B-cell leukemia homeobox 1 (PBX1) that lacks its DNA-binding and Hox-binding domains, and functions as a dominant negative. We have shown that PBX1-d expression in CD4+ T cells is associated with systemic lupus erythematosus (SLE) in a mouse model as well as in human subjects. More specifically, PBX1-d expression leads to the production of autoreactive activated CD4+ T cells, a reduced frequency and function of Foxp3+ regulatory T (Treg) cells and an expansion of follicular helper T (Tfh) cells. Very little is known about the function of PBX1 in T cells, except that it directly regulates the expression of miRNAs associated with Treg and Tfh homeostasis. In the present study, we show that PBX1 directly regulated the expression of CD44, a marker of T cell activation. Two PBX1 binding sites in the promoter directly regulated CD44 expression, with PBX1-d driving a higher expression than the normal isoform PBX1-b. In addition, mutations in each of the two binding sites had different effects of PBX1-b and PBX1-d. Finally, we showed that an enhanced recruitment of co-factor MEIS by PBX1-d over PBX1-b, while there was no difference for co-factor PREP1 recruitment. Therefore, this study demonstrates that the lupus-associated PBX1-d isoform directly transactivates CD44, a marker of CD44 activation and memory, and that it has different DNA binding and co-factor recruitment relative to the normal isoform. Taken together, these results confirm that PBX1 directly regulates genes related to T cell activation and show that the lupus-associated isoform PBX1-d has unique molecular functions. PMID:28257976

Predictive markers of chemoresistance in advanced stages epithelial ovarian carcinoma.

PubMed

Bonneau, Claire; Rouzier, Roman; Geyl, Caroline; Cortez, Annie; Castela, Mathieu; Lis, Raphael; Daraï, Emile; Touboul, Cyril

2015-01-01

DNA repair mechanisms, environment-mediated drug resistance and cancer initiating cells (CIC) are three major research concepts that can explain the chemoresistance of epithelial ovarian cancer (EOC). The objective was to test if changes in the expression of potential markers associated with drug resistance before and after chemotherapy would correlate with platinum resistance, defined as a recurrence within the first year after chemotherapy cessation, and with survival, in advanced EOC. We included 32 patients with stage IIIC-IV EOC who underwent laparoscopy to evaluate the extent of carcinomatosis, neoadjuvant chemotherapy (carboplatin/taxol) and interval surgery. Biopsies taken during the initial laparoscopies and interval surgeries were evaluated using immunohistochemistry for the expression of 7 proteins: CD117, CD44 and ALDH1 to evaluate CIC; IL-6, IL-8 and BMP2 to evaluate environment-mediated drug resistance; and ERCC1 to evaluate DNA repair. Expression measurements were correlated with platin resistance and survival. The markers' relevance was confirmed in vitro using chemoresistance tests and flow cytometric measurements of the proportion of CD44+ cells. 17 patients were chemoresistant and 15 patients were chemosensitive. We observed increases in CD44, IL-6 and ERCC1 expression and stable ALDH1, CD117, IL-8, and BMP2 expression. Reduced expression of cancer initiating cell markers and increased expression of environment-mediated drug resistance markers were associated with poor prognosis. We also demonstrated that CD44+ cells had survival advantages in vitro. Changes in CD44 and IL-8 expression on tumor cells appeared to correlate with overall survival and should be further tested as predictors of chemoresistance using larger cohort. Copyright © 2014 Elsevier Inc. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Motegi, Atsushi, E-mail: amotegi@east.ncc.go.jp; Fujii, Satoshi; Zenda, Sadamoto

Purpose: To investigate the significance of CD44 protein expression on the treatment outcomes of radiation therapy in patients with oropharyngeal squamous cell carcinoma (OPSCC) with or without p16 protein expression in the tumor tissue. Methods and Materials: We reviewed the medical records of 58 OPSCC patients who had undergone radiation therapy and examined the tumor tissue expressions of CD44 and p16 protein by immunohistochemical staining. The correlations between the expressions of these proteins and the patients' treatment outcomes were analyzed. Results: The data of 58 consecutive OPSCC patients who had undergone definitive intensity modulated radiation therapy were analyzed. The male/femalemore » ratio was 55:3, and the median age was 64 years. The clinical stage of the disease was stage II in 7 patients, stage III in 5 patients, stage IVA in 35 patients, and stage IVB in 11 patients. Of the patients, 79% received additional induction and/or concurrent chemotherapy. The median follow-up duration was 34 months. The 3-year overall survival, progression-free survival (PFS) and locoregional control (LRC) rates of all the patients, regardless of the results of immunohistochemistry, were 73%, 64% and 76%, respectively. The PFS and LRC rates in the CD44{sup −} patients (86% and 93%, respectively) were significantly higher than those in the CD44{sup +} patients (57% and 70%, respectively). The PFS and LRC rates in the p16{sup +} patients (83% and 90%, respectively) were significantly higher than those in the p16{sup −} patients (45% and 61%, respectively). Patients who were CD44{sup −}/p16{sup +} showed the best LRC rates, and those who were CD44{sup +}/p16{sup −} showed the worst PFS and LRC rates among all the groups. Conclusions: Profiling of CD44 and p16 protein expressions by immunohistochemical staining is useful for predicting the treatment outcomes in patients with OPSCC undergoing definitive intensity modulated radiation therapy.« less

In vivo characterization of the novel CD44v6-targeting Fab fragment AbD15179 for molecular imaging of squamous cell carcinoma: a dual-isotope study

PubMed Central

2014-01-01

Background Patients with squamous cell carcinoma in the head and neck region (HNSCC) offer a diagnostic challenge due to difficulties to detect small tumours and metastases. Imaging methods available are not sufficient, and radio-immunodiagnostics could increase specificity and sensitivity of diagnostics. The objective of this study was to evaluate, for the first time, the in vivo properties of the radiolabelled CD44v6-targeting fragment AbD15179 and to assess its utility as a targeting agent for radio-immunodiagnostics of CD44v6-expressing tumours. Methods The fully human CD44v6-targeting Fab fragment AbD15179 was labelled with 111In or 125I, as models for radionuclides suitable for imaging with SPECT or PET. Species specificity, antigen specificity and internalization properties were first assessed in vitro. In vivo specificity and biodistribution were then evaluated in tumour-bearing mice using a dual-tumour and dual-isotope setup. Results Both species-specific and antigen-specific binding of the conjugates were demonstrated in vitro, with no detectable internalization. The in vivo studies demonstrated specific tumour binding and favourable tumour targeting properties for both conjugates, albeit with higher tumour uptake, slower tumour dissociation, higher tumour-to-blood ratio and higher CD44v6 sensitivity for the 111In-labelled fragment. In contrast, the 125I-Fab demonstrated more favourable tumour-to-organ ratios for liver, spleen and kidneys. Conclusions We conclude that AbD15179 efficiently targets CD44v6-expressing squamous cell carcinoma xenografts, and particularly, the 111In-Fab displayed high and specific tumour uptake. CD44v6 emerges as a suitable target for radio-immunodiagnostics, and a fully human antibody fragment such as AbD15179 can enable further clinical imaging studies. PMID:24598405

FGFR signaling regulates resistance of head and neck cancer stem cells to cisplatin.

PubMed

McDermott, Sarah C; Rodriguez-Ramirez, Christie; McDermott, Sean P; Wicha, Max S; Nör, Jacques E

2018-05-18

Patients with recurrent or metastatic head and neck squamous cell carcinoma (HNSCC) have poor prognosis with less than 1-year median survival. Platinum-based chemotherapy remains the first-line treatment for HNSCC. The cancer stem cell (CSC) hypothesis postulates that tumors are maintained by a self-renewing CSC population that is also capable of differentiating into non-self renewing cell populations that constitute the bulk of the tumor. A small population of CSC exists within HNSCC that are relatively resistant to chemotherapy and clinically predicted to contribute to tumor recurrence. These head and neck CSCs (HNCSC) are identified by high cell-surface expression of CD44 and high intracellular activity of aldehyde dehydrogenase (ALDH) and termed ALDH high CD44 high . Here, we performed microarray analysis in two HNSCC cell lines (UM-SCC-1, UM-SCC-22B) to investigate molecular pathways active in untreated and cisplatin-resistant ALDH high CD44 high cells. Gene set enrichment analysis and iPathway analysis identified signaling pathways with major implications to the pathobiology of cancer (e.g. TNFα, IFN, IL6/STAT, NF-κB) that are enriched in cisplatin-resistant ALDH high CD44 high cells, when compared to control cells. FGF2 was also enriched in cisplatin-resistant ALDH high CD44 high , which was confirmed by ELISA analysis. Inhibition of FGF signaling using BGJ398, a pan-FGF receptor (FGFR) small-molecule inhibitor, decreased ALDH high CD44 high alone in UM-SCC-1 and preferentially targeted cisplatin-resistant ALDH high CD44 high cells in UM-SCC-22B. These findings suggest that FGFR signaling might play an important role in the resistance of head and neck CSC to cisplatin. Collectively, this work suggests that some head and neck cancer patients might benefit from the combination of cisplatin and a FGFR inhibitor.

A Missing PD-L1/PD-1 Coinhibition Regulates Diabetes Induction by Preproinsulin-Specific CD8 T-Cells in an Epitope-Specific Manner

PubMed Central

Schuster, Cornelia; Brosi, Helen; Stifter, Katja; Boehm, Bernhard O.; Schirmbeck, Reinhold

2013-01-01

Coinhibitory PD-1/PD-L1 (B7-H1) interactions provide critical signals for the regulation of autoreactive T-cell responses. We established mouse models, expressing the costimulator molecule B7.1 (CD80) on pancreatic beta cells (RIP-B7.1 tg mice) or are deficient in coinhibitory PD-L1 or PD-1 molecules (PD-L1−/− and PD-1−/− mice), to study induction of preproinsulin (ppins)-specific CD8 T-cell responses and experimental autoimmune diabetes (EAD) by DNA-based immunization. RIP-B7.1 tg mice allowed us to identify two CD8 T-cell specificities: pCI/ppins DNA exclusively induced Kb/A12–21-specific CD8 T-cells and EAD, whereas pCI/ppinsΔA12–21 DNA (encoding ppins without the COOH-terminal A12–21 epitope) elicited Kb/B22–29-specific CD8 T-cells and EAD. Specific expression/processing of mutant ppinsΔA12–21 (but not ppins) in non-beta cells, targeted by intramuscular DNA-injection, thus facilitated induction of Kb/B22–29-specific CD8 T-cells. The A12–21 epitope binds Kb molecules with a very low avidity as compared with B22–29. Interestingly, immunization of coinhibition-deficient PD-L1−/− or PD-1−/− mice with pCI/ppins induced Kb/A12–21-monospecific CD8 T-cells and EAD but injections with pCI/ppinsΔA12–21 did neither recruit Kb/B22–29-specific CD8 T-cells into the pancreatic target tissue nor induce EAD. PpinsΔA12–21/(Kb/B22–29)-mediated EAD was efficiently restored in RIP-B7.1+/PD-L1−/− mice, differing from PD-L1−/− mice only in the tg B7.1 expression in beta cells. Alternatively, an ongoing beta cell destruction and tissue inflammation, initiated by ppins/(Kb/A12–21)-specific CD8 T-cells in pCI/ppins+pCI/ppinsΔA12–21 co-immunized PD-L1−/− mice, facilitated the expansion of ppinsΔA12–21/(Kb/B22–29)-specific CD8 T-cells. CD8 T-cells specific for the high-affinity Kb/B22–29- (but not the low-affinity Kb/A12–21)-epitope thus require stimulatory ´help from beta cells or inflamed islets to expand in PD-L1-deficient mice. The new PD-1/PD-L1 diabetes models may be valuable tools to study under well controlled experimental conditions distinct hierarchies of autoreactive CD8 T-cell responses, which trigger the initial steps of beta cell destruction or emerge during the pathogenic progression of EAD. PMID:23977133

Bioaccumulation, morphological changes, and induction of metallothionein gene expression in the digestive system of the freshwater crab Sinopotamon henanense after exposure to cadmium.

PubMed

Wu, Hao; Li, Yingjun; Lang, Xingping; Wang, Lan

2015-08-01

To study the responses of digestive system of the freshwater crab Sinopotamon henanense to the exposure with cadmium (Cd), crabs were acutely exposed to 7.25, 14.50, and 29.00 mg/l Cd for 96 h and subchronically exposed to 0.725, 1.450, and 2.900 mg/l for 21 days. Cd bioaccumulation in the hepatopancreas and digestive tract (esophagus and intestine) was examined. Furthermore, histopathological alterations of the esophagus, midgut, hindgut, and hepatopancreas were assessed in animals from the 29.0 and 2.90 mg/l Cd treatment groups, and expression of metallothionein messenger RNA (MT mRNA) in the hepatopancreas and intestine was measured in all treatment groups. The results showed difference in the middle and high concentrations between acute and subchronic treatment groups. Cd content in digestive tract after acute 14.5 and 29.0 mg/l Cd exposure was significantly higher than that at subchronic 1.45 and 2.90 mg/l exposure, but Cd levels in hepatopancreas were not significantly different under the same condition. Acute exposure to Cd induced greater morphological damage than subchronic exposure: large areas of epithelial cells were necrotic in hepatopancreas and midgut, which detached from the basal lamina. Vacuolated muscle cells were observed in the hindgut of animals from the acute exposure group, but the changes of esophageal morphology were not obvious after acute or subchronic treatments. The expression of MT mRNA increased with increasing Cd concentration, and MT mRNA level in acute exposure groups was significantly lower when compared to the subchronic exposure groups. Higher Cd content and lower MT mRNA expression in the acutely exposed groups may be responsible for more severe damage of digestive system in these exposure groups.

Circulating CD4+CD28null and extra-thymic CD4+CD8+ double positive T cells are independently associated with disease damage in systemic lupus erythematosus patients.

PubMed

Ugarte-Gil, M F; Sánchez-Zúñiga, C; Gamboa-Cárdenas, R V; Aliaga-Zamudio, M; Zevallos, F; Tineo-Pozo, G; Cucho-Venegas, J M; Mosqueira-Riveros, A; Medina, M; Perich-Campos, R A; Alfaro-Lozano, J L; Rodriguez-Bellido, Z; Alarcón, G S; Pastor-Asurza, C A

2016-03-01

To determine whether circulating CD4+CD28null and extra-thymic CD4+CD8+ double positive (DP) T cells are independently associated with damage accrual in systemic lupus erythematosus (SLE) patients. This cross-sectional study was conducted between September 2013 and April 2014 in consecutive SLE patients from our Rheumatology Department. CD4+CD28null and CD4+CD8+ DP T-cell frequencies were analyzed by flow-cytometry. The association of damage (SLICC/ACR Damage Index, SDI) and CD4+CD28null and CD4+CD8+ DP T cells was examined by univariable and multivariable Poisson regression models, adjusting for possible confounders. All analyses were performed using SPSS 21.0. Patients' (n = 133) mean (SD) age at diagnosis was 35.5 (16.8) years, 124 (93.2%) were female; all were mestizo (mixed Caucasian and Amerindian ancestry). Disease duration was 7.4 (6.8) years. The SLE Disease Activity Index was 5.5 (4.2), and the SDI 0.9 (1.2). The percentages of CD4+CD28null and CD4+CD8+ DP T cells were 17.1 (14.4) and 0.4 (1.4), respectively. The percentage of CD4+CD28null and CD4+CD8+ DP T cells were positively associated with a higher SDI in both univariable (rate ratio (RR) 1.02, 95% confidence interval (CI): 1.01-1.03 and 1.17, 95% CI: 1.07-1.27, respectively; p < 0.001 for both) and multivariable analyses RR 1.02, 95% CI: 1.01-1.03, p = 0.001 for CD4+CD28null T cells and 1.28, 95% CI: 1.13-1.44, p < 0.001 for CD4+CD8+ DP T cells). Only the renal domain remained associated with CD4+CD28null in multivariable analyses (RR 1.023 (1.002-1.045); p = 0.034). In SLE patients, CD4+CD28null and CD4+CD8+ DP T cells are independently associated with disease damage. Longitudinal studies are warranted to determine the predictive value of these associations. © The Author(s) 2015.

A novel population of local pericyte precursor cells in tumor stroma that require Notch signaling for differentiation.

PubMed

Patenaude, Alexandre; Woerher, Stefan; Umlandt, Patricia; Wong, Fred; Ibrahim, Rawa; Kyle, Alastair; Unger, Sandy; Fuller, Megan; Parker, Jeremy; Minchinton, Andrew; Eaves, Connie J; Karsan, Aly

2015-09-01

Pericytes are perivascular support cells, the origin of which in tumor tissue is not clear. Recently, we identified a Tie1(+) precursor cell that differentiates into vascular smooth muscle, in a Notch-dependent manner. To understand the involvement of Notch in the ontogeny of tumor pericytes we used a novel flow immunophenotyping strategy to define CD146(+)/CD45(-)/CD31(-/lo) pericytes in the tumor stroma. This strategy combined with ex vivo co-culture experiments identified a novel pericyte progenitor cell population defined as Sca1(hi)/CD146(-)/CD45(-)/CD31(-). The differentiation of these progenitor cells was stimulated by co-culture with endothelial cells. Overexpression of the Notch ligand Jagged1 in endothelial cells further stimulated the differentiation of Sca1(hi)/CD146(-)/CD45(-)/CD31(-) cells into pericytes, while inhibition of Notch signaling with a γ-secretase inhibitor reduced this differentiation. However, Notch inhibition specifically in Tie1-expressing cells did not change the abundance of pericytes in tumors, suggesting that the pericyte precursor is distinct from the vascular smooth muscle cell precursor. Transplant experiments showed that the bone marrow contributes minimally to tumor pericytes. Immunophenotyping revealed that Sca1(hi)/CD146(-)/CD45(-)/CD31(-) cells have greater potential to differentiate into pericytes and have increased expression of classic mesenchymal stem cell markers (CD13, CD44, Nt5e and Thy-1) compared to Sca1(-/lo)/CD146(-)/CD45(-)/CD31(-) cells. Our results suggest that a local Sca1(hi)/CD146(-)/CD45(-)/CD31(-) pericyte progenitor resides in the tumor microenvironment and requires Notch signaling for differentiation into mature pericytes. Copyright © 2015 Elsevier Inc. All rights reserved.

Comparison of the properties of human CD146+ and CD146- periodontal ligament cells in response to stimulation with tumour necrosis factor α.

PubMed

Zhu, Wenjun; Tan, Yuanyuan; Qiu, Qihong; Li, Xiting; Huang, Zixian; Fu, Yun; Liang, Min

2013-12-01

Periodontal ligament stem cells (PDLSCs) can be used in periodontal regeneration. Tumour necrosis factor-alpha (TNF-α) participates in the regulation of cell proliferation, apoptosis, differentiation, and migration. However, whether TNF-α can affect the biological features of PDLSCs is still unclear. The objective of this study was to illustrate the biological effects (proliferation, apoptosis, osteogenesis and migration) of TNF-α on human CD146 positive periodontal ligament cells (CD146+PLDCs) and CD146 negative periodontal ligament cells (CD146-PDLCs). CD146±PDLCs were isolated from human PDLCs and analyzed using a fluorescence-activated cell sorter. The biological effects of TNF-α on CD146±PDLCs were evaluated by CCK-8 assay (proliferation), DAPI staining (apoptosis), alizarin red staining and alkaline phosphatase activities assay (osteogenesis), and wounding assay and transwell assay (migration). CD146+PDLCs, which expressed MSC surface markers CD105, CD90, CD73, CD44, and Stro-1, showed higher proliferative and osteogenic potential than CD146-PDLCs. TNF-α at a dose of 2.5ng/ml was found to enhance both proliferation and osteogenesis in CD146+PDLCs. At 5ng/ml, TNF-α promoted proliferation, osteogenesis, and apoptosis in CD146+PDLCs and enhanced osteogenesis in CD146-PDLCs. At 10ng/ml, TNF-α only aggravated apoptosis in CD146+PDLCs. The migratory ability of both CD146+PDLCs and CD146-PDLCs was not altered by TNF-α. CD146+PDLCs were subpopulation of MSC. It showed greater proliferative and osteogenic potential than CD146-PDLCs. At low concentration, TNF-α was beneficial to CD146+PDLCs on proliferation and osteogenesis, and at high concentration it was detrimental. CD146-PDLCs were found to be less sensitive to TNF-α. Copyright © 2013 Elsevier Ltd. All rights reserved.

Pre-transplant immune state defined by serum markers and alloreactivity predicts acute rejection after living donor kidney transplantation.

PubMed

Vondran, Florian W R; Timrott, Kai; Kollrich, Sonja; Steinhoff, Ann-Kristin; Kaltenborn, Alexander; Schrem, Harald; Klempnauer, Juergen; Lehner, Frank; Schwinzer, Reinhard

2014-09-01

Acute rejection (AR) remains a major cause for long-term kidney allograft failure. Reliable immunological parameters suitable to define the pre-transplant immune state and hence the individual risk of graft rejection are highly desired to preferably adapt the immunosuppressive regimen in advance. Donor and third party alloreactivities were determined by mixed lymphocyte cultures. Soluble forms of CD25, CD30, and CD44 were detected in patients' serum by ELISA. Various lymphocyte subpopulations were measured using flow cytometry. All patients received triple immunosuppression (tacrolimus/mycophenolate mofetil/steroids) and were grouped according to biopsy results within the first year: rejection-free (RF, n = 13), borderline (BL, n = 5), or acute rejection (AR, n = 7). Patients with AR showed the highest pre-transplant alloreactivities and serum levels (sCD25/sCD30/sCD44) according to the pattern RF < BL < AR. Relying on serum analysis only, multivariate logistic regression (logit link function) yielded a prognostic score for prediction of rejection with 75.0% sensitivity and 69.2% specificity. Patients with rejection showed markedly higher pre-transplant frequencies of CD4(+) /CD8(+) T cells lacking CD28, but lower numbers of CD8(+) CD161(bright) T cells and NK cells than RF individuals. Pre-transplant immune state defined by alloreactivity, serum markers, and particular lymphocyte subsets seems to correlate with occurrence of graft rejection after kidney transplantation. A prognostic score based on pre-transplant serum levels has shown great potential for prediction of rejection episodes and should be further evaluated. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Ex vivo expansion of canine cytotoxic large granular lymphocytes exhibiting characteristics of natural killer cells.

PubMed

Shin, Dong-Jun; Park, Ji-Yun; Jang, Youn-Young; Lee, Je-Jung; Lee, Youn-Kyung; Shin, Myung-Geun; Jung, Ji-Youn; Carson, William E; Cho, Duck; Kim, Sang-Ki

2013-06-15

Canine NK cells still are not well-characterized due to the lack of information concerning specific NK cell markers and the fact that NK cells are not an abundant cell population. In this study, we selectively expanded the canine cytotoxic large granular lymphocytes (CLGLs) that exhibit morphologic, genetic, and functional characteristics of NK cells from normal donor PBMCs. The cultured CLGLs were characterized by a high proportion of CD5(dim) expressing cells, of which the majority of cells co-expressed CD3 and CD8, but did not express TCRαβ and TCRγδ. The phenotype of the majority of the CLGLs was CD5(dim)CD3(+)CD8(+) TCRαβ(-)TCRγδ(-)CD4(-)CD21(-)CD11c(+/-)CD11d(+/-)CD44(+). The expression of mRNAs for NK cell-associated receptors (NKG2D, NKp30, NKp44, Ly49, perforin, and granzyme B) were highly upregulated in cultured CLGLs. Specifically, NKp46 was remarkably upregulated in the cultured CLGLs compared to PBMCs. The mRNAs for the NKT-associated iTCRα gene in CLGLs was present at a basal level. The cytotoxic activity of the CLGLs against canine NK cell-sensitive CTAC cells was remarkably elevated in a dose-dependent manner, and the CLGLs produced large amounts of IFN-γ. The antitumor activity of CLGLs extended to different types of canine tumor cells (CF41.Mg and K9TCC-pu-AXC) without specific antigen recognition. These results are consistent with prior reports, and strongly suggest that the selectively expanded CLGLs represent a population of canine NK cells. The results of this study will contribute to future research on canine NK cells as well as NK cell-based immunotherapy. Copyright © 2013 Elsevier B.V. All rights reserved.

Intra-operative electron beam radiotherapy and abdomino-pelvic surgery for cancer: influence on immunological parameters.

PubMed

Bussières, E; Richaud, P; Gualde, N

1992-10-01

Evolution of some immunological parameters was observed during the first month in 20 patients with different abdomino-pelvic cancers who underwent surgery with intra-operative radiation therapy (IORT) (mean dose of 19.44 Gy, range 15, 25). Observed parameters before (DO-) and after procedure (DO+), on seventh (D7) and fourteenth (D14) days and fifth week (D30) were: lymphocyte count, lymphocyte subsets (CD19, CD3, CD4, CD8, CD56), natural killer (NK) activity, immunoglobulins, C3 and C4b fractions of complement, soluble receptor for interleukin 2 (sIL2-R). Results showed a decrease of circulating lymphocytes (DO-: 1189 +/- 168 cells/mm3; D7: 889 +/- 91; P = 0.011), of absolute number of CD3 lymphocytes (DO-: 785 +/- 114 cells/mm3; D7: 563 +/- 86; P = 0.025), of CD4 lymphocytes (DO-: 441 +/- 70 cells/mm3; DO+: 299 +/- 43; P = 0.013) and of CD8 lymphocytes (DO-:361 +/- 50 cells/mm3, D7:250 +/- 44; P = 0.006). All values returned towards preoperative levels by D30. Absolute number of NK cells was unchanged but NK activity was significantly diminished (effector target ratio 5:1 DO-:33 +/- 5%; DO+:44 +/- 7%; D7:18 +/- 3%; D14:21 +/- 4%; D30:25 +/- 4%). sIL2-R was significantly enhanced from D7 to D30. All these impairments are moderate and these observations provide some evidence of satisfactory tolerance to IORT for abdomino-pelvic cancers during the immediate postoperative period.

Syntheses, crystal structures and photoluminescence properties of five Cd/Zn-organic frameworks

NASA Astrophysics Data System (ADS)

Li, Qing; Xue, Dong-Xu; Zhang, Yu-Feng; Zhang, Zong-Hui; Gao, Ziwei

2018-07-01

Luminescent metal-organic frameworks (MOFs) have displayed extensively potential applications for photocatalysis, photoluminescence, electroluminescence, chemical sensors et al. Herein, five new Cd/Zn-organic frameworks of [Cd(HL)C2H5OH] (1), [Cd(HL)(2,2‧-Bpy)H2O] (2), [Cd2(HL)2(Phen)2] (3), [Zn(HL)BIMB] (4), [Cd3(HL)3(4,4‧-Bpy)DMF]·(H2O) (5) have been deliberately constructed via solvothermal reactions of d10 transition metal salts, i.e. Cd(NO3)2•4H2O or Zn(NO3)2·6H2O, and a V-shaped semi-rigid organic linker of 4,4'-(hydroxymethanediyl) dibenzoic acid (H3L) along with the auxiliary poly-nitrogen ligands of 2,2‧-Bpy(2,2‧-bipyridine), Phen(phenanthroline), BIMB(1,1‧-benzene-1,4-diyldimethanediyl-bis-1H-imidazole) and 4,4‧-Bpy(4,4‧-bipyridine). The crystal structures of compounds 1-5 were precisely determined by single-crystal X-ray diffraction (SC-XRD), Powder X-ray diffraction (PXRD), Fourier transform infrared spectroscopy (FT-IR) and Thermogravimetic analysis (TGA). As revealed by SC-XRD, the isolated 1 presents a 2-periodic framework encompassing side-by-side channel-typed helical tubes. Compounds 2-4 display Z-shaped 1-periodic single chains, concomitant with twin chains and tubular structure, respectively. Interestingly, compound 5 demonstrates a two-fold interpenetrated 3-periodic skeleton in the presence of a rigid pillar of 4,4‧-Bpy. Additionally, photoluminescence properties of 1-5 were lastly investigated.

O-glycans direct selectin ligands to lipid rafts on leukocytes.

PubMed

Shao, Bojing; Yago, Tadayuki; Setiadi, Hendra; Wang, Ying; Mehta-D'souza, Padmaja; Fu, Jianxin; Crocker, Paul R; Rodgers, William; Xia, Lijun; McEver, Rodger P

2015-07-14

Palmitoylated cysteines typically target transmembrane proteins to domains enriched in cholesterol and sphingolipids (lipid rafts). P-selectin glycoprotein ligand-1 (PSGL-1), CD43, and CD44 are O-glycosylated proteins on leukocytes that associate with lipid rafts. During inflammation, they transduce signals by engaging selectins as leukocytes roll in venules, and they move to the raft-enriched uropods of polarized cells upon chemokine stimulation. It is not known how these glycoproteins associate with lipid rafts or whether this association is required for signaling or for translocation to uropods. Here, we found that loss of core 1-derived O-glycans in murine C1galt1(-/-) neutrophils blocked raft targeting of PSGL-1, CD43, and CD44, but not of other glycosylated proteins, as measured by resistance to solubilization in nonionic detergent and by copatching with a raft-resident sphingolipid on intact cells. Neuraminidase removal of sialic acids from wild-type neutrophils also blocked raft targeting. C1galt1(-/-) neutrophils or neuraminidase-treated neutrophils failed to activate tyrosine kinases when plated on immobilized anti-PSGL-1 or anti-CD44 F(ab')2. Furthermore, C1galt1(-/-) neutrophils incubated with anti-PSGL-1 F(ab')2 did not generate microparticles. In marked contrast, PSGL-1, CD43, and CD44 moved normally to the uropods of chemokine-stimulated C1galt1(-/-) neutrophils. These data define a role for core 1-derived O-glycans and terminal sialic acids in targeting glycoprotein ligands for selectins to lipid rafts of leukocytes. Preassociation of these glycoproteins with rafts is required for signaling but not for movement to uropods.

Maternal serum soluble CD30 is increased in pregnancies complicated with acute pyelonephritis.

PubMed

Kusanovic, Juan Pedro; Romero, Roberto; Esoinoza, Jimmy; Gotsch, Francesca; Edwin, Samuel; Chaiworapongsa, Tinnakorn; Mittal, Pooja; Soto, Eleazar; Erez, Offer; Mazaki-Tovi, Shali; Than, Nandor Gabor; Friel, Lara A; Yoon, Bo Hyun; Mazor, Moshe; Hassan, Sonia S

2007-11-01

Normal pregnancy is characterized by activation of the innate immunity and suppression of the adaptive limb of the immune response. However, pregnant women are more susceptible to the effects of infection and microbial products than non-pregnant women. CD30 is a member of the tumor necrosis factor receptor superfamily and is preferentially expressed by activated T cells producing Th2-type cytokines. Its soluble form (sCD30) is proposed to be an index of Th2 immune response. High serum concentrations of sCD30 have been found in the acute phase of viral infections, such as HIV-1 and hepatitis B. There is, however, conflicting evidence about serum sCD30 concentration in patients with bacterial infections. The objective of this study was to determine whether there are changes in the serum concentration of sCD30 in pregnant women with pyelonephritis. This cross-sectional study included normal pregnant women (N = 89) and pregnant women with pyelonephritis (N = 41). Maternal serum concentration of sCD30 was measured by a specific and sensitive enzyme-linked immunoassay. Non-parametric tests were used for comparisons. A p value <0.05 was considered statistically significant. (1) Pregnant women with pyelonephritis had a significantly higher median serum concentration of sCD30 than those with a normal pregnancy (median 44.3 U/mL, range 16-352.5 vs. median 29.7 U/mL, range 12.2-313.2, respectively; p < 0.001), and (2) No significant differences were found in the median maternal serum concentration of sCD30 between pregnant women with pyelonephritis who had a positive blood culture compared to those with a negative blood culture (median 47.7 U/mL, range 17.1-118.8 vs. median 42.6 U/mL, range 16-352.5, respectively; p = 0.86). Acute pyelonephritis during pregnancy is associated with a higher maternal serum concentration of sCD30 than normal pregnancy. This finding is novel and suggests that pregnant women with pyelonephritis may have a complex immune state in which there is activation of some components of what is considered a Th2 immune response.

Characteristics of Labial Gland Mesenchymal Stem Cells of Healthy Individuals and Patients with Sjögren's Syndrome: A Preliminary Study.

PubMed

Wang, Shi-Qin; Wang, Yi-Xiang; Hua, Hong

2017-08-15

Sjögren's syndrome (SS) is a systemic autoimmune disease that is characterized by focal lymphocytic infiltration into exocrine organs such as salivary and lacrimal glands, resulting in dry mouth and eyes, and other systemic injuries. There is no curative clinical therapy for SS, and stem cell therapy has shown great potential in this area. The mesenchymal stem cells (MSCs) in the salivary glands of healthy individuals and in patients with SS have not been extensively studied. The aim of this study was to elucidate the characteristics of MSCs from the labial glands of healthy controls and of those from patients with SS to elucidate the related pathogenesis and to uncover potential avenues for novel clinical interventions. Labial glands from patients with SS and healthy subjects were obtained, and MSCs were isolated and cultured by using the tissue adherent method. The MSC characteristics of the cultured cells were confirmed by using morphology, proliferation, colony forming-unit (CFU) efficiency, and multipotentiality, including osteogenic, adipogenic, and salivary gland differentiation. The MSCs from the healthy controls and SS patients expressed characteristic MSC markers, including CD29, CD44, CD73, CD90, and CD105; they were negative for CD34, CD45, and CD106, and also negative for the salivary gland epithelium markers (CD49f and CD117). Labial gland MSCs from both groups were capable of osteogenic and adipogenic differentiation. The CFU efficiency and adipogenic differentiation potential of MSCs were significantly lower in the SS group compared with the healthy controls. Cells from both groups could also be induced into salivary gland-like cells. Real-time polymerase chain reaction and immunofluorescence staining showed that the gene and protein expression of AMY1, AQP5, and ZO-1 in cells from the SS group was lower than that in cells from the healthy group. Thus, MSCs from the labial glands in patients with SS could lack certain characteristics and functions, especially related to salivary secretion. These preliminary data provided insights that could lead to the development of novel therapeutic strategies for the treatment of SS.

Immunohistochemical Analysis of p53, Ki-67, CD44, HER-2/neu Expression Patterns in Gastric Cancer, and Their Association with One Year Survival in North-West of Iran

PubMed Central

Sanaat, Zohreh; Halimi, Monireh; Ghojezadeh, Morteza; Pirovi, Amir Hossein; Gharamaleki, Jalil Vaez; Ziae, Ali Esfahani Jamal Eivazi; Kermani, Iraj Aswadi

2013-01-01

Introduction Gastric cancer remains the second most common cause of cancer-related deaths worldwide. In many malignancies like, lung and breast, multiple prognostic factors are known, such as mutations in Ki-67, HER-2/neu, p53. In this study, we evaluated immunohistochemical protein expression patterns of cell-cycle-regulators p53, proliferation marker Ki-67, surface expression of CD44, HER-2/neu oncogene proposed as useful prognostic factors. Methods In this descriptive-analytic study, we evaluate 100 patients with gastric cancer who were referred to Shahid Ghazi Hospital or other oncology clinics of Tabriz University of Medical Sciences in 2005-2010. Patients with pathologic confirmation of gastric cancer were selected. Expression of p53, ki-67, CD-44, HER-2/neu were detected by immunohistochemical staining. Results In this study, 100 patients with gastric cancer participated. 76(76%) were men and 24(24%) were women with mean age of 64.02(8.05) years. Seventy two samples were intestinal type and 28 were diffuse type. CD44 was positive in 27(27%) patients. P53 was positive in 35(35%) patients. Ki-67 was positive in 53(53%) patients. HER-2/neu was positive in 51(51%) patients. Conclusion The frequency of positive p53, Ki-67, CD44 and HER-2/neu varied in different studies. Positive Ki-67 and HER-2/neu were not associated with changes in survival but positive p53 and CD44 were significantly associated with improved survival. PMID:24505530

Characterization of CD44-Mediated Cancer Cell Uptake and Intracellular Distribution of Hyaluronan-Grafted Liposomes

PubMed Central

Qhattal, Hussaini Syed Sha; Liu, Xinli

2011-01-01

Hyaluronan (HA) is a biocompatible and biodegradable linear polysaccharide which is of interest for tumor targeting through cell surface CD44 receptors. HA binds with high affinity to CD44 receptors, which are overexpressed in many tumors and involved in cancer metastasis. In the present study, we investigated the impact of HA molecular weight (MW), grafting density, and CD44 receptor density on endocytosis of HA-grafted liposomes (HA-liposomes) by cancer cells. Additionally, the intracellular localization of the HA-liposomes was determined. HAs of different MWs (5-8, 10-12, 175-350, and 1600 kDa) were conjugated to liposomes with varying degrees of grafting density. HA surface density was quantified using the hexadecyltrimethylammonium bromide turbidimetric method. Cellular uptake and subcellular localization of HA-liposomes were evaluated by flow cytometry and fluorescence microscopy. Mean particle sizes of HA-liposomes ranged from 120 to 180 nm and increased with the bigger size of HA. HA-liposome uptake correlated with HA MW (5-8 < 10-12 < 175-350 kDa), grafting density, and CD44 receptor density and exceeded that obtained with unconjugated plain liposomes. HA-liposomes were taken up into cells via lipid raft-mediated endocytosis, which is both energy- and cholesterol-dependent. Once within cells, HA-liposomes localized primarily to endosomes and lysosomes. The results demonstrate that cellular targeting efficiency of HA-liposomes depends strongly upon HA MW, grafting density, and cell surface receptor CD44 density. The results support a role of HA-liposomes for targeted drug delivery. PMID:21696190

Novel Single-Cell Analysis Platform Based on a Solid-State Zinc-Coadsorbed Carbon Quantum Dots Electrochemiluminescence Probe for the Evaluation of CD44 Expression on Breast Cancer Cells.

PubMed

Qiu, Youyi; Zhou, Bin; Yang, Xiaojuan; Long, Dongping; Hao, Yan; Yang, Peihui

2017-05-24

A novel single-cell analysis platform was fabricated using solid-state zinc-coadsorbed carbon quantum dot (ZnCQDs) nanocomposites as an electrochemiluminescence (ECL) probe for the detection of breast cancer cells and evaluation of the CD44 expression level. Solid-state ZnCQDs nanocomposite probes were constructed through the attachment of ZnCQDs to gold nanoparticles and then the loading of magnetic beads to amplify the ECL signal, exhibiting a remarkable 120-fold enhancement of the ECL intensity. Hyaluronic acid (HA)-functionalized solid-state probes were used to label a single breast cancer cell by the specific recognition of HA with CD44 on the cell surface, revealing more stable, sensitive, and effective tagging in comparison with the water-soluble CQDs. This strategy exhibited a good analytical performance for the analysis of MDA-MB-231 and MCF-7 single cells with linear range from 1 to 18 and from 1 to 12 cells, respectively. Furthermore, this single-cell analysis platform was used for evaluation of the CD44 expression level of these two cell lines, in which the MDA-MB-231 cells revealed a 2.8-5.2-fold higher CD44 expression level. A total of 20 single cells were analyzed individually, and the distributions of the ECL intensity revealed larger variations, indicating the high cellular heterogeneity of the CD44 expression level on the same cell line. The as-proposed single-cell analysis platform might provide a novel protocol to effectively study the individual cellular function and cellular heterogeneity.

Universal monitoring of minimal residual disease in acute myeloid leukemia.

PubMed

Coustan-Smith, Elaine; Song, Guangchun; Shurtleff, Sheila; Yeoh, Allen Eng-Juh; Chng, Wee Joo; Chen, Siew Peng; Rubnitz, Jeffrey E; Pui, Ching-Hon; Downing, James R; Campana, Dario

2018-05-03

Optimal management of acute myeloid leukemia (AML) requires monitoring of treatment response, but minimal residual disease (MRD) may escape detection. We sought to identify distinctive features of AML cells for universal MRD monitoring. We compared genome-wide gene expression of AML cells from 157 patients with that of normal myeloblasts. Markers encoded by aberrantly expressed genes, including some previously associated with leukemia stem cells, were studied by flow cytometry in 240 patients with AML and in nonleukemic myeloblasts from 63 bone marrow samples. Twenty-two (CD9, CD18, CD25, CD32, CD44, CD47, CD52, CD54, CD59, CD64, CD68, CD86, CD93, CD96, CD97, CD99, CD123, CD200, CD300a/c, CD366, CD371, and CX3CR1) markers were aberrantly expressed in AML. Leukemia-associated profiles defined by these markers extended to immature CD34+CD38- AML cells; expression remained stable during treatment. The markers yielded MRD measurements matching those of standard methods in 208 samples from 52 patients undergoing chemotherapy and revealed otherwise undetectable MRD. They allowed MRD monitoring in 129 consecutive patients, yielding prognostically significant results. Using a machine-learning algorithm to reduce high-dimensional data sets to 2-dimensional data, the markers allowed a clear visualization of MRD and could detect 1 leukemic cell among more than 100,000 normal cells. The markers uncovered in this study allow universal and sensitive monitoring of MRD in AML. In combination with contemporary analytical tools, the markers improve the discrimination between leukemic and normal cells, thus facilitating data interpretation and, hence, the reliability of MRD results. National Cancer Institute (CA60419 and CA21765); American Lebanese Syrian Associated Charities; National Medical Research Council of Singapore (1299/2011); Viva Foundation for Children with Cancer, Children's Cancer Foundation, Tote Board & Turf Club, and Lee Foundation of Singapore.

Soluble CD30 serum level in HCV-positive chronic active hepatitis: A surrogate marker of disease activity?

PubMed

Foschi, F G; Gramenzi, A; Castelli, E; Cursaro, C; Pagani, S; Margotti, M; D'Errico, A; Andreone, P; Stefanini, G F; Bernardi, M

2000-06-01

In the present study, high levels of CD30s, a glycoprotein preferentially expressed and released by T lymphocytes producing Th(2)-type cytokines, were seen in the sera of patients with chronic hepatitis C, and a correlation with histological activity of the disease was found. CD30s levels were assayed in the sera of 29 HCV RNA-positive patients with histologically proven chronic active hepatitis and in 30 healthy blood donors. Thirteen of 29 (45%) HCV patients had CD30s serum levels above the normal range (>20 U/ml). Mean CD30s serum levels were significantly higher in HCV patients than in controls (P<0.0005). A positive correlation was found between serum CD30s levels and both the histological activity index (r=0.59, P=0.001) and ALT serum levels (r=0.5; P=0.006). The raised CD30s level found in more severe HCV liver disease indirectly suggests activation and expansion of Th(2)cells. CD30s levels could represent a useful surrogate marker of activity in chronic HCV infections. Copyright 2000 Academic Press.

CD45RA, a specific marker for leukaemia stem cell sub-populations in acute myeloid leukaemia.

PubMed

Kersten, Bas; Valkering, Matthijs; Wouters, Rolf; van Amerongen, Rosa; Hanekamp, Diana; Kwidama, Zinia; Valk, Peter; Ossenkoppele, Gert; Zeijlemaker, Wendelien; Kaspers, Gertjan; Cloos, Jacqueline; Schuurhuis, Gerrit J

2016-04-01

Chemotherapy resistant leukaemic stem cells (LSC) are thought to be responsible for relapses after therapy in acute myeloid leukaemia (AML). Flow cytometry can discriminate CD34(+) CD38(-) LSC and normal haematopoietic stem cells (HSC) by using aberrant expression of markers and scatter properties. However, not all LSC can be identified using currently available markers, so new markers are needed. CD45RA is expressed on leukaemic cells in the majority of AML patients. We investigated the potency of CD45RA to specifically identify LSC and HSC and improve LSC quantification. Compared to our best other markers (CLL-1, also termed CLEC12A, CD33 and CD123), CD45RA was the most reliable marker. Patients with high percentages (>90%) of CD45RA on CD34(+) CD38(-) LSC have 1·69-fold higher scatter values compared to HSC (P < 0·001), indicating a more mature CD34(+) CD38(-) phenotype. Patients with low (<10%) or intermediate (10-90%) CD45RA expression on LSC showed no significant differences to HSC (1·12- and 1·15-fold higher, P = 0·31 and P = 0·44, respectively). CD45RA-positive LSC tended to represent more favourable cytogenetic/molecular markers. In conclusion, CD45RA contributes to more accurate LSC detection and is recommended for inclusion in stem cell tracking panels. CD45RA may contribute to define new LSC-specific therapies and to monitor effects of anti-LSC treatment. © 2016 John Wiley & Sons Ltd.

Influence of heat treatment on hole transfer dynamics in core-shell quantum dot/organic hole conductor hybrid films

NASA Astrophysics Data System (ADS)

Sun, Mingye; Zheng, Youjin; Zhang, Lei; Zhao, Liping; Zhang, Bing

2017-08-01

The influence of heat treatment on hole transfer (HT) processes from the CdSe/ZnS and CdSe/CdS/ZnS quantum dots (QDs) to 4,4‧,4″-Tris(carbazol-9-yl)-triphenylamine (TCTA) in QD/TCTA hybrid films has been researched with time-resolved photoluminescence (PL) spectroscopy. The PL dynamic results demonstrated a heat-treatment-temperature-dependent HT process from the core-shell CdSe QDs to TCTA. The HT rates and efficiencies can be effectively increased due to reduced distance between core-shell CdSe QDs and TCTA after heat treatment. The CdS shell exhibited a more obvious effect on HT from the core-shell CdSe QDs to TCTA than on electron transfer to TiO2, due to higher barrier for holes to tunnel through CdS shell and larger effective mass of holes in CdS than electrons. These results indicate that heat treatment would be an effective means to further optimize solid-state QD sensitized solar cells and rational design of CdS shell is significant.

AFM force measurements of the gp120-sCD4 and gp120 or CD4 antigen-antibody interactions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Yong, E-mail: dr_yongchen@hotmail.com; Department of Microbiology and Immunology, University of Illinois at Chicago, Chicago, IL 60612; Zeng, Gucheng

2011-04-08

Highlights: {yields} The unbinding force of sCD4-gp120 interaction was 25.45 {+-} 20.46 pN. {yields} The unbinding force of CD4 antigen-antibody interaction was 51.22 {+-} 34.64 pN. {yields} The unbinding force of gp120 antigen-antibody interaction was 89.87 {+-} 44.63 pN. {yields} The interaction forces between various HIV inhibitors and the target molecules are significantly different. {yields} Functionalizing on AFM tip or substrate of an interaction pair caused different results. -- Abstract: Soluble CD4 (sCD4), anti-CD4 antibody, and anti-gp120 antibody have long been regarded as entry inhibitors in human immunodeficiency virus (HIV) therapy. However, the interactions between these HIV entry inhibitors andmore » corresponding target molecules are still poorly understood. In this study, atomic force microscopy (AFM) was utilized to investigate the interaction forces among them. We found that the unbinding forces of sCD4-gp120 interaction, CD4 antigen-antibody interaction, and gp120 antigen-antibody interaction were 25.45 {+-} 20.46, 51.22 {+-} 34.64, and 89.87 {+-} 44.63 pN, respectively, which may provide important mechanical information for understanding the effects of viral entry inhibitors on HIV infection. Moreover, we found that the functionalization of an interaction pair on AFM tip or substrate significantly influenced the results, implying that we must perform AFM force measurement and analyze the data with more caution.« less

Isolation and characterization of equine endometrial mesenchymal stromal cells.

PubMed

Rink, B Elisabeth; Amilon, Karin R; Esteves, Cristina L; French, Hilari M; Watson, Elaine; Aurich, Christine; Donadeu, F Xavier

2017-07-12

Equine mesenchymal stromal/stem cells (MSCs) are most commonly harvested from bone marrow (BM) or adipose tissue, requiring the use of surgical procedures. By contrast, the uterus can be accessed nonsurgically, and may provide a more readily available cell source. While human endometrium is known to harbor mesenchymal precursor cells, MSCs have not been identified in equine endometrium. This study reports the isolation, culture, and characterization of MSCs from equine endometrium. The presence of MSC and pericyte markers in endometrial sections was determined using immunohistochemistry. Stromal cells were harvested and cultured after separation of epithelial cells from endometrial fragments using Mucin-1-bound beads. For comparison, MSCs were also harvested from BM. The expression of surface markers in endometrial and BM-derived MSCs was characterized using flow cytometry and quantitative polymerase chain reaction. MSCs were differentiated in vitro into adipogenic, chondrogenic, osteogenic, and smooth muscle lineages. Typical markers of MSCs (CD29, CD44, CD90, and CD105) and pericytes (NG2 and CD146) were localized in the equine endometrium. Both endometrial and BM MSCs grew clonally and robustly expressed MSC and pericyte markers in culture while showing greatly reduced or negligible expression of hematopoietic markers (CD45, CD34) and MHC-II. Additionally, both endometrial and BM MSCs differentiated into adipogenic, osteogenic, and chondrogenic lineages in vitro, and endometrial MSCs had a distinct ability to undergo smooth muscle differentiation. We have demonstrated for the first time the presence of cells in equine endometrium that fulfill the definition of MSCs. The equine endometrium may provide an alternative, easily accessible source of MSCs, not only for therapeutic regeneration of the uterus, but also for other tissues where MSCs from other sources are currently being used therapeutically.

Isolation and hepatocyte differentiation of mesenchymal stem cells from porcine bone marrow--"surgical waste" as a novel MSC source.

PubMed

Brückner, S; Tautenhahn, H-M; Winkler, S; Stock, P; Jonas, S; Dollinger, M; Christ, B

2013-06-01

Mesenchymal stem cells (MSC) isolated from bone marrow and differentiated into hepatocyte-like cells have increasingly gained attention for clinical cell therapy of liver diseases because of their high regenerative capacity. They are available from bone marrow aspirates of the os coxae after puncture of the crista iliaca or from bone marrow "surgical waste" gained from amputations or knee and hip operations. Thus, the aim of the study was to demonstrate whether these pBM-MSC (porcine bone marrow-derived mesenchymal stem cells) displayed mesenchymal features and hepatocyte differentiation potential. MSC were isolated either from crista iliaca punctures or after sampling and collagenase digestion of bone marrow from the os femoris. Mesenchymal features were assessed by flow cytometry for specific surface antigens and their ability to differentiate into at least 3 lineages. Functional properties, such as urea or glycogen synthesis and cytochrome P450 activity, as well as the cell morphology were examined during hepatocyte differentiation. pBM-MSC from both sources lacked the hematopoietic markers CD14 and CD45 but expressed the typical mesenchymal markers CD44, CD29, CD90, and CD105. Both cell types could differentiate into adipocyte, osteocyte, and hepatocyte lineages. After hepatocyte differentiation, CD105 expression decreased significantly and cells changed morphology from fibroblastoid into polygonal, displaying significantly increased glycogen storage, urea synthesis, and cytochrome activity. pBM-MSC from various sources were identical in respect to their mesenchymal features and their hepatocyte differentiation potential. Hence, long bones might be a particularly useful resource to isolate bone marrow mesenchymal stem cells for transplantation. Copyright © 2013 Elsevier Inc. All rights reserved.

Expansion of mesenchymal stem cells from human pancreatic ductal epithelium.

PubMed

Seeberger, Karen L; Dufour, Jannette M; Shapiro, Andrew M James; Lakey, Jonathan R T; Rajotte, Ray V; Korbutt, Gregory S

2006-02-01

Fibroblast-like cells emerging from cultured human pancreatic endocrine and exocrine tissue have been reported. Although a thorough phenotypic characterization of these cells has not yet been carried out, these cells have been hypothesized to be contaminating fibroblasts, mesenchyme and/or possibly beta-cell progenitors. In this study, we expanded fibroblast-like cells from adult human exocrine pancreas following islet isolation and characterized these cells as mesenchymal stem cells (MSCs) based on their cell surface antigen expression and ability to differentiate into mesoderm. Analysis by flow cytometry demonstrated that pancreatic MSCs express cell surface antigens used to define MSCs isolated from bone marrow such as CD13, CD29, CD44, CD49b, CD54, CD90 and CD105. In addition, utilizing protocols used to differentiate MSCs isolated from other somatic tissues, we successfully differentiated pancreatic MSCs into: (1) osteocytes that stained positive for alkaline phosphatase, collagen, mineralization (calcification) and expressed osteocalcin, (2) adipocytes that contained lipid inclusions and expressed fatty acid binding protein 4 and (3) chondrocytes that expressed aggrecan. We also demonstrated that pancreatic MSCs are multipotent and capable of deriving cells of endodermal origin. Pancreatic MSCs were differentiated into hepatocytes that stained positive for human serum albumin and expressed endoderm and liver-specific genes such as GATA 4 and tyrosine aminotransferase. In addition, preliminary protocols used to differentiate these cells into insulin-producing cells resulted in the expression of genes necessary for islet and beta-cell development such as Pax4 and neurogenin 3. Therefore, multipotent MSCs residing within the adult exocrine pancreas could represent a progenitor cell, which when further manipulated could result in the production of functional islet beta-cells.

Chitosan promotes cancer progression and stem cell properties in association with Wnt signaling in colon and hepatocellular carcinoma cells

PubMed Central

Chang, Po-Hsiang; Sekine, Keisuke; Chao, Hsiao-Mei; Hsu, Shan-hui; Chern, Edward

2017-01-01

Cancer stem cells (CSCs), a small population of cancer cells, have been considered to be the origin of cancer initiation, recurrence, and metastasis. Tumor microenvironment provides crucial signals for CSCs to maintain stem cell properties and promotes tumorigenesis. Therefore, establishment of an appropriate cell culture system to mimic the microenvironment for CSC studies is an important issue. In this study, we grew colon and hepatocellular carcinoma (HCC) cells on chitosan membranes and evaluated the tumor progression and the CSC properties. Experimental results showed that culturing cancer cells on chitosan increased cell motility, drug resistance, quiescent population, self-renewal capacity, and the expression levels of stemness and CSC marker genes, such as OCT4, NANOG, CD133, CD44, and EpCAM. Furthermore, we demonstrated that chitosan might activate canonical Wnt/β-catenin-CD44 axis signaling in CD44positive colon cancer cells and noncanonical Wnt-STAT3 signaling in CD44negative HCC cells. In conclusion, chitosan as culture substrates activated the essential signaling of CSCs and promoted CSC properties. The chitosan culture system provides a convenient platform for the research of CSC biology and screening of anticancer drugs. PMID:28367998

MicroRNA-126 deficiency enhanced the activation and function of CD4+ T cells by elevating IRS-1 pathway.

PubMed

Chu, F; Hu, Y; Zhou, Y; Guo, M; Lu, J; Zheng, W; Xu, H; Zhao, J; Xu, L

2018-02-01

Recent evidence has shown that microRNA-126 (miR-126) has been involved in the development and function of immune cells, which contributed to the pathogenesis of related clinical diseases. However, the potential role of miR-126 in the development and function of CD4 + T cells remains largely unknown. Here we first found that the activation and proliferation, as well as the expression of interferon (IFN)-γ, of CD4 + T cells from miR-126 knock-down (KD) mice using the miRNA-sponge technique were enhanced significantly in vitro, compared with those in CD4 + T cells from wild-type (WT) mice. To monitor further the possible effect of miR-126 deficiency on the function of CD4 + T cells in vivo, we used dextran sulphate sodium (DSS)-induced murine model of acute autoimmune colitis and found that miR-126 deficiency could elevate the pathology of colitis. Importantly, the proportion of CD4 + T cells in splenocytes increased significantly in miR-126KD mice. Moreover, the expression levels of CD69 and CD44 on CD4 + T cells increased significantly and the expression level of CD62L decreased significantly. Of note, adoptive cell transfer assay showed that the pathology of colitis was more serious in carboxyfluorescein succinimidyl ester (CFSE)-labelled miR-126KD CD4 + T cell-transferred group, compared with that in the CFSE-labelled WT CD4 + T cells transferred group. Consistently, the expression levels of CD69 and CD44 on CFSE + cells increased significantly. Furthermore, both the proliferation and IFN-γ secretion of CFSE + cells also increased significantly in the CFSE-labelled miR-126KD CD4 + T cell-transferred group. Mechanistic evidence showed that the expression of insulin receptor substrate 1 (IRS-1), as a functional target of miR-126, was elevated in CD4 + T cells from miR-126KD mice, accompanied by altered transduction of the extracellular regulated kinase, protein B (AKT) and nuclear factor kappa B (NF-κB) pathway. Our data revealed a novel role in which miR-126 was an intrinsic regulator in the function of CD4 + T cells, which provided preliminary basis for exploring further the role of miR-126 in the development, function of CD4 + T cells and related clinical diseases. © 2017 British Society for Immunology.

Cadmium accumulation is enhanced by ammonium compared to nitrate in two hyperaccumulators, without affecting speciation

PubMed Central

Cheng, Miaomiao; Wang, Peng; Kopittke, Peter M.; Wang, Anan; Sale, Peter W.G.

2016-01-01

Nitrogen fertilization could improve the efficiency of Cd phytoextraction in contaminated soil and thus shorten the remediation time. However, limited information is available on the effect of N form on Cd phytoextraction and associated mechanisms in plants. This study examined the effect of N form on Cd accumulation, translocation, and speciation in Carpobrotus rossii and Solanum nigrum. Plants were grown in nutrient solution with 5–15 μM Cd in the presence of 1000 µM NH4 + or NO3 −. Plant growth and Cd uptake were measured, and Cd speciation was analyzed using synchrotron-based X-ray absorption spectroscopy. Shoot Cd accumulation was 30% greater with NH4 + than NO3 − supply. Carpobrotus rossii accumulated three times more Cd than S. nigrum. However, Cd speciation in the plants was not influenced by N form, but it did vary with species and tissues. In C. rossii, up to 91% of Cd was bound to S-containing ligands in all tissues except the xylem sap where 87–95% were Cd-OH complexes. Furthermore, the proportion of Cd-S in shoots was substantially lower in S. nigrum (44–69%) than in C. rossii (60–91%). It is concluded that the application of NH4 + (instead of NO3 −) increased shoot Cd accumulation by increasing uptake and translocation, rather than changing Cd speciation, and is potentially an effective approach for increasing Cd phytoextraction. PMID:27385767

Solvent-Induced Cadmium(II) Metal-Organic Frameworks with Adjustable Guest-Evacuated Porosity: Application in the Controllable Assembly of MOF-Derived Porous Carbon Materials for Supercapacitors.

PubMed

Yue, Man-Li; Jiang, Yi-Fan; Zhang, Lin; Yu, Cheng-Yan; Zou, Kang-Yu; Li, Zuo-Xi

2017-11-07

In this work, five new cadmium metal-organic frameworks (Cd-MOFs 1-5) have been synthesized from solvothermal reactions of Cd(NO 3 ) 2 ⋅4 H 2 O with isophthalic acid and 1,4-bis(imidazol-1-yl)-benzene under different solvent systems of CH 3 OH, C 2 H 5 OH, (CH 3 ) 2 CHOH, DMF, and N-methyl-2-pyrrolidone (NMP), respectively. Cd-MOF 1 shows a 3D diamondoid framework with 1D rhombic and hexagonal channels, and the porosity is 12.9 %. Cd-MOF 2 exhibits a 2D (4,4) layer with a 1D parallelogram channel and porosity of 23.6 %. Cd-MOF 3 has an 8-connected dense network with the Schäfli symbol of [4 24 ⋅6 4 ] based on the Cd 6 cluster. Cd-MOFs 4-5 are isomorphous, and display an absolutely double-bridging 2D (4,4) layer with 1D tetragonal channels and porosities of 29.2 and 28.2 %, which are occupied by DMF and NMP molecules, respectively. Followed by the calcination-thermolysis procedure, Cd-MOFs 1-5 are employed as precursors to prepare MOF-derived porous carbon materials (labeled as PC-me, PC-eth, PC-ipr, PC-dmf and PC-nmp), which have the BET specific surface area of 23, 51, 10, 122, and 96 m 2  g -1 , respectively. The results demonstrate that the specific surface area of PCs is tuned by the porosity of Cd-MOFs, where the later is highly dependent on the solvent. Thereby, the specific surface area of PCs could be adjusted by the solvent used in the synthese of MOF precusors. Significantly, PCs have been further activated by KOH to obtain activated carbon materials (APCs), which possess even higher specific surface area and larger porosity. After a series of characterization and electrochemical investigations, the APC-dmf electrode exhibits the best porous properties and largest specific capacitances (153 F g -1 at 5 mV s -1 and 156 F g -1 at 0.5 Ag -1 ). Meanwhile, the APC-dmf electrode shows excellent cycling stability (ca. 84.2 % after 5000 cycles at 1 Ag -1 ), which can be applied as a suitable electrode material for supercapacitors. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Light- and transmission-electron-microscopic investigations on distribution of CD44, connexin 43 and actin cytoskeleton during the foreign body reaction to a nanoparticular hydroxyapatite in mini-pigs.

PubMed

Wenisch, Sabine; Cavalcanti-Adam, E Ada; Tryankowski, Eva; Raabe, Oksana; Kilian, Olaf; Heiss, Christian; Alt, Volker; Arnhold, Stefan; Schnettler, Reinhard

2012-07-01

Foreign body giant cells (FBGCs) are formed by fusion of mononucleated macrophages during the foreign body response to a nanoparticulate hydroxyapatite (HA) implanted in defects of mini-pig femura. The molecular mechanisms underlying the formation of FBGCs are still largely obscure. Here we propose connexin 43 (cx43) and CD44 as candidate molecules involved in the fusion process. Immunohistochemistry and ultrastructural immunogold labeling indicated that cx43 is present within the ruffled border of FBGCs and is the main component of gap junctions formed between fusing macrophages. CD44 was strongly expressed during clustering and fusion of mononucleated macrophages. FBGCs adhering apically at the implanted HA showed CD44 reactivity only along the basolateral aspects of the plasma membranes, while podosome formation was observed within the sealing zone and ruffled border. Taken together, these findings demonstrate that cx43 and CD44 are part of the fusion machinery responsible for the formation of FBGCs. Furthermore, the results of microfilament and cx43 labeling suggest a functional role for podosomes and hemi-channels in biomaterial degradation. Copyright © 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

The CD4+/CD8+ Ratio in Pulmonary Tuberculosis: Systematic and Meta-Analysis Article.

PubMed

Yin, Yongmei; Qin, Jie; Dai, Yaping; Zeng, Fanwei; Pei, Hao; Wang, Jun

2015-02-01

The ratio of CD4+/CD8+ has been used as a clinically index to evaluate patients' immunity. Numerous researchers have studied CD4+/CD8+ ratio in pulmonary tuberculosis (PTB) patients. However, the change of CD4+/CD8+ ratio remains controversial. We present a meta-analysis of 15 case-control studies to identify the change of CD4+/CD8+ ratio in PTB patients. We assessed heterogeneity of effect estimates within each group using I(2) test. Subgroup analysis was performed to explore the potential source of heterogeneity. To investigate further the potential publication bias, we visually examined the funnel plots. For robustness of results, we performed sensitivity analysis by removing studies. Data entry and analyses were carried out with RevMan 5.2 (The Nordic Cochrane Centre). Twelve peripheral blood studies were categorized into two subgroups. Eight studies presented a significant decrease of CD4+/CD8+ ratio in PTB cases compared to healthy subjects (SMD: -0.45; 95% CI -0.65--0.25; I(2) = 7%). Other four studies researched on the newly diagnosed patients presented a more seriously and significantly decrease (SMD: -2.17; 95% CI -2.61--1.74; I(2) = 37%). The pooled analysis of bronchoalveolar lavage fluid (BALF) studies showed a significant increase of CD4+/CD8+ ratio using Flow Cytometry (FCM) (SMD: 4.75; 95% CI 3.44-6.05; I(2) =0%). The present meta-analysis indicated that there was a synthetic evidence for the reduced CD4+/CD8+ ratio in peripheral blood of PTB patients, especially newly diagnosed cases. However, the CD4+/CD8+ ratio in BALF was increased using method of FCM.

Nondestructive Investigation of Heterojunction Interfacial Properties Using Two-Wavelength Raman Spectroscopy on Thin-Film CdS/CdTe Solar Cells.

PubMed

Zeng, Guanggen; Harrison, Paul; Kidman, Ali; Al-Mebir, Alaa; Feng, Lianghuan; Wu, Judy

2016-09-01

Raman spectra specific to CdS and CdTe were obtained on the CdS/CdTe heterojunction interface by employing two excitation wavelengths of λ1 = 488 nm and λ2 = 633 nm, respectively, from the glass side of Glass/FTO/CdS/CdTe/HgTe:Cu:graphite/Ag solar cells fabricated using pulsed-laser deposition (PLD). This two-wavelength Raman spectroscopy approach, with one wavelength selected below the absorption edge of the window layer (λ2 in this case), allows nondestructive characterization of the CdS/CdTe heterojunction and therefore correlation of the interfacial properties with the solar cell performance. In this study, the evolution of the interfacial strain relaxation during cell fabrication process was found to be affected not only by the inter-diffusion of S and Te corresponding to the formation of CdSxTe1-x ternary alloy with a various x from ∼0.01 to ∼0.067, but also by the variation in misfit dislocations (MDs) at CdS/CdTe interface from Raman TO/LO ratio ∼2.85 for as-deposited sample to TO/LO ∼4.44 for the cells post treatment. This is consistent with the change of the Urbach energy from 0.03 eV to 0.09 eV, indicative of the deterioration of crystalline quality of CdTe at interface although improved CdTe crystalline quality was observed away from the interface after the CdCl2 annealing. This difference crucially impacted on the rectification characteristics of the CdS/CdTe heterojunction and therefore the solar cell performance. © The Author(s) 2016.

Walnut Phenolic Extract and Its Bioactive Compounds Suppress Colon Cancer Cell Growth by Regulating Colon Cancer Stemness.

PubMed

Lee, Jisoo; Kim, Yoo-Sun; Lee, JaeHwan; Heo, Seung Chul; Lee, Kook Lae; Choi, Sang-Woon; Kim, Yuri

2016-07-21

Walnut has been known for its health benefits, including anti-cardiovascular disease and anti-oxidative properties. However, there is limited evidence elucidating its effects on cancer stem cells (CSCs) which represent a small subset of cancer cells that provide resistance against chemotherapy. This study aimed to evaluate the anti-CSCs potential of walnut phenolic extract (WPE) and its bioactive compounds, including (+)-catechin, chlorogenic acid, ellagic acid, and gallic acid. In the present study, CD133⁺CD44⁺ cells were isolated from HCT116 cells using fluorescence-activated cell sorting (FACS) and then treated with WPE. As a result, survival of the CD133⁺CD44⁺ HCT116 cells was inhibited and cell differentiation was induced by WPE. In addition, WPE down-regulated the CSC markers, CD133, CD44, DLK1, and Notch1, as well as the β-catenin/p-GSK3β signaling pathway. WPE suppressed the self-renewal capacity of CSCs. Furthermore, the WPE exhibited stronger anti-CSC effects than its individual bioactive compounds. Finally, the WPE inhibited specific CSC markers in primary colon cancer cells isolated from primary colon tumor. These results suggest that WPE can suppress colon cancer by regulating the characteristics of colon CSCs.

Walnut Phenolic Extract and Its Bioactive Compounds Suppress Colon Cancer Cell Growth by Regulating Colon Cancer Stemness

PubMed Central

Lee, Jisoo; Kim, Yoo-Sun; Lee, JaeHwan; Heo, Seung Chul; Lee, Kook Lae; Choi, Sang-Woon; Kim, Yuri

2016-01-01

Walnut has been known for its health benefits, including anti-cardiovascular disease and anti-oxidative properties. However, there is limited evidence elucidating its effects on cancer stem cells (CSCs) which represent a small subset of cancer cells that provide resistance against chemotherapy. This study aimed to evaluate the anti-CSCs potential of walnut phenolic extract (WPE) and its bioactive compounds, including (+)-catechin, chlorogenic acid, ellagic acid, and gallic acid. In the present study, CD133+CD44+ cells were isolated from HCT116 cells using fluorescence-activated cell sorting (FACS) and then treated with WPE. As a result, survival of the CD133+CD44+ HCT116 cells was inhibited and cell differentiation was induced by WPE. In addition, WPE down-regulated the CSC markers, CD133, CD44, DLK1, and Notch1, as well as the β-catenin/p-GSK3β signaling pathway. WPE suppressed the self-renewal capacity of CSCs. Furthermore, the WPE exhibited stronger anti-CSC effects than its individual bioactive compounds. Finally, the WPE inhibited specific CSC markers in primary colon cancer cells isolated from primary colon tumor. These results suggest that WPE can suppress colon cancer by regulating the characteristics of colon CSCs. PMID:27455311

Does the liposuction method influence the phenotypic characteristic of human adipose-derived stem cells?

PubMed

Bajek, Anna; Gurtowska, Natalia; Gackowska, Lidia; Kubiszewska, Izabela; Bodnar, Magdalena; Marszałek, Andrzej; Januszewski, Rafał; Michalkiewicz, Jacek; Drewa, Tomasz

2015-05-14

Adipose-derived stem cells (ASCs) possess a high differentiation and proliferation potential. However, the phenotypic characterization of ASCs is still difficult. Until now, there is no extensive analysis of ASCs markers depending on different liposuction methods. Therefore, the aim of the present study was to analyse 242 surface markers and determine the differences in the phenotypic pattern between ASCs obtained during mechanical and ultrasound-assisted liposuction. ASCs were isolated from healthy donors, due to mechanical and ultrasound-assisted liposuction and cultured in standard medium to the second passage. Differentiation potential and markers expression was evaluated to confirm the mesenchymal nature of cells. Then, the BD LyoplateTM Human Cell Surface Marker Screening Panel was used. Results shown that both population of ASCs are characterized by high expression of markers specific for ASCs: cluster of differentiation (CD)9, CD10, CD34, CD44, CD49d, CD54, CD55, CD59, CD71 and low expression of CD11a, CD11c and CD144. Moreover, we have noticed significant differences in antigen expression in 58 markers from the 242 studied. Presented study shows for the first time that different liposuction methods are not a significant factor which can influence the expression of human ASCs surface markers. © 2015 The Authors.

Spontaneous and natural cytotoxicity receptor-mediated cytotoxicity are effector functions of distinct natural killer subsets in hepatitis C virus-infected chimpanzees.

PubMed

Verstrepen, B E; Nieuwenhuis, I G; Mooij, P; Bogers, W M; Boonstra, A; Koopman, G

2016-07-01

In humans, CD16 and CD56 are used to identify functionally distinct natural killer (NK) subsets. Due to ubiquitous CD56 expression, this marker cannot be used to distinguish between NK cell subsets in chimpanzees. Therefore, functional analysis of distinct NK subsets during hepatitis C virus (HCV) infection has never been performed in these animals. In the present study an alternative strategy was used to identify four distinct NK subsets on the basis of the expression of CD16 and CD94. The expression of activating and inhibiting surface receptors showed that these subsets resemble human NK subsets. CD107 expression was used to determine degranulation of the different subsets in naive and HCV-infected chimpanzees. In HCV-infected chimpanzees increased spontaneous cytotoxicity was observed in CD94(high/dim) CD16(pos) and CD94(low) CD16(pos) subsets. By contrast, increased natural cytotoxicity receptor (NCR)- mediated degranulation after NKp30 and NKp44 triggering was demonstrated in the CD94(dim) CD16(neg) subset. Our findings suggest that spontaneous and NCR-mediated cytotoxicity are effector functions of distinct NK subsets in HCV-infected chimpanzees. © 2016 British Society for Immunology.

Regulation of inflammatory chemokine receptors on blood T cells associated to the circulating versus liver chemokines in dengue fever.

PubMed

de-Oliveira-Pinto, Luzia Maria; Marinho, Cíntia Ferreira; Povoa, Tiago Fajardo; de Azeredo, Elzinandes Leal; de Souza, Luiza Assed; Barbosa, Luiza Damian Ribeiro; Motta-Castro, Ana Rita C; Alves, Ada M B; Ávila, Carlos André Lins; de Souza, Luiz José; da Cunha, Rivaldo Venâncio; Damasco, Paulo Vieira; Paes, Marciano Viana; Kubelka, Claire Fernandes

2012-01-01

Little is known about the role of chemokines/chemokines receptors on T cells in natural DENV infection. Patients from DENV-2 and -3- outbreaks were studied prospectively during the acute or convalescent phases. Expression of chemokine receptor and activation markers on lymphocyte subpopulations were determined by flow cytometry analysis, plasma chemokine ligands concentrations were measured by ELISA and quantification of CCL5/RANTES(+) cells in liver tissues from fatal dengue cases was performed by immunochemistry. In the acute DENV-infection, T-helper/T-cytotoxic type-1 cell (Th1/Tc1)-related CCR5 is significantly higher expressed on both CD4 and CD8 T cells. The Th1-related CXCR3 is up-regulated among CD4 T cells and Tc2-related CCR4 is up-regulated among CD8 T cells. In the convalescent phase, all chemokine receptor or chemokine ligand expression tends to reestablish control healthy levels. Increased CCL2/MCP-1 and CCL4/MIP-1β but decreased CCL5/RANTES levels were observed in DENV-patients during acute infection. Moreover, we showed an increased CD107a expression on CCR5 or CXCR3-expressing T cells and higher expression of CD29, CD44(HIGH) and CD127(LOW) markers on CCR4-expressing CD8 T cells in DENV-patients when compared to controls. Finally, liver from dengue fatal patients showed increased number of cells expressing CCL5/RANTES in three out of four cases compared to three death from a non-dengue patient. In conclusion, both Th1-related CCR5 and CXCR3 among CD4 T cells have a potential ability to exert cytotoxicity function. Moreover, Tc1-related CCR5 and Tc2-related CCR4 among CD8 T cells have a potential ability to exert effector function and migration based on cell markers evaluated. The CCR5 expression would be promoting an enhanced T cell recruitment into liver, a hypothesis that is corroborated by the CCL5/RANTES increase detected in hepatic tissue from dengue fatal cases. The balance between protective and pathogenic immune response mediated by chemokines during dengue fever will be discussed.

Regulation of Inflammatory Chemokine Receptors on Blood T Cells Associated to the Circulating Versus Liver Chemokines in Dengue Fever

PubMed Central

Povoa, Tiago Fajardo; de Azeredo, Elzinandes Leal; de Souza, Luiza Assed; Barbosa, Luiza Damian Ribeiro; Motta-Castro, Ana Rita C.; Alves, Ada M. B.; Ávila, Carlos André Lins; de Souza, Luiz José; da Cunha, Rivaldo Venâncio; Damasco, Paulo Vieira; Paes, Marciano Viana; Kubelka, Claire Fernandes

2012-01-01

Little is known about the role of chemokines/chemokines receptors on T cells in natural DENV infection. Patients from DENV-2 and -3- outbreaks were studied prospectively during the acute or convalescent phases. Expression of chemokine receptor and activation markers on lymphocyte subpopulations were determined by flow cytometry analysis, plasma chemokine ligands concentrations were measured by ELISA and quantification of CCL5/RANTES+ cells in liver tissues from fatal dengue cases was performed by immunochemistry. In the acute DENV-infection, T-helper/T-cytotoxic type-1 cell (Th1/Tc1)-related CCR5 is significantly higher expressed on both CD4 and CD8 T cells. The Th1-related CXCR3 is up-regulated among CD4 T cells and Tc2-related CCR4 is up-regulated among CD8 T cells. In the convalescent phase, all chemokine receptor or chemokine ligand expression tends to reestablish control healthy levels. Increased CCL2/MCP-1 and CCL4/MIP-1β but decreased CCL5/RANTES levels were observed in DENV-patients during acute infection. Moreover, we showed an increased CD107a expression on CCR5 or CXCR3-expressing T cells and higher expression of CD29, CD44HIGH and CD127LOW markers on CCR4-expressing CD8 T cells in DENV-patients when compared to controls. Finally, liver from dengue fatal patients showed increased number of cells expressing CCL5/RANTES in three out of four cases compared to three death from a non-dengue patient. In conclusion, both Th1-related CCR5 and CXCR3 among CD4 T cells have a potential ability to exert cytotoxicity function. Moreover, Tc1-related CCR5 and Tc2-related CCR4 among CD8 T cells have a potential ability to exert effector function and migration based on cell markers evaluated. The CCR5 expression would be promoting an enhanced T cell recruitment into liver, a hypothesis that is corroborated by the CCL5/RANTES increase detected in hepatic tissue from dengue fatal cases. The balance between protective and pathogenic immune response mediated by chemokines during dengue fever will be discussed. PMID:22815692

Self-assembly of high-nuclearity lanthanide-based nanoclusters for potential bioimaging applications

NASA Astrophysics Data System (ADS)

Yang, Xiaoping; Wang, Shiqing; Schipper, Desmond; Zhang, Lijie; Li, Zongping; Huang, Shaoming; Yuan, Daqiang; Chen, Zhongning; Gnanam, Annie J.; Hall, Justin W.; King, Tyler L.; Que, Emily; Dieye, Yakhya; Vadivelu, Jamuna; Brown, Katherine A.; Jones, Richard A.

2016-05-01

Two series of Cd-Ln and Ni-Ln clusters [Ln8Cd24L12(OAc)44(48)Cl4(0)] and [Ln8Ni6L6(OAc)24(EtOH)6(H2O)2] were constructed using a flexible ligand. The Cd-Ln clusters exhibit interesting nano-drum-like structures which allows direct visualization by TEM. Luminex MicroPlex Microspheres loaded with the Cd-Sm cluster were visualized using epifluorescence microscopy. Cytotoxicity studies on A549 and AGS cancer cell lines showed that the materials have mild to moderate cytotoxicity.Two series of Cd-Ln and Ni-Ln clusters [Ln8Cd24L12(OAc)44(48)Cl4(0)] and [Ln8Ni6L6(OAc)24(EtOH)6(H2O)2] were constructed using a flexible ligand. The Cd-Ln clusters exhibit interesting nano-drum-like structures which allows direct visualization by TEM. Luminex MicroPlex Microspheres loaded with the Cd-Sm cluster were visualized using epifluorescence microscopy. Cytotoxicity studies on A549 and AGS cancer cell lines showed that the materials have mild to moderate cytotoxicity. Electronic supplementary information (ESI) available: Full experimental and characterization details for 1-5. CCDC 1007468, 1007469 and 1007472-1007474. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c6nr00642f

Double heterojunction nanowire photocatalysts for hydrogen generation

NASA Astrophysics Data System (ADS)

Tongying, P.; Vietmeyer, F.; Aleksiuk, D.; Ferraudi, G. J.; Krylova, G.; Kuno, M.

2014-03-01

Charge separation and charge transfer across interfaces are key aspects in the design of efficient photocatalysts for solar energy conversion. In this study, we investigate the hydrogen generating capabilities and underlying photophysics of nanostructured photocatalysts based on CdSe nanowires (NWs). Systems studied include CdSe, CdSe/CdS core/shell nanowires and their Pt nanoparticle-decorated counterparts. Femtosecond transient differential absorption measurements reveal how semiconductor/semiconductor and metal/semiconductor heterojunctions affect the charge separation and hydrogen generation efficiencies of these hybrid photocatalysts. In turn, we unravel the role of surface passivation, charge separation at semiconductor interfaces and charge transfer to metal co-catalysts in determining photocatalytic H2 generation efficiencies. This allows us to rationalize why Pt nanoparticle decorated CdSe/CdS NWs, a double heterojunction system, performs best with H2 generation rates of ~434.29 +/- 27.40 μmol h-1 g-1 under UV/Visible irradiation. In particular, we conclude that the CdS shell of this double heterojunction system serves two purposes. The first is to passivate CdSe NW surface defects, leading to long-lived charges at the CdSe/CdS interface capable of carrying out reduction chemistries. Upon photoexcitation, we also find that CdS selectively injects charges into Pt NPs, enabling simultaneous reduction chemistries at the Pt NP/solvent interface. Pt nanoparticle decorated CdSe/CdS NWs thus enable reduction chemistries at not one, but rather two interfaces, taking advantage of each junction's optimal catalytic activities.Charge separation and charge transfer across interfaces are key aspects in the design of efficient photocatalysts for solar energy conversion. In this study, we investigate the hydrogen generating capabilities and underlying photophysics of nanostructured photocatalysts based on CdSe nanowires (NWs). Systems studied include CdSe, CdSe/CdS core/shell nanowires and their Pt nanoparticle-decorated counterparts. Femtosecond transient differential absorption measurements reveal how semiconductor/semiconductor and metal/semiconductor heterojunctions affect the charge separation and hydrogen generation efficiencies of these hybrid photocatalysts. In turn, we unravel the role of surface passivation, charge separation at semiconductor interfaces and charge transfer to metal co-catalysts in determining photocatalytic H2 generation efficiencies. This allows us to rationalize why Pt nanoparticle decorated CdSe/CdS NWs, a double heterojunction system, performs best with H2 generation rates of ~434.29 +/- 27.40 μmol h-1 g-1 under UV/Visible irradiation. In particular, we conclude that the CdS shell of this double heterojunction system serves two purposes. The first is to passivate CdSe NW surface defects, leading to long-lived charges at the CdSe/CdS interface capable of carrying out reduction chemistries. Upon photoexcitation, we also find that CdS selectively injects charges into Pt NPs, enabling simultaneous reduction chemistries at the Pt NP/solvent interface. Pt nanoparticle decorated CdSe/CdS NWs thus enable reduction chemistries at not one, but rather two interfaces, taking advantage of each junction's optimal catalytic activities. Electronic supplementary information (ESI) available: Details of NW syntheses, processing and characterization. Additional TEM images of CdS, CdSe and CdSe/CdS core/shell NWs. NW concentration and cross section estimates. Details of the Pt NP decoration. Additional TEM images of Pt NP decorated CdS, CdSe and CdSe/CdS core/shell NWs. Size distribution of Pt NPs for CdSe/Pt NP and CdSe/CdS/Pt NP NWs. Xe arc lamp spectrum. Details of H2 generation experiments. Estimated photon absorption rate. Details of TDA measurements. TDA spectra and kinetics of CdS and CdS/Pt NP NWs. Plot illustrating CdSe NW band edge bleach kinetics. Comparison of CdSe band edge bleach kinetics in CdSe/CdS core/shell NWs when excited at λexc = 387 nm and λexc = 560 nm. Comparison of CdSe band edge bleach kinetics in CdSe/Pt NP NWs when excited at λexc = 387 nm and λexc = 560 nm. Bar graph showing H2 generation efficiencies of CdS and CdS/Pt NP NWs. Bleach kinetics of CdSe/CdS/Pt NP NWs at λexc = 387 nm and λexc = 560 nm. Comparison of CdS band edge bleach kinetics in CdS/Pt NP, and CdSe/CdS core/shell NWs when excited at λexc = 387 nm. See DOI: 10.1039/c4nr00298a

Human palatine tonsil: a new potential tissue source of multipotent mesenchymal progenitor cells

PubMed Central

Janjanin, Sasa; Djouad, Farida; Shanti, Rabie M; Baksh, Dolores; Gollapudi, Kiran; Prgomet, Drago; Rackwitz, Lars; Joshi, Arjun S; Tuan, Rocky S

2008-01-01

Introduction Mesenchymal progenitor cells (MPCs) are multipotent progenitor cells in adult tissues, for example, bone marrow (BM). Current challenges of clinical application of BM-derived MPCs include donor site morbidity and pain as well as low cell yields associated with an age-related decrease in cell number and differentiation potential, underscoring the need to identify alternative sources of MPCs. Recently, MPC sources have diversified; examples include adipose, placenta, umbilicus, trabecular bone, cartilage, and synovial tissue. In the present work, we report the presence of MPCs in human tonsillar tissue. Methods We performed comparative and quantitative analyses of BM-MPCs with a subpopulation of adherent cells isolated from this lymphoid tissue, termed tonsil-derived MPCs (T-MPCs). The expression of surface markers was assessed by fluorescent-activated cell sorting analysis. Differentiation potential of T-MPCs was analyzed histochemically and by reverse transcription-polymerase chain reaction for the expression of lineage-related marker genes. The immunosuppressive properties of MPCs were determined in vitro in mixed lymphocyte reactions. Results Surface epitope analysis revealed that T-MPCs were negative for CD14, CD31, CD34, and CD45 expression and positive for CD29, CD44, CD90, and CD105 expression, a characteristic phenotype of BM-MPCs. Similar to BM-MPCs, T-MPCs could be induced to undergo adipogenic differentiation and, to a lesser extent, osteogenic and chondrogenic differentiation. T-MPCs did not express class II major histocompatibility (MHC) antigens, and in a similar but less pronounced manner compared with BM-MPCs, T-MPCs were immunosuppressive, inhibiting the proliferation of T cells stimulated by allogeneic T cells or by non-specific mitogenic stimuli via an indoleamine 2,3-dioxygenase-dependent mechanism. Conclusion Human palatine T-MPCs represent a new source of progenitor cells, potentially applicable for cell-based therapies. PMID:18662393

Effects of low-doses of Bacillus spp. from permafrost on differentiation of bone marrow cells.

PubMed

Kalyonova, L F; Novikova, M A; Kostolomova, E G

2015-01-01

The effects of a new microorganism species (Bacillus spp., strain M3) isolated from permafrost specimens from Central Yakutia (Mamontova Mountain) on the bone marrow hemopoiesis were studied on laboratory mice. Analysis of the count and immunophenotype of bone marrow cells indicated that even in low doses (1000-5000 microbial cells) these microorganisms modulated hemopoiesis and lymphopoiesis activity. The percentage of early hemopoietic precursors (CD117(+)CD34(-)) increased, intensity of lymphocyte precursor proliferation and differentiation (CD25(+)CD44(-)) decreased, and the percentage of lymphocytes released from the bone marrow (CD25(+)CD44(+)) increased on day 21 after injection of the bacteria. These changes in activity of hemopoiesis were associated with changes in the level of regulatory T lymphocytes (reduced expression of TCRαβ) and were most likely compensatory. The possibility of modulating hemopoiesis activity in the bone marrow by low doses of one microorganism strain isolated from the permafrost could be useful for evaluating the effects of other low dose bacteria on the bone marrow hemopoiesis.

Regulation of cytokine signaling by B cell antigen receptor and CD40-controlled expression of heparan sulfate proteoglycans.

PubMed

van der Voort, R; Keehnen, R M; Beuling, E A; Spaargaren, M; Pals, S T

2000-10-16

Recently, biochemical, cell biological, and genetic studies have converged to reveal that integral membrane heparan sulfate proteoglycans (HSPGs) are critical regulators of growth and differentiation of epithelial and connective tissues. As a large number of cytokines involved in lymphoid tissue homeostasis or inflammation contain potential HS-binding domains, HSPGs presumably also play important roles in the regulation of the immune response. In this report, we explored the expression, regulation, and function of HSPGs on B lymphocytes. We demonstrate that activation of the B cell antigen receptor (BCR) and/or CD40 induces a strong transient expression of HSPGs on human tonsillar B cells. By means of these HSPGs, the activated B cells can bind hepatocyte growth factor (HGF), a cytokine that regulates integrin-mediated B cell adhesion and migration. This interaction with HGF is highly selective since the HSPGs did not bind the chemokine stromal cell-derived factor (SDF)-1 alpha, even though the affinities of HGF and SDF-1alpha for heparin are similar. On the activated B cells, we observed induction of a specific HSPG isoform of CD44 (CD44-HS), but not of other HSPGs such as syndecans or glypican-1. Interestingly, the expression of CD44-HS on B cells strongly promotes HGF-induced signaling, resulting in an HS-dependent enhanced phosphorylation of Met, the receptor tyrosine kinase for HGF, as well as downstream signaling molecules including Grb2-associated binder 1 (Gab1) and Akt/protein kinase B (PKB). Our results demonstrate that the BCR and CD40 control the expression of HSPGs, specifically CD44-HS. These HSPGs act as functional coreceptors that selectively promote cytokine signaling in B cells, suggesting a dynamic role for HSPGs in antigen-specific B cell differentiation.

O-glycans direct selectin ligands to lipid rafts on leukocytes

PubMed Central

Shao, Bojing; Yago, Tadayuki; Setiadi, Hendra; Wang, Ying; Mehta-D’souza, Padmaja; Fu, Jianxin; Crocker, Paul R.; Rodgers, William; Xia, Lijun; McEver, Rodger P.

2015-01-01

Palmitoylated cysteines typically target transmembrane proteins to domains enriched in cholesterol and sphingolipids (lipid rafts). P-selectin glycoprotein ligand-1 (PSGL-1), CD43, and CD44 are O-glycosylated proteins on leukocytes that associate with lipid rafts. During inflammation, they transduce signals by engaging selectins as leukocytes roll in venules, and they move to the raft-enriched uropods of polarized cells upon chemokine stimulation. It is not known how these glycoproteins associate with lipid rafts or whether this association is required for signaling or for translocation to uropods. Here, we found that loss of core 1-derived O-glycans in murine C1galt1−/− neutrophils blocked raft targeting of PSGL-1, CD43, and CD44, but not of other glycosylated proteins, as measured by resistance to solubilization in nonionic detergent and by copatching with a raft-resident sphingolipid on intact cells. Neuraminidase removal of sialic acids from wild-type neutrophils also blocked raft targeting. C1galt1−/− neutrophils or neuraminidase-treated neutrophils failed to activate tyrosine kinases when plated on immobilized anti–PSGL-1 or anti-CD44 F(ab′)2. Furthermore, C1galt1−/− neutrophils incubated with anti–PSGL-1 F(ab′)2 did not generate microparticles. In marked contrast, PSGL-1, CD43, and CD44 moved normally to the uropods of chemokine-stimulated C1galt1−/− neutrophils. These data define a role for core 1-derived O-glycans and terminal sialic acids in targeting glycoprotein ligands for selectins to lipid rafts of leukocytes. Preassociation of these glycoproteins with rafts is required for signaling but not for movement to uropods. PMID:26124096

The mandibular ridge oral mucosa model of stromal influences on the endothelial tip cells: an immunohistochemical and TEM study.

PubMed

Rusu, Mugurel Constantin; Didilescu, Andreea Cristiana; Stănescu, Ruxandra; Pop, Florinel; Mănoiu, Valentina Mariana; Jianu, Adelina Maria; Vâlcu, Marek

2013-02-01

This study aimed to evaluate by immunohistochemistry and transmission electron microscopy (TEM) the morphological features of the oral mucosa endothelial tip cells (ETCs) and to determine the immune and ultrastructural patterns of the stromal nonimmune cells which could influence healing processes. Immune labeling was performed on bioptic samples obtained from six edentulous patients undergoing surgery for dental implants placement; three normal samples were collected from patients prior to the extraction of the third mandibular molar. The antibodies were tested for CD34, CD117(c-kit), platelet derived growth factor receptor-alpha (PDGFR-α), Mast Cell Tryptase, CD44, vimentin, CD45, CD105, alpha-smooth muscle actin, FGF2, Ki67. In light microscopy, while stromal cells (StrCs) of the reparatory and normal oral mucosa, with a fibroblastic appearance, were found positive for a CD34/CD44/CD45/CD105/PDGFR-α/vimentin immune phenotype, the CD117/c-kit labeling led to a positive stromal reaction only in the reparatory mucosa. In TEM, non-immune StrCs presenting particular ultrastructural features were identified as circulating fibrocytes (CFCs). Within the lamina propria CFCs were in close contact with ETCs. Long processes of the ETCs were moniliform, and hook-like collaterals were arising from the dilated segments, suggestive for a different stage migration. Maintenance and healing of oral mucosa are so supported by extensive processes of angiogenesis, guided by ETCs that, in turn, are influenced by the CFCs that populate the stromal compartment both in normal and reparatory states. Therefore, CFCs could be targeted by specific therapies, with pro- or anti-angiogenic purposes. Copyright © 2012 Wiley Periodicals, Inc.

CD19+ B cell subsets in the peripheral blood and skin lesions of psoriasis patients and their correlations with disease severity

PubMed Central

Lu, J.; Ding, Y.; Yi, X.; Zheng, J.

2016-01-01

T lymphocytes are important in the pathogenesis of psoriasis, and increasing evidence indicates that B cells also play an important role. The mechanisms of action, however, remain unclear. We evaluated the ratios of CD19+ B cells in peripheral blood mononuclear cells (PBMCs) from 157 patients with psoriasis (65 patients with psoriasis vulgaris, 32 patients with erythrodermic psoriasis, 30 patients with arthropathic psoriasis, and 30 patients with pustular psoriasis) and 35 healthy controls (HCs). Ratios of CD19+ B cells in skin lesions were compared with non-lesions in 7 erythrodermic psoriasis patients. The Psoriasis Area Severity Index (PASI) was used to measure disease severity. CD19+ B cell ratios in PBMCs from psoriasis vulgaris (at both the active and stationary stage) and arthropathic psoriasis patients were higher compared with HCs (P<0.01), but ratios were lower in erythrodermic and pustular psoriasis patients (P<0.01). CD19+ B cell ratios in erythrodermic psoriasis skin lesions were higher than in non-lesion areas (P<0.001). Different subsets of CD19+CD40+, CD19+CD44+, CD19+CD80+, CD19+CD86+, CD19+CD11b+, and CD19+HLA-DR+ B cells in PBMCs were observed in different psoriasis clinical subtypes. PASI scores were positively correlated with CD19+ B cell ratios in psoriasis vulgaris and arthropathic psoriasis cases (r=0.871 and r=0.692, respectively, P<0.01), but were negatively correlated in pustular psoriasis (r=-0.569, P<0.01). The results indicated that similar to T cells, B cells activation may also play important roles in different pathological stages of psoriasis. PMID:27532281

A phase IIa study of HA-irinotecan, formulation of hyaluronic acid and irinotecan targeting CD44 in extensive-stage small cell lung cancer.

PubMed

Alamgeer, Muhammad; Neil Watkins, D; Banakh, Ilia; Kumar, Beena; Gough, Daniel J; Markman, Ben; Ganju, Vinod

2018-04-01

Preclinical studies in small cell lung cancer (SCLC) have shown that hyaluronic acid (HA) can be effectively used to deliver chemotherapy and selectively decrease CD44 expressing (stem cell-like) tumour cells. The current study aimed to replicate these findings and obtain data on safety and activity of HA-irinotecan (HA-IR). Eligible patients with extensive stage SCLC were consented. A safety cohort (n = 5) was treated with HA-IR and Carboplatin (C). Subsequently, the patients were randomised 1:1 to receive experimental (HA-IR + C) or standard (IR + C) treatment, to a maximum of 6 cycles. The second line patients were added to the study and treated with open label HA-IR + C. Tumour response was measured after every 2 cycles. Baseline tumour specimens were stained for CD44s and CD44v6 expression. Circulating tumour cells (CTCs) were enumerated before each treatment cycle. Out of 39 patients screened, 34 were evaluable for the study. The median age was 66 (range 39-83). The overall response rates were 69% and 75% for experimental and standard arms respectively. Median progression free survival was 42 and 28 weeks, respectively (p = 0.892). The treatments were well tolerated. The incidence of grade III/IV diarrhea was more common in the standard arm, while anaemia was more common in the experimental arm. IHC analysis suggested that the patients with CD44s positive tumours may gain survival benefit from HA-IR. HA-IR is well tolerated and active in ES-SCLC. The effect of HA-IR on CD44s + cancer stem-like cells provide an early hint towards a potential novel target.

Analysis of CD44-Hyaluronan Interactions in an Artificial Membrane System

PubMed Central

Wolny, Patricia M.; Banerji, Suneale; Gounou, Céline; Brisson, Alain R.; Day, Anthony J.; Jackson, David G.; Richter, Ralf P.

2010-01-01

CD44 is a major cell surface receptor for the large polydisperse glycosaminoglycan hyaluronan (HA). Binding of the long and flexible HA chains is thought to be stabilized by the multivalent nature of the sugar molecule. In addition, high and low molecular weight forms of HA provoke distinct proinflammatory and anti-inflammatory effects upon binding to CD44 and can deliver either proliferative or antiproliferative signals in appropriate cell types. Despite the importance of such interactions, however, neither the stoichiometry of multivalent HA binding at the cell surface nor the molecular basis for functional distinction between different HA size categories is understood. Here we report on the design of a supported lipid bilayer system that permits quantitative analysis of multivalent binding through presentation of CD44 in a stable, natively oriented manner and at controlled density. Using this system in combination with biophysical techniques, we show that the amount of HA binding to bilayers that are densely coated with CD44 increases as a function of HA size, with half-maximal saturation at ∼30 kDa. Moreover, reversible binding was confined to the smaller HA species (molecular weight of ≤10 kDa), whereas the interaction was essentially irreversible with larger polymers. The amount of bound HA decreased with decreasing receptor surface density, but the stability of binding was not affected. From a physico-chemical perspective, the binding properties of HA share many similarities with the typical behavior of a flexible polymer as it adsorbs onto a homogeneously attractive surface. These findings provide new insight into the multivalent nature of CD44-HA interactions and suggest a molecular basis for the distinct biological properties of different size fractions of hyaluronan. PMID:20663884

CD44 and TLR4 mediate hyaluronic acid regulation of Lgr5+ stem cell proliferation, crypt fission, and intestinal growth in postnatal and adult mice.

PubMed

Riehl, Terrence E; Santhanam, Srikanth; Foster, Lynne; Ciorba, Matthew; Stenson, William F

2015-12-01

Hyaluronic acid, a glycosaminoglycan in the extracellular matrix, binds to CD44 and Toll-like receptor 4 (TLR4). We previously addressed the role of hyaluronic acid in small intestinal and colonic growth in mice. We addressed the role of exogenous hyaluronic acid by giving hyaluronic acid intraperitoneally and the role of endogenous hyaluronic acid by giving PEP-1, a peptide that blocks hyaluronic acid binding to its receptors. Exogenous hyaluronic acid increased epithelial proliferation but had no effect on intestinal length. PEP-1 resulted in a shortened small intestine and colon and diminished epithelial proliferation. In the current study, we sought to determine whether the effects of hyaluronic acid on growth were mediated by signaling through CD44 or TLR4 by giving exogenous hyaluronic acid or PEP-1 twice a week from 3-8 wk of age to wild-type, CD44(-/-), and TLR4(-/-) mice. These studies demonstrated that signaling through both CD44 and TLR4 were important in mediating the effects of hyaluronic acid on growth in the small intestine and colon. Extending our studies to early postnatal life, we assessed the effects of exogenous hyaluronic acid and PEP-1 on Lgr5(+) stem cell proliferation and crypt fission. Administration of PEP-1 to Lgr5(+) reporter mice from postnatal day 7 to day 14 decreased Lgr5(+) cell proliferation and decreased crypt fission. These studies indicate that endogenous hyaluronic acid increases Lgr5(+) stem cell proliferation, crypt fission, and intestinal lengthening and that these effects are dependent on signaling through CD44 and TLR4. Copyright © 2015 the American Physiological Society.

Metastatic Potential of Cancer Stem Cells in Head and Neck Squamous Cell Carcinoma

PubMed Central

Davis, Samantha J.; Divi, Vasu; Owen, John H.; Papagerakis, Silvana; Bradford, Carol R.; Carey, Thomas E.; Prince, Mark E. P.

2012-01-01

Objective Subpopulations of highly tumorigenic cells, which have the unique capacity to self-renew and produce differentiated progeny, have been identified in multiple malignancies. In head and neck squamous cell carcinoma (HNSCC), this subpopulation of cells, termed cancer stem cells (CSCs) are contained within the population with high CD44 expression. It has been postulated that CSCs play a role in invasion and metastasis; however, there is little evidence to support this theory. We designed in vitro and in vivo models of metastasis to study the behavior of CSCs in HNSCC. Design Cells were sorted for CD44 expression using flow cytometry. Sorted cells were used in an in vitro invasion assay. For in vivo studies, CSCs and non-CSCs were injected into the tail veins of mice, and lungs were either harvested or imaged to evaluate for metastases. Results In vitro, CD44high cells were more motile but less invasive than CD44low cells. In vivo, 4/5 mice injected with CD44high cells and 0/5 mice injected with CD44low cells formed lung metastases. Two of the metastases arose from CSCs from a primary tumor and three from CSCs from HNSCC cell lines. Conclusions In vitro, CSCs do not have an increased ability to invade through basement membrane, but they do migrate more efficiently through a porous barrier. In contrast, CSCs formed metastases quite efficiently in vivo, whereas non-CSCs did not form metastases at all. This phenomenon could be due to enhanced migratory capacity of CSCs, which may be more important than basement membrane degradation in vivo. PMID:21173377

Determinants of Risk Infection During Therapy with Anti TNF-Alpha Blocking Agents in Rheumatoid Arthritis

PubMed Central

Benucci, M; Saviola, G; Baiardi, P; Manfredi, M; Sarzi Puttini, P; Atzeni, Fabiola

2012-01-01

The use of TNF-alpha antagonists (infliximab, etanercept, adalimumab) has changed the course of many rheumatic diseases including rheumatoid arthritis (RA). Since their approval, some questions regarding their safety including infections have been observed. The aim of the study was to evaluate the changes in cytokines levels and cells subsets in patients with RA during anti TNF blocking agents treatment and the possible effect on infections’ development. We evaluated in 89 RA patients [39 treated with etanercept (ETN), 29 with adalimumab (ADA) and 21 with infliximab (IFN)] at baseline and after 6 months the following parameters: procalcitonin, ESR, CRP, cytokines as TNF, IL-6, IL-10, IL-8 and the TNF/IL-10 ratio, and peripheral mononuclear cells as CD3+, CD3+/CD4+, CD3+/CD8+, CD19+, CD3- /CD16+/56+, CD14+HLADR+, CD20+, CD19+/CD38+. Peripheral mononuclear cells were detected by flow cytometric system Cytomics FC500 and cytokines circulating levels by a quantitative sandwich enzyme immunoassay technique (Human IL-8 Instant ELISAe Bioscience, Human IL-6 Instant ELISA e Bioscience, Human IL-10 Instant ELISAe Bioscience and Human TNF-a Quantikine immunoassay RD system). A lower reduction of CD14+HLADR+ in ADA group 54.6±10.4% vs ETA 48.4±15.7% vs INF 40.7±16.5%, p<0.039 was found. No differences in all three groups on peripheral mononuclear cells CD3+, CD3+/CD4+, CD3+/CD8+, CD19+, CD 20+, CD19+/CD38+, CD3-/CD16+/56+, and cytokine circulating levels were found. The number of infections at 6 months was: 10.3% in ADA group, 12.8% in ETN group and 19.04% in IFN group. A correlation was found between the reduction in CD14+HLADR+ cells and IFN treatment. Our data showed that the level of CD14+HLADR+ cells was reduced during therapy with IFN. ADA and ETN don’t reduce lymphocyte populations and their subsets such as CD14+HLADR+ cells that play an important role host defence. PMID:22655000

A functional glycoproteomics approach identifies CD13 as a novel E-selectin ligand in breast cancer.

PubMed

Carrascal, M A; Silva, M; Ferreira, J A; Azevedo, R; Ferreira, D; Silva, A M N; Ligeiro, D; Santos, L L; Sackstein, R; Videira, P A

2018-05-17

The glycan moieties sialyl-Lewis-X and/or -A (sLe X/A ) are the primary ligands for E-selectin, regulating subsequent tumor cell extravasation into distant organs. However, the nature of the glycoprotein scaffolds displaying these glycans in breast cancer remains unclear and constitutes the focus of the present investigation. We isolated glycoproteins that bind E-selectin from the CF1_T breast cancer cell line, derived from a patient with ductal carcinoma. Proteins were identified using bottom-up proteomics approach by nanoLC-orbitrap LTQ-MS/MS. Data were curated using bioinformatics tools to highlight clinically relevant glycoproteins, which were validated by flow cytometry, Western blot, immunohistochemistry and in-situ proximity ligation assays in clinical samples. We observed that the CF1_T cell line expressed sLe X , but not sLe A and the E-selectin reactivity was mainly on N-glycans. MS and bioinformatics analysis of the targeted glycoproteins, when narrowed down to the most clinically relevant species in breast cancer, identified CD44 glycoprotein (HCELL) and CD13 as key E-selectin ligands. Additionally, the co-expression of sLe X -CD44 and sLe X -CD13 was confirmed in clinical breast cancer tissue samples. Both CD44 and CD13 glycoforms display sLe X in breast cancer and bind E-selectin, suggesting a key role in metastasis development. Such observations provide a novel molecular rationale for developing targeted therapeutics. While HCELL expression in breast cancer has been previously reported, this is the first study indicating that CD13 functions as an E-selectin ligand in breast cancer. This observation supports previous associations of CD13 with metastasis and draws attention to this glycoprotein as an anti-cancer target. Copyright © 2018 Elsevier B.V. All rights reserved.

Proceedings of the 8th Matched-Field Processing Workshop, 12-14 June 1996,

DTIC Science & Technology

1996-10-01

and M. B. Porter Active Matched-Field Tracking (AMFT) ............................................ 29 Homer Bucker Matched-Field Track - Before - Detect (TBD...CD I- z - .4 U) - U :T 0 4,) 0j w CfI -ID 0 ci) CD) CD CD o0 0 C 0D CD 0C o 00 Matched-Field Track - Before - Detect (TBD) Processing using SWellEX...surfaces are used in a source-track search. Track - before - detect (TBD) processing makes use of this technique to extract source track information so that the

Dietary determinants of cadmium exposure in the Strong Heart Family Study

PubMed Central

Olmedo, Pablo; Grau-Perez, Maria; Fretts, Amanda; Tellez-Plaza, Maria; Gil, Fernando; Yeh, Fawn; Umans, Jason G.; Francesconi, Kevin A.; Goessler, Walter; Franceschini, Nora; Lee, Elisa T.; Best, Lyle G.; Cole, Shelley A.; Howard, Barbara V.; Navas-Acien, Ana

2017-01-01

Urinary cadmium (Cd) concentrations in the Strong Heart Family Study (SHFS) participants are higher than in the general US population. This difference is unlikely to be related to tobacco smoking. We evaluated the association of consumption of processed meats and other dietary products with urinary Cd concentrations in the SHFS, a family-based study conducted in American Indian communities. We included 1725 participants with urine Cd concentrations (standardized to urine creatinine) and food frequency questionnaire data grouped in 24 categories, including processed meat. Median (IQR) urinary Cd concentrations were 0.44 (0.20–0.85) μg/g creatinine. The age, sex, smoking, education, center, body mass index, and total kcal adjusted geometric mean ratio (GMR) (95%CI) of urinary cadmium concentrations per IQR increase in each dietary category was 1.16 (1.04–1.29) for processed meat, 1.10 (1.00–1.21) for fries and chips, 0.87 (0.80–0.95) for dairy products, and 0.89 (0.82–0.97) for fruit juices. The results remained similar after further adjustment for the dietary categories associated with urinary Cd in the previous model except for fries and chips, which was no longer statistically significant. These findings revealed the potential importance of processed meat products as a dietary source of cadmium. PMID:28012896

Immune Cell Subsets Evaluation as a Predictive Tool for Hepatitis B Infection Outcome and Treatment Responsiveness.

PubMed

Kandilarova, Snezhina M; Georgieva, Atanaska I; Mihaylova, Anastasiya P; Baleva, Marta P; Atanasova, Valentina K; Petrova, Diana V; Popov, Georgi T; Naumova, Elissaveta J

2017-03-01

The patient's immune response is one of the major factors influencing HBV eradication or chronification, and it is thought to be responsible for the treatment success. Our study aimed to investigate whether cellular defense mechanisms are associated with the course of HBV infection (spontaneous recovery [SR] or chronification [CHB]) and with the therapeutic approach. A total of 139 patients (118 with CHB, 21 SR) and 29 healthy individuals (HI) were immunophenotyped by flowcytometry. Fifty-six patients were treatment-naïve, 20 were treated with interferons and 42 with nucleoside/ nucleotide analogues. Deficiency of T lymphocytes, helper-inducer (CD3+CD4+), suppressorcytotoxic (CD8+CD3+) and cytotoxic (CD8+CD11b-, CD8+CD28+) subsets, activated T cells (CD3+HLA-DR+, CD8+CD38+) and increased CD57+CD8- cells, elevated percentages of B lymphocytes and NKT cells were observed in CHB patients compared with HI. In SR patients, elevated CD8+CD11b+, NKT and activated T cells were found in comparison with controls. The higher values of T cells and their subsets in SR patients than in CHB patients reflect a recovery of cellular immunity in resolved HBV infection individuals. In both groups of treated patients, reduced T lymphocytes, CD3+CD4+ and CD8+CD38+ subsets were found in comparison with HI. Higher proportions of cytotoxic subsets were observed in treated patients compared with treatment-naïve CHB patients, more pronounced in the group with interferon therapy. Our data demonstrate that cellular immune profiles may be of prognostic value in predicting the clinical course of HBV infection, and the determination of the therapeutic response.

Nutritional status, growth and disease management in children with single and dual diagnosis of type 1 diabetes mellitus and coeliac disease.

PubMed

Mackinder, Mary; Allison, Gavin; Svolos, Vaios; Buchanan, Elaine; Johnston, Alison; Cardigan, Tracey; Laird, Nicola; Duncan, Hazel; Fraser, Karen; Edwards, Christine A; Craigie, Ian; McGrogan, Paraic; Gerasimidis, Konstantinos

2014-05-28

The consequences of subclinical coeliac disease (CD) in Type 1 diabetes mellitus (T1DM) remain unclear. We looked at growth, anthropometry and disease management in children with dual diagnosis (T1DM + CD) before and after CD diagnosis. Anthropometry, glycated haemoglobin (HbA1c) and IgA tissue transglutaminase (tTg) were collected prior to, and following CD diagnosis in 23 children with T1DM + CD. This group was matched for demographics, T1DM duration, age at CD diagnosis and at T1DM onset with 23 CD and 44 T1DM controls. No differences in growth or anthropometry were found between children with T1DM + CD and controls at any time point. Children with T1DM + CD, had higher BMI z-score two years prior to, than at CD diagnosis (p < 0.001). BMI z-score change one year prior to CD diagnosis was lower in the T1DM + CD than the T1DM group (p = 0.009). At two years, height velocity and change in BMI z-scores were similar in all groups. No differences were observed in HbA1c between the T1DM + CD and T1DM groups before or after CD diagnosis. More children with T1DM + CD had raised tTg levels one year after CD diagnosis than CD controls (CDx to CDx + 1 yr; T1DM + CD: 100% to 71%, p = 0.180 and CD: 100% to 45%, p < 0.001); by two years there was no difference. No major nutrition or growth deficits were observed in children with T1DM + CD. CD diagnosis does not impact on T1DM glycaemic control. CD specific serology was comparable to children with single CD, but those with dual diagnosis may need more time to adjust to gluten free diet.

EPR hyperfine structure of the Mo-related defect in CdWO4

NASA Astrophysics Data System (ADS)

Elsts, E.; Rogulis, U.

2005-01-01

The hyperfine structure (hf) of the electron paramagnetic resonance (EPR) spectrum of Mo-related impurity defects in CdWO4 crystals observed previously (U. Rogulis, Radiat. Meas. 29, 287 (1998) [1]) is reconsidered taking into account interactions with two different groups of neighbouring Cd nuclei. The best fit calculated EPR spectrum to the experimental is obtained considering 2 groups of 3 and 2 equivalent Cd nuclei, respectively.

Allergen-specific responses of CD19(+)CD5(+)Foxp3(+) regulatory B cells (Bregs) and CD4(+)Foxp3(+) regulatory T cell (Tregs) in immune tolerance of cow milk allergy of late eczematous reactions.

PubMed

Noh, Joonyong; Noh, Geunwoong; Kim, Hyuk Soon; Kim, A-Ram; Choi, Wahn Soo

2012-01-01

Foxp3-expressing cells among CD19(+)CD5(+) B cells were identified as regulatory B cells. Food allergy manifesting as late eczematous reactions is regarded as a non-IgE-mediated food allergy. The diagnosis for milk allergy manifesting as late eczematous reactions was made on the basis of the findings obtained from a double-blind placebo-controlled food challenge in patients with atopic dermatitis. Twelve patients with milk allergy and 12 patients who could tolerate milk were selected. On casein stimulation, the CD19(+)CD5(+)Foxp3(+) B cell (Breg) fraction in CD5(+) B cells decreased from 4.4±1.1% to 3.1±0.7% (P=0.047, n=12) in the milk allergy group and increased from 4.4±1.3% to 5.2±1.4% (P=0.001, n=10) in the milk-tolerant group. On the other hand, on allergen stimulation, the number of CD4(+)Foxp3(+) regulatory T cells (Tregs) in the milk allergy group and milk-tolerant group increased from 2.6±0.7% to 3.4±0.6% (P=0.014, n=9) and from 2.7±1.0% to 3.5±1.0% (P=0.038, n=10), respectively. In conclusion, allergen-specific responses of Bregs, rather than those of Tregs, seem to influence the immune responses (i.e., allergy or tolerance) to a food allergen. Copyright © 2012 Elsevier Inc. All rights reserved.

Doxorubicin attached to HPMA copolymer via amide bond modifies the glycosylation pattern of EL4 cells.

PubMed

Kovar, Lubomir; Etrych, Tomas; Kabesova, Martina; Subr, Vladimir; Vetvicka, David; Hovorka, Ondrej; Strohalm, Jiri; Sklenar, Jan; Chytil, Petr; Ulbrich, Karel; Rihova, Blanka

2010-08-01

To avoid the side effects of the anti-cancer drug doxorubicin (Dox), we conjugated this drug to a N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer backbone. Dox was conjugated via an amide bond (Dox-HPMA(AM), PK1) or a hydrazone pH-sensitive bond (Dox-HPMA(HYD)). In contrast to Dox and Dox-HPMA(HYD), Dox-HPMA(AM) accumulates within the cell's intracellular membranes, including those of the Golgi complex and endoplasmic reticulum, both involved in protein glycosylation. Flow cytometry was used to determine lectin binding and cell death, immunoblot to characterize the presence of CD7, CD43, CD44, and CD45, and high-performance anion exchange chromatography with pulsed amperometric detector analysis for characterization of plasma membrane saccharide composition. Incubation of EL4 cells with Dox-HPMA(AM) conjugate, in contrast to Dox or Dox-HPMA(HYD), increased the amounts of membrane surface-associated glycoproteins, as well as saccharide moieties recognized by peanut agglutinin, Erythrina cristagalli, or galectin-1 lectins. Only Dox-HPMA(AM) increased expression of the highly glycosylated membrane glycoprotein CD43, while expression of others (CD7, CD44, and CD45) was unaffected. The binding sites for galectin-1 are present on CD43 molecule. Furthermore, we present that EL4 treated with Dox-HPMA(AM) possesses increased sensitivity to galectin-1-induced apoptosis. In this study, we demonstrate that Dox-HPMA(AM) treatment changes glycosylation of the EL4 T cell lymphoma surface and sensitizes the cells to galectin-1-induced apoptosis.

Nationwide prevalence and drug treatment practices of inflammatory bowel diseases in Hungary: A population-based study based on the National Health Insurance Fund database.

PubMed

Kurti, Zsuzsanna; Vegh, Zsuzsanna; Golovics, Petra A; Fadgyas-Freyler, Petra; Gecse, Krisztina B; Gonczi, Lorant; Gimesi-Orszagh, Judit; Lovasz, Barbara D; Lakatos, Peter L

2016-11-01

Crohn's disease (CD) and ulcerative colitis (UC) are chronic inflammatory diseases associated with a substantial healthcare utilization. Our aim was to estimate the national prevalence of inflammatory bowel disease (IBD), CD and UC and to describe current drug treatment practices in CD and UC. Patients and drug dispensing events were identified according to international classification codes for UC and CD in in-patient care, non-primary out-patient care and drug prescription databases (2011-2013) of the National Health Insurance Fund. A total of 55,039 individuals (men: 44.6%) with physician-diagnosed IBD were alive in Hungary in 2013, corresponding to a prevalence of 0.55% (95% CI, 0.55-0.56). The prevalence of CD 0.20% (95% CI, 0.19-0.20), and UC was 0.34% (95% CI, 0.33-0.34). The prevalence both in men and women was the highest in the 20-39 year-olds in CD. Current use of immunosuppressives and biological therapy was highest in the pediatric CD population (44% and 15%) followed by adult CD (33% and 9%), while their use was lowest in elderly patients. Interestingly, current use of 5-ASA (5-aminosalicylates) was high in both UC and CD irrespective of the age group. The Hungarian IBD prevalence based on nationwide database of the National Health Insurance Fund was high. We identified significant differences in the drug prescription practices according to age-groups. Copyright © 2016 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.

T cell-intrinsic factors contribute to the differential ability of CD8+ T cells to rapidly secrete IFN-γ in the absence of antigen.

PubMed

Bou Ghanem, Elsa N; Nelson, Christina C; D'Orazio, Sarah E F

2011-02-01

A subset of CD44(hi)CD8(+) T cells isolated from C57BL/6/J (B6) mice, but not BALB/c/By/J (BALB/c) mice, rapidly secrete IFN-γ within 16 h of infection with Listeria monocytogenes. This Ag-independent response requires the presence of both IL-12 and IL-18. Previous studies showed that dendritic cells from B6 mice produced more Th1-type cytokines such as IL-12 than did those from BALB/c mice in response to L. monocytogenes infection. In this report, we demonstrate that the microenvironment in L. monocytogenes-infected BALB/c mice is sufficient to induce responsive B6 CD8(+) T cells to rapidly secrete IFN-γ. Furthermore, BALB/c CD8(+) T cells did not rapidly secrete IFN-γ even when they were exposed to high concentrations of IL-12 plus IL-18 in vitro. In the presence of IL-12 and IL-18, B6 CD44(hi)CD8(+) T cells upregulated expression of the receptor subunits for these cytokines more rapidly than did BALB/c T cells. In comparing particular subsets of memory phenotype CD8(+) T cells, we found that virtual memory cells, rather than true Ag-experienced cells, had the greatest level of impairment in BALB/c mice. These data suggest that the degree of cytokine-driven bystander activation of CD8(+) T cells that occurs during infection depends on both APCs and T cell-intrinsic properties that can vary among mouse strains.

Anion dependent self-assembly of 56-metal Cd-Ln nanoclusters with enhanced near-infrared luminescence properties

NASA Astrophysics Data System (ADS)

Yang, Xiaoping; Schipper, Desmond; Zhang, Lijie; Yang, Keqin; Huang, Shaoming; Jiang, Jijun; Su, Chengyong; Jones, Richard A.

2014-08-01

Two series of Cd-Ln clusters: nano-drum [Ln8Cd24L12(OAc)48] and nano-double-drum [Ln12Cd44L20Cl30(OAc)54] (Ln = Nd and Yb) were prepared using a flexible Schiff base ligand bearing two aryl-Br groups. Chloride (Cl-) ions, together with the interactions of Br with other electronegative atoms, play a key role in the formation of the nano-double-drums. The structures were studied by TEM and photophysical properties were determined.Two series of Cd-Ln clusters: nano-drum [Ln8Cd24L12(OAc)48] and nano-double-drum [Ln12Cd44L20Cl30(OAc)54] (Ln = Nd and Yb) were prepared using a flexible Schiff base ligand bearing two aryl-Br groups. Chloride (Cl-) ions, together with the interactions of Br with other electronegative atoms, play a key role in the formation of the nano-double-drums. The structures were studied by TEM and photophysical properties were determined. Electronic supplementary information (ESI) available: Full experimental and characterization details for 1-4. CCDC 972369-972372. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c4nr03075c

Use of clay to remediate cadmium contaminated soil under different water management regimes.

PubMed

Li, Jianrui; Xu, Yingming

2017-07-01

We examined in situ remediation of sepiolite on cadmium-polluted soils with diverse water regimes, and several variables including brown rice Cd, exchangeable Cd, pH, and available Fe/P. pH, available Fe/P in soils increased gradually during continuous flooding, which contributed to Cd absorption on colloids. In control group (untreated soils), compared to conventional irrigation, brown rice Cd in continuous flooding reduced by 37.9%, and that in wetting irrigation increased by 31.0% (p<0.05). In contrast to corresponding controls, brown rice Cd in sepiolite treated soils reduced by 44.4%, 34.5% and 36.8% under continuous flooding, conventional irrigation and wetting irrigation (p<0.05), and exchangeable Cd in amended soils reduced by 27.5-49.0%, 14.3-40.5%, and 24.9-32.8% under three water management regimes (p<0.05). Compared to corresponding controls, decreasing amplitudes of exchangeable Cd and brown rice Cd in sepiolite treated soils were higher in continuous flooding than in conventional irrigation and wetting irrigation. Continuous flooding management promoted soil Cd immobilization by sepiolite. Copyright © 2017. Published by Elsevier Inc.

Comparative analysis of rat mesenchymal stem cells derived from slow and fast skeletal muscle in vitro.

PubMed

Okumachi, Etsuko; Lee, Sang Yang; Niikura, Takahiro; Iwakura, Takashi; Dogaki, Yoshihiro; Waki, Takahiro; Takahara, Shunsuke; Ueha, Takeshi; Sakai, Yoshitada; Kuroda, Ryosuke; Kurosaka, Masahiro

2015-03-01

Skeletal muscle comprises different kinds of muscle fibres that can be classified as slow and fast fibres. The purpose of this study was to compare the yield, proliferation, and multi-potentiality of rat mesenchymal stem cells (MSCs) from the tibialis anterior (TA; fast muscle) and soleus (SO; slow muscle) in vitro. The TA and SO muscles were harvested, and isolated cells were plated. After two hours, the cells were washed extensively to remove any cell that did not adhere to the cell culture plate. The adherent cells, namely MSCs, were then cultured. Both types of MSCs were differentiated toward the osteogenic, chondrogenic and adipogenic lineages using lineage specific induction factors. The colony-forming unit fibroblast (CFU-F) assay revealed that the SO contained significantly higher quantities of MSCs than the TA. The self-renewal capacity of MSCs derived from the TA was significantly higher at later passages (passage 9-11). Both types of MSCs exhibited similar cell surface antigens to bone marrow (BM)-derived MSCs and were positive for CD29, CD44, and CD90 and negative for CD11b, CD34, and CD45. TA-derived MSCs were superior in terms of osteogenic differentiation capacity, but there was no significant difference in chondrogenic and adipogenic differentiation capacity. Our results demonstrated significant differences in the properties of muscle-derived MSCs from different muscle types (i.e. fast or slow muscles). The greater expandability and osteogenic differentiation ability of TA-derived MSCs suggests that fast muscle may be a better source for generating large numbers of MSCs for bone regeneration.

Lower respiratory tract infections among human immunodeficiency virus-exposed, uninfected infants

PubMed Central

Mussi-Pinhata, Marisa M; Motta, Fabrizio; Freimanis-Hance, Laura; de Souza, Ricardo; Szyld, Edgardo; Succi, Regina CM; Christie, Celia DC; Rolon, Maria J.; Ceriotto, Mariana; Read, Jennifer S.

2010-01-01

Objectives To evaluate whether maternal HIV disease severity during pregnancy is associated with an increased likelihood of lower respiratory tract infections (LRTIs) in HIV-exposed, uninfected infants. Methods HIV-exposed, uninfected, singleton, term infants enrolled in the NISDI Perinatal Study, with birth weight ≥ 2500 grams were followed from birth until six months of age. LRTI diagnoses, hospitalizations, and associated factors were assessed. Results Of 547 infants, 103 (18.8%) experienced 116 episodes of LRTIs (incidence=0.84 LRTIs/100 child-weeks). Most (81%) episodes were bronchiolitis. Forty-nine (9.0%) infants were hospitalized at least once with an LRTI. There were 53 hospitalizations (45.7%) for 116 LRTI episodes. None of these infants were breastfed. The odds of LRTI in infants whose mothers had CD4%<14 were 4.4 times than that of those whose mothers had CD4%≥29 (p=0.003). The odds of LRTI were 16.0 times that of infants with a CD4+ count [cells/mm3] < 750 at birth compared to those with CD4+≥750 (p=0.002). Maternal CD4+ decline and Infant hemoglobin at the 6-12 week visit were associated with infant LRTIs after 6-12 weeks and before six months of age. Conclusions Acute bronchiolitis is common and frequently severe among HIV-exposed, uninfected infants aged six months or less. Lower maternal and infant CD4+ values were associated with a higher risk of infant LRTIs. Further understanding of the immunological mechanisms of severe LRTIs is needed. PMID:20452798

Dual-targeting hybrid nanoparticles for the delivery of SN38 to Her2 and CD44 overexpressed human gastric cancer

NASA Astrophysics Data System (ADS)

Yang, Zhe; Luo, Huiyan; Cao, Zhong; Chen, Ya; Gao, Jinbiao; Li, Yingqin; Jiang, Qing; Xu, Ruihua; Liu, Jie

2016-06-01

Gastric cancer (GC), particularly of the type with high expression of both human epidermal growth factor receptor 2 (Her2) and cluster determinant 44 (CD44), is one of the most malignant human tumors which causes a high mortality rate due to rapid tumor growth and metastasis. To develop effective therapeutic treatments, a dual-targeting hybrid nanoparticle (NP) system was designed and constructed to deliver the SN38 agent specifically to human solid gastric tumors bearing excessive Her2 and CD44. The hybrid NPs consist of a particle core made of the biodegradable polymer PLGA and a lipoid shell prepared by conjugating the AHNP peptides and n-hexadecylamine (HDA) to the carboxyl groups of hyaluronic acid (HA). Upon encapsulation of the SN38 agent in the NPs, the AHNP peptides and HA on the NP surface allow preferential delivery of the drug to gastric cancer cells (e.g., HGC27 cells) by targeting Her2 and CD44. Cellular uptake and in vivo biodistribution experiments verified the active targeting and prolonged in vivo circulation properties of the dual-targeting hybrid NPs, leading to enhanced accumulation of the drug in tumors. Furthermore, the anti-proliferation mechanism studies revealed that the inhibition of the growth and invasive activity of HGC27 cells was not only attributed to the enhanced cellular uptake of dual-targeting NPs, but also benefited from the suppression of CD44 and Her2 expression by HA and AHNP moieties. Finally, intravenous administration of the SN38-loaded dual-targeting hybrid NPs induced significant growth inhibition of HGC27 tumor xenografted in nude mice compared with a clinical antitumor agent, Irinotecan (CPT-11), and the other NP formulations. These results demonstrate that the designed dual-targeting hybrid NPs are promising for targeted anti-cancer drug delivery to treat human gastric tumors over-expressing Her2 and CD44.Gastric cancer (GC), particularly of the type with high expression of both human epidermal growth factor receptor 2 (Her2) and cluster determinant 44 (CD44), is one of the most malignant human tumors which causes a high mortality rate due to rapid tumor growth and metastasis. To develop effective therapeutic treatments, a dual-targeting hybrid nanoparticle (NP) system was designed and constructed to deliver the SN38 agent specifically to human solid gastric tumors bearing excessive Her2 and CD44. The hybrid NPs consist of a particle core made of the biodegradable polymer PLGA and a lipoid shell prepared by conjugating the AHNP peptides and n-hexadecylamine (HDA) to the carboxyl groups of hyaluronic acid (HA). Upon encapsulation of the SN38 agent in the NPs, the AHNP peptides and HA on the NP surface allow preferential delivery of the drug to gastric cancer cells (e.g., HGC27 cells) by targeting Her2 and CD44. Cellular uptake and in vivo biodistribution experiments verified the active targeting and prolonged in vivo circulation properties of the dual-targeting hybrid NPs, leading to enhanced accumulation of the drug in tumors. Furthermore, the anti-proliferation mechanism studies revealed that the inhibition of the growth and invasive activity of HGC27 cells was not only attributed to the enhanced cellular uptake of dual-targeting NPs, but also benefited from the suppression of CD44 and Her2 expression by HA and AHNP moieties. Finally, intravenous administration of the SN38-loaded dual-targeting hybrid NPs induced significant growth inhibition of HGC27 tumor xenografted in nude mice compared with a clinical antitumor agent, Irinotecan (CPT-11), and the other NP formulations. These results demonstrate that the designed dual-targeting hybrid NPs are promising for targeted anti-cancer drug delivery to treat human gastric tumors over-expressing Her2 and CD44. Electronic supplementary information (ESI) available. See DOI: 10.1039/c6nr01749e

Low CD1c + myeloid dendritic cell counts correlated with a high risk of rapid disease progression during early HIV-1 infection.

PubMed

Diao, Yingying; Geng, Wenqing; Fan, Xuejie; Cui, Hualu; Sun, Hong; Jiang, Yongjun; Wang, Yanan; Sun, Amy; Shang, Hong

2015-08-19

During early HIV-1 infection (EHI), the interaction between the immune response and the virus determines disease progression. Although CD1c + myeloid dendritic cells (mDCs) can trigger the immune response, the relationship between CD1c + mDC alteration and disease progression has not yet been defined. EHI changes in CD1c + mDC counts, surface marker (CD40, CD86, CD83) expression, and IL-12 secretion were assessed by flow cytometry in 29 patients. When compared with the normal controls, patients with EHI displayed significantly lower CD1c + mDC counts and IL-12 secretion and increased surface markers. CD1c + mDC counts were positively correlated with CD4+ T cell counts and inversely associated with viral loads. IL-12 secretion was only positively associated with CD4+ T cell counts. Rapid progressors had lower counts, CD86 expression, and IL-12 secretion of CD1c + mDCs comparing with typical progressors. Kaplan-Meier analysis and Cox regression models suggested patients with low CD1c + mDC counts (<10 cells/μL) had a 4-fold higher risk of rapid disease progression than those with high CD1c + mDC counts. However, no relationship was found between surface markers or IL-12 secretion and disease progression. During EHI, patients with low CD1c + mDC counts were more likely to experience rapid disease progression than those with high CD1c + mDC counts.

Human mesenchymal stem cells derived from limb bud can differentiate into all three embryonic germ layers lineages.

PubMed

Jiao, Fei; Wang, Juan; Dong, Zhao-Lun; Wu, Min-Juan; Zhao, Ting-Bao; Li, Dan-Dan; Wang, Xin

2012-08-01

Mesenchymal stem cells (MSCs) have been isolated from many sources, including adults and fetuses. Previous studies have demonstrated that, compared with their adult counterpart, fetal MSCs with several remarkable advantages may be a better resource for clinical applications. In this study, we successfully isolated a rapidly proliferating cell population from limb bud of aborted fetus and termed them "human limb bud-derived mesenchymal stem cells" (hLB-MSCs). Characteristics of their morphology, phenotype, cell cycle, and differentiation properties were analyzed. These adherent cell populations have a typically spindle-shaped morphology. Flow cytometry analysis showed that hLB-MSCs are positive for CD13, CD29, CD90, CD105, and CD106, but negative for CD3, CD4, CD5, CD11b, CD14, CD15, CD34, CD45, CD45RA, and HLA-DR. The detection of cell cycle from different passages indicated that hLB-MSCs have a similar potential for propagation during long culture in vitro. The most novel finding here is that, in addition to their mesodermal differentiation (osteoblasts and adipocytes), hLB-MSCs can also differentiated into extramesenchymal lineages, such as neural (ectoderm) and hepatic (endoderm) progenies. These results indicate that hLB-MSCs have a high level of plasticity and can differentiate into cell lineages from all three embryonic layers in vitro.

Human Mesenchymal Stem Cells Derived From Limb Bud Can Differentiate into All Three Embryonic Germ Layers Lineages

PubMed Central

Jiao, Fei; Wang, Juan; Dong, Zhao-lun; Wu, Min-juan; Zhao, Ting-bao; Li, Dan-dan

2012-01-01

Abstract Mesenchymal stem cells (MSCs) have been isolated from many sources, including adults and fetuses. Previous studies have demonstrated that, compared with their adult counterpart, fetal MSCs with several remarkable advantages may be a better resource for clinical applications. In this study, we successfully isolated a rapidly proliferating cell population from limb bud of aborted fetus and termed them “human limb bud–derived mesenchymal stem cells” (hLB-MSCs). Characteristics of their morphology, phenotype, cell cycle, and differentiation properties were analyzed. These adherent cell populations have a typically spindle-shaped morphology. Flow cytometry analysis showed that hLB-MSCs are positive for CD13, CD29, CD90, CD105, and CD106, but negative for CD3, CD4, CD5, CD11b, CD14, CD15, CD34, CD45, CD45RA, and HLA-DR. The detection of cell cycle from different passages indicated that hLB-MSCs have a similar potential for propagation during long culture in vitro. The most novel finding here is that, in addition to their mesodermal differentiation (osteoblasts and adipocytes), hLB-MSCs can also differentiated into extramesenchymal lineages, such as neural (ectoderm) and hepatic (endoderm) progenies. These results indicate that hLB-MSCs have a high level of plasticity and can differentiate into cell lineages from all three embryonic layers in vitro. PMID:22775353

CD44v6 expression in human skin keratinocytes as a possible mechanism for carcinogenesis associated with chronic arsenic exposure.

PubMed

Huang, S; Guo, S; Guo, F; Yang, Q; Xiao, X; Murata, M; Ohnishi, S; Kawanishi, S; Ma, N

2013-01-14

Inorganic arsenic is a well-known human skin carcinogen. Chronic arsenic exposure results in various types of human skin lesions, including squamous cell carcinoma (SCC). To investigate whether mutant stem cells participate in arsenic-associated carcinogenesis, we repeatedly exposed the HaCaT cells line to an environmentally relevant level of arsenic (0.05 ppm) in vitro for 18 weeks. Following sodium arsenic arsenite administration, cell cycle, colony-forming efficiency (CFE), cell tumorigenicity, and expression of CD44v6, NF-κB and p53, were analyzed at different time points (0, 5, 10, 15, 20, 25 and 30 passages). We found that a chronic exposure of HaCaT cells to a low level of arsenic induced a cancer stem- like phenotype. Furthermore, arsenic-treated HaCaT cells also became tumorigenic in nude mice, their growth cycle was predominantly in G2/M and S phases. Relative to nontreated cells, they exhibited a higher growth rate and a significant increase in CFE. Western blot analysis found that arsenic was capable of increasing cell proliferation and sprouting of cancer stem-like phenotype. Additionally, immunohistochemical analysis demonstrated that CD44v6 expression was up-regulated in HaCaT cells exposed to a low level of arsenic during early stages of induction. The expression of CD44v6 in arsenic-treated cells was positively correlated with their cloning efficiency in soft agar (r=0.949, P=0.01). Likewise, the expressions of activating transcription factor NF-κB and p53 genes in the arsenic-treated HaCaT cells were significantly higher than that in non-treated cells. Higher expressions of CD44v6, NF-κB and p53 were also observed in tumor tissues isolated from Balb/c nude mice. The present results suggest that CD44v6 may be a biomarker of arsenic-induced neoplastic transformation in human skin cells, and that arsenic promotes malignant transformation in human skin lesions through a NF-κB signaling pathway-stimulated expression of CD44v6.

Monoamine Oxidase Deficiency Causes Prostate Atrophy and Reduces Prostate Progenitor Cell Activity.

PubMed

Yin, Lijuan; Li, Jingjing; Liao, Chun-Peng; Jason Wu, Boyang

2018-04-10

Monoamine oxidases (MAOs) degrade a number of biogenic and dietary amines, including monoamine neurotransmitters, and play an essential role in many biological processes. Neurotransmitters and related neural events have been shown to participate in the development, differentiation, and maintenance of diverse tissues and organs by regulating the specialized cellular function and morphological structures of innervated organs such as the prostate. Here we show that mice lacking both MAO isoforms, MAOA and MAOB, exhibit smaller prostate mass and develop epithelial atrophy in the ventral and dorsolateral prostates. The cellular composition of prostate epithelium showed reduced CK5 + or p63 + basal cells, accompanied by lower Sca-1 expression in p63 + basal cells, but intact differentiated CK8 + luminal cells in MAOA/B-deficient mouse prostates. MAOA/B ablation also decreased epithelial cell proliferation without affecting cell apoptosis in mouse prostates. Using a human prostate epithelial cell line, we found that stable knockdown of MAOA and MAOB impaired the capacity of prostate stem cells to form spheres, coinciding with a reduced CD133 + /CD44 + /CD24 - stem cell population and less expression of CK5 and select stem cell markers, including ALDH1A1, TROP2, and CD166. Alternative pharmacological inhibition of MAOs also repressed prostate cell stemness. In addition, we found elevated expression of MAOA and MAOB in epithelial and/or stromal components of human prostate hyperplasia samples compared with normal prostate tissues. Taken together, our findings reveal critical roles for MAOs in the regulation of prostate basal progenitor cells and prostate maintenance. Stem Cells 2018. © AlphaMed Press 2018.

Label retention identifies a multipotent mesenchymal stem cell-like population in the postnatal thymus.

PubMed

Osada, Masako; Singh, Varan J; Wu, Kenmin; Sant'Angelo, Derek B; Pezzano, Mark

2013-01-01

Thymic microenvironments are essential for the proper development and selection of T cells critical for a functional and self-tolerant adaptive immune response. While significant turnover occurs, it is unclear whether populations of adult stem cells contribute to the maintenance of postnatal thymic epithelial microenvironments. Here, the slow cycling characteristic of stem cells and their property of label-retention were used to identify a K5-expressing thymic stromal cell population capable of generating clonal cell lines that retain the capacity to differentiate into a number of mesenchymal lineages including adipocytes, chondrocytes and osteoblasts suggesting a mesenchymal stem cell-like phenotype. Using cell surface analysis both culture expanded LRCs and clonal thymic mesenchymal cell lines were found to express Sca1, PDGFRα, PDGFRβ,CD29, CD44, CD49F, and CD90 similar to MSCs. Sorted GFP-expressing stroma, that give rise to TMSC lines, contribute to thymic architecture when reaggregated with fetal stroma and transplanted under the kidney capsule of nude mice. Together these results show that the postnatal thymus contains a population of mesenchymal stem cells that can be maintained in culture and suggests they may contribute to the maintenance of functional thymic microenvironments.

Role of capsule endoscopy in suspected celiac disease: A European multi-centre study

PubMed Central

Luján-Sanchis, Marisol; Pérez-Cuadrado-Robles, Enrique; García-Lledó, Javier; Juanmartiñena Fernández, José-Francisco; Elli, Luca; Jiménez-García, Victoria-Alejandra; Egea-Valenzuela, Juan; Valle-Muñoz, Julio; Carretero-Ribón, Cristina; Fernández-Urién-Sainz, Ignacio; López-Higueras, Antonio; Alonso-Lázaro, Noelia; Sanjuan-Acosta, Mileidis; Sánchez-Ceballos, Francisco; Rosa, Bruno; González-Vázquez, Santiago; Branchi, Federica; Ruano-Díaz, Lucía; Prieto-de-Frías, César; Pons-Beltrán, Vicente; Borque-Barrera, Pilar; González-Suárez, Begoña; Xavier, Sofía; Argüelles-Arias, Federico; Herrerías-Gutiérrez, Juan-Manuel; Pérez-Cuadrado-Martínez, Enrique; Sempere-García-Argüelles, Javier

2017-01-01

AIM To analyze the diagnostic yield (DY), therapeutic impact (TI) and safety of capsule endoscopy (CE). METHODS This is a multi-centre, observational, analytical, retrospective study. A total of 163 patients with suspicion of celiac disease (CD) (mean age = 46.4 ± 17.3 years, 68.1% women) who underwent CE from 2003 to 2015 were included. Patients were divided into four groups: seronegative CD with atrophy (Group-I, n = 19), seropositive CD without atrophy (Group-II, n = 39), contraindication to gastroscopy (Group-III, n = 6), seronegative CD without atrophy, but with a compatible context (Group-IV, n = 99). DY, TI and the safety of CE were analysed. RESULTS The overall DY was 54% and the final diagnosis was villous atrophy (n = 65, 39.9%), complicated CD (n = 12, 7.4%) and other enteropathies (n = 11, 6.8%; 8 Crohn’s). DY for groups I to IV was 73.7%, 69.2%, 50% and 44.4%, respectively. Atrophy was located in duodenum in 24 cases (36.9%), diffuse in 19 (29.2%), jejunal in 11 (16.9%), and patchy in 10 cases (15.4%). Factors associated with a greater DY were positive serology (68.3% vs 49.2%, P = 0.034) and older age (P = 0.008). On the other hand, neither sex nor clinical presentation, family background, positive histology or HLA status were associated with DY. CE results changed the therapeutic approach in 71.8% of the cases. Atrophy was associated with a greater TI (92.3% vs 45.3%, P < 0.001) and 81.9% of the patients responded to diet. There was one case of capsule retention (0.6%). Agreement between CE findings and subsequent histology was 100% for diagnosing normal/other conditions, 70% for suspected CD and 50% for complicated CD. CONCLUSION CE has a high DY in cases of suspicion of CD and it leads to changes in the clinical course of the disease. CE is safe procedure with a high degree of concordance with histology and it helps in the differential diagnosis of CD. PMID:28216978

A dual-potential electrochemiluminescence ratiometric sensor for sensitive detection of dopamine based on graphene-CdTe quantum dots and self-enhanced Ru(II) complex.

PubMed

Fu, Xiaomin; Tan, Xingrong; Yuan, Ruo; Chen, Shihong

2017-04-15

A novel dual-potential ratiometric electrochemiluminescence (ECL) sensor was designed for detecting dopamine (DA) based on graphene-CdTe quantum dots (G-CdTe QDs) as the cathodic emitter and self-enhanced Ru(II) composite (TAEA-Ru) as the anodic emitter. TAEA-Ru was prepared by linking ruthenium(II) tris(2,2'-bipyridyl-4,4'-dicarboxylato) with tris(2-aminoethyl)amine. Firstly, 3-aminopropyltriethoxysilane founctionalized G-CdTe QDs was used as the substrate for capturing target DA via the specific recognition of the diol of DA to the oxyethyl group of APTES. Then, Cu 2 O nanocrystals supported TAEA-Ru was further bound by the strong interaction between amino groups of DA and carboxyl groups of the Cu 2 O-TAEA-Ru. With the increase in DA concentration, the loading of Cu 2 O-TAEA-Ru at the electrode increased. As a result, the anodic ECL signal from TAEA-Ru increased, and the cathodic ECL signal from G-CdTe QDs/O 2 system decreased correspondingly. Such a decrease was resulted from the ECL resonance energy transfer (RET) from G-CdTe QDs to TAEA-Ru as well as the dual quenching effects of Cu 2 O to G-CdTe QDs, namely the ECL-RET from G-CdTe QDs to Cu 2 O and the consumption of coreactant O 2 by Cu 2 O. Based on the ratio of two ECL signals, the determination of DA was achieved with a linear range from 10.0 fM to 1.0nM and a detection limit low to 2.9 fM (S/N=3). The combination of G-CdTe QDs/O 2 and TAEA-Ru would break the limitation of the same coreatant shared in previous ECL ratiometric systems and provide a potential application of ECL ratiometric sensor in the detection of biological small molecules with the assistance of the dual molecular recognition strategy. Copyright © 2016 Elsevier B.V. All rights reserved.

The PBX1 lupus susceptibility gene regulates CD44 expression.

PubMed

Niu, Yuxin; Sengupta, Mayami; Titov, Anton A; Choi, Seung-Chul; Morel, Laurence

2017-05-01

PBX1-d is novel splice isoform of pre-B-cell leukemia homeobox 1 (PBX1) that lacks its DNA-binding and Hox-binding domains, and functions as a dominant negative. We have shown that PBX1-d expression in CD4 + T cells is associated with systemic lupus erythematosus (SLE) in a mouse model as well as in human subjects. More specifically, PBX1-d expression leads to the production of autoreactive activated CD4+ T cells, a reduced frequency and function of Foxp3+ regulatory T (Treg) cells and an expansion of follicular helper T (Tfh) cells. Very little is known about the function of PBX1 in T cells, except that it directly regulates the expression of miRNAs associated with Treg and Tfh homeostasis. In the present study, we show that PBX1 directly regulated the expression of CD44, a marker of T cell activation. Two PBX1 binding sites in the promoter directly regulated CD44 expression, with PBX1-d driving a higher expression than the normal isoform PBX1-b. In addition, mutations in each of the two binding sites had different effects of PBX1-b and PBX1-d. Finally, we showed that an enhanced recruitment of co-factor MEIS by PBX1-d over PBX1-b, while there was no difference for co-factor PREP1 recruitment. Therefore, this study demonstrates that the lupus-associated PBX1-d isoform directly transactivates CD44, a marker of CD44 activation and memory, and that it has different DNA binding and co-factor recruitment relative to the normal isoform. Taken together, these results confirm that PBX1 directly regulates genes related to T cell activation and shows that the lupus-associated isoform PBX1-d has unique molecular functions. Copyright © 2017 Elsevier Ltd. All rights reserved.

Sexually immature male ERE-Luc reporter mice to assess low dose estrogen-like effects of CdCl2 versus dietary Cd

PubMed Central

Ramachandran, Balaji; Rizzi, Nicoletta; Maggi, Adriana

2014-01-01

CdCl2 salt is widely used in exposure oriented studies, while the biological exposure of Cadmium (Cd) occurs mostly through diet. Hence, we designed a in vivo imaging methodology with sexually immature male ERE-Luc reporter mice to test the estrogen-like (EL) effects of Cd as a natural component in wheat and flax bread based diets (containing 17.57 and 49.22 ug/kg Cd concentrations respectively) and CdCl2 per-oral dose of 1 ug/kg/bw/day. Total exposure of ingested and % bioaccumulation of Cd in selected organs were estimated as 547 ng (4.4%), 776 ng (0.3%) and 2131.8 ng (0.1%) corresponding to CdCl2, wheat and flax bread based diet treatments respectively. Cd from CdCl2 bioaccumulated more readily, despite the exposure of Cd is higher with bread based diets. Longitudinal in vivo imaging did not reveal significant changes in luciferase activity. White adipose tissue (WAT) and prostate were identified as novel target organs of Cd. Indeed, the rest of the observed EL effects, endogenous target gene expression and necropsy findings are not consistent to any particular organ or treatment. This implies that, the observed EL effects due to low doses of Cd (either from CdCl2 or dietary form) occur only as subtle changes at the molecular level, but inadequate to cause significant changes at the anatomo-pathological level during the 21 day exposure period. The study demonstrates the sensitivity of the methodology to assess EL effects of food embedded Cd and underlines the limitations of directly extrapolating the results of suspected chemicals in their pure form to dietary exposure scenarios. PMID:24795841

In Vivo, Fatty Acid Translocase (CD36) Critically Regulates Skeletal Muscle Fuel Selection, Exercise Performance, and Training-induced Adaptation of Fatty Acid Oxidation*

PubMed Central

McFarlan, Jay T.; Yoshida, Yuko; Jain, Swati S.; Han, Xioa-Xia; Snook, Laelie A.; Lally, James; Smith, Brennan K.; Glatz, Jan F. C.; Luiken, Joost J. F. P.; Sayer, Ryan A.; Tupling, A. Russell; Chabowski, Adrian; Holloway, Graham P.; Bonen, Arend

2012-01-01

For ∼40 years it has been widely accepted that (i) the exercise-induced increase in muscle fatty acid oxidation (FAO) is dependent on the increased delivery of circulating fatty acids, and (ii) exercise training-induced FAO up-regulation is largely attributable to muscle mitochondrial biogenesis. These long standing concepts were developed prior to the recent recognition that fatty acid entry into muscle occurs via a regulatable sarcolemmal CD36-mediated mechanism. We examined the role of CD36 in muscle fuel selection under basal conditions, during a metabolic challenge (exercise), and after exercise training. We also investigated whether CD36 overexpression, independent of mitochondrial changes, mimicked exercise training-induced FAO up-regulation. Under basal conditions CD36-KO versus WT mice displayed reduced fatty acid transport (−21%) and oxidation (−25%), intramuscular lipids (less than or equal to −31%), and hepatic glycogen (−20%); but muscle glycogen, VO2max, and mitochondrial content and enzymes did not differ. In acutely exercised (78% VO2max) CD36-KO mice, fatty acid transport (−41%), oxidation (−37%), and exercise duration (−44%) were reduced, whereas muscle and hepatic glycogen depletions were accelerated by 27–55%, revealing 2-fold greater carbohydrate use. Exercise training increased mtDNA and β-hydroxyacyl-CoA dehydrogenase similarly in WT and CD36-KO muscles, but FAO was increased only in WT muscle (+90%). Comparable CD36 increases, induced by exercise training (+44%) or by CD36 overexpression (+41%), increased FAO similarly (84–90%), either when mitochondrial biogenesis and FAO enzymes were up-regulated (exercise training) or when these were unaltered (CD36 overexpression). Thus, sarcolemmal CD36 has a key role in muscle fuel selection, exercise performance, and training-induced muscle FAO adaptation, challenging long held views of mechanisms involved in acute and adaptive regulation of muscle FAO. PMID:22584574

Topological identification of the first uninodal 8-connected lsz MOF built from 2,2'-difluorobiphenyl-4,4'-dicarboxylate pillars and cadmium(II)-triazolate layers.

PubMed

Zhang, Yuchi; Wu, Yuanhua; He, Xin; Ma, Junhan; Shen, Xuan; Zhu, Dunru

2018-03-01

Using polynuclear metal clusters as nodes, many high-symmetry high-connectivity nets, like 8-connnected bcu and 12-connected fcu, have been attained in metal-organic frameworks (MOFs). However, construction of low-symmetry high-connected MOFs with a novel topology still remains a big challenge. For example, a uninodal 8-connected lsz network, observed in inorganic ZrSiO 4 , has not been topologically identified in MOFs. Using 2,2'-difluorobiphenyl-4,4'-dicarboxylic acid (H 2 L) as a new linker and 1,2,4-triazole (Htrz) as a coligand, a novel three-dimensional Cd II -MOF, namely poly[tetrakis(μ 4 -2,2'-difluorobiphenyl-4,4'-dicarboxylato-κ 5 O 1 ,O 1' :O 1' :O 4 :O 4' )tetrakis(N,N-dimethylformamide-κO)tetrakis(μ 3 -1,2,4-triazolato-κ 3 N 1 :N 2 :N 4 )hexacadmium(II)], [Cd 6 (C 14 H 6 F 2 O 4 ) 4 (C 2 H 2 N 3 ) 4 (C 3 H 7 NO) 4 ] n , (I), has been prepared. Single-crystal structure analysis indicates that six different Cd II ions co-exist in (I) and each Cd II ion displays a distorted [CdO 4 N 2 ] octahedral geometry with four equatorial O atoms and two axial N atoms. Three Cd II ions are connected by four carboxylate groups and four trz - ligands to form a linear trinuclear [Cd 3 (COO) 4 (trz) 4 ] cluster, as do the other three Cd II ions. Two Cd 3 clusters are linked by trz - ligands in a μ 1,2,4 -bridging mode to produce a two-dimensional Cd II -triazolate layer with (6,3) topology in the ab plane. These two-dimensional layers are further pillared by the L 2- ligands along the c axis to generate a complicated three-dimensional framework. Topologically, regarding the Cd 3 cluster as an 8-connected node, the whole architecture of (I) is a uninodal 8-connected lsz framework with the Schläfli symbol (4 22 ·6 6 ). Complex (I) was further characterized by elemental analysis, IR spectroscopy, powder X-ray diffraction, thermogravimetric analysis and a photoluminescence study. MOF (I) has a high thermal and water stability.

Deep sequencing and flow cytometric characterization of expanded effector memory CD8+CD57+ T cells frequently reveals T-cell receptor Vβ oligoclonality and CDR3 homology in acquired aplastic anemia.

PubMed

Giudice, Valentina; Feng, Xingmin; Lin, Zenghua; Hu, Wei; Zhang, Fanmao; Qiao, Wangmin; Ibanez, Maria Del Pilar Fernandez; Rios, Olga; Young, Neal S

2018-05-01

Oligoclonal expansion of CD8 + CD28 - lymphocytes has been considered indirect evidence for a pathogenic immune response in acquired aplastic anemia. A subset of CD8 + CD28 - cells with CD57 expression, termed effector memory cells, is expanded in several immune-mediated diseases and may have a role in immune surveillance. We hypothesized that effector memory CD8 + CD28 - CD57 + cells may drive aberrant oligoclonal expansion in aplastic anemia. We found CD8 + CD57 + cells frequently expanded in the blood of aplastic anemia patients, with oligoclonal characteristics by flow cytometric Vβ usage analysis: skewing in 1-5 Vβ families and frequencies of immunodominant clones ranging from 1.98% to 66.5%. Oligoclonal characteristics were also observed in total CD8 + cells from aplastic anemia patients with CD8 + CD57 + cell expansion by T-cell receptor deep sequencing, as well as the presence of 1-3 immunodominant clones. Oligoclonality was confirmed by T-cell receptor repertoire deep sequencing of enriched CD8 + CD57 + cells, which also showed decreased diversity compared to total CD4 + and CD8 + cell pools. From analysis of complementarity-determining region 3 sequences in the CD8 + cell pool, a total of 29 sequences were shared between patients and controls, but these sequences were highly expressed in aplastic anemia subjects and also present in their immunodominant clones. In summary, expansion of effector memory CD8 + T cells is frequent in aplastic anemia and mirrors Vβ oligoclonal expansion. Flow cytometric Vβ usage analysis combined with deep sequencing technologies allows high resolution characterization of the T-cell receptor repertoire, and might represent a useful tool in the diagnosis and periodic evaluation of aplastic anemia patients. (Registered at clinicaltrials.gov identifiers: 00001620, 01623167, 00001397, 00071045, 00081523, 00961064 ). Copyright © 2018 Ferrata Storti Foundation.

The effects of CD147 on the cell proliferation, apoptosis, invasion, and angiogenesis in glioma.

PubMed

Yin, Haoyuan; Shao, Ying; Chen, Xuan

2017-01-01

To analyze the effects of extracellular matrix metalloproteinase inducer (CD147) on glioma proliferation, apoptosis, invasion, and angiogenesis. Tissue samples were obtained from 101 glioma cases while normal brain tissues were obtained from 30 brain injury cases. Immunohistochemical assay was performed to detect the expressions of CD147, CD34, and VEGF in tissue samples. QRT-PCR was performed to detect the relative expression of CD147 mRNA in human glioma cell lines. CD147 siRNA was transfected into glioma cell line U251. Cell proliferation, apoptosis, invasion, and angiogenesis were tested by MTT, flow cytometry, Transwell assay, and vasculogenic mimicry assay, respectively. Expressions of relative proteins were analyzed with western blot. CD147 was positively expressed with the percentage of 0, 37.5, 44.8, 67.9, and 85.7 % in normal tissues and glioma tissues with WHO grades I-IV, respectively, and the scores of MVDand VEGF were associated with the expression of CD147. CD147 was significantly upregulated in the human glioma cell lines (P < 0.05). Downregulated the expression of CD147 suppressed cell proliferation, blocked cell cycle, induced apoptosis, inhibited cell invasion and angiogenesis in glioma cells in vitro. The expression of CD147 was significantly associated with WHO tumor grade and angiogenesis; silencing of CD147 contributed to inhibition of glioma proliferation, invasion, and angiogenesis. Our study provided firm evidence that CD 147 is a potential glioma target for anti-angiogenic therapies.

[Key effect genes responding to nerve injury identified by gene ontology and computer pattern recognition].

PubMed

Pan, Qian; Peng, Jin; Zhou, Xue; Yang, Hao; Zhang, Wei

2012-07-01

In order to screen out important genes from large gene data of gene microarray after nerve injury, we combine gene ontology (GO) method and computer pattern recognition technology to find key genes responding to nerve injury, and then verify one of these screened-out genes. Data mining and gene ontology analysis of gene chip data GSE26350 was carried out through MATLAB software. Cd44 was selected from screened-out key gene molecular spectrum by comparing genes' different GO terms and positions on score map of principal component. Function interferences were employed to influence the normal binding of Cd44 and one of its ligands, chondroitin sulfate C (CSC), to observe neurite extension. Gene ontology analysis showed that the first genes on score map (marked by red *) mainly distributed in molecular transducer activity, receptor activity, protein binding et al molecular function GO terms. Cd44 is one of six effector protein genes, and attracted us with its function diversity. After adding different reagents into the medium to interfere the normal binding of CSC and Cd44, varying-degree remissions of CSC's inhibition on neurite extension were observed. CSC can inhibit neurite extension through binding Cd44 on the neuron membrane. This verifies that important genes in given physiological processes can be identified by gene ontology analysis of gene chip data.

Diverse Cd(II) compounds based on N-benzoyl-L-glutamic acid and N-donor ligands: Structures and photoluminescent properties

NASA Astrophysics Data System (ADS)

Ma, Ning; Guo, Wei-Ying; Song, Hui-Hua; Yu, Hai-Tao

2016-01-01

Five new Cd(II) coordination polymers with N-benzoyl-L-glutamic acid (H2bzgluO) and different N-donor ligands, [Cd(bzgluO)(2,2‧-bipy)(H2O)]n (1), [Cd(bzgluO)(2,4‧-bipy)2(H2O)·3H2O]n (2), [Cd(bzgluO)(phen)·H2O]n (3), [Cd(bzgluO)(4,4‧-bipy)(H2O)]n (4), [Cd(bzgluO)(bpp)(H2O)·2H2O]n (5) were synthesized (2,2‧-bipy=2,2‧-bipyridine, 2,4‧-bipy=2,4‧-bipyridine, phen=1,10-phenanthroline, 4,4‧-bipy=4,4‧-bipyridine, bpp=1,3-di(4-pyridyl)propane). Compounds 1-2 exhibit a 1D single-chain structure. Compound 1 generates a 2D supramolecular structure via π-π stacking and hydrogen bonding, 3D architecture of compound 2 is formed by hydrogen bonding. Compound 3 features a 1D double-chain structure, which are linked by π-π interactions into a 2D supramolecular layer. Compounds 4-5 display a 2D network structure. Neighboring layers of 4 are extended into a 3D supramolecular architecture through hydrogen bonding. The structural diversity of these compounds is attributed to the effect of ancillary N-donor ligands and coordination modes of H2bzgluO. Luminescent properties of 1-5 were studied at room temperature. Circular dichroism of compounds 1, 2 and 5 were investigated.

The impact of donor characteristics on the immune cell composition of mixture allografts of granulocyte-colony-stimulating factor-mobilized marrow harvests and peripheral blood harvests.

PubMed

Wang, Yu-Tong; Zhao, Xiang-Yu; Zhao, Xiao-Su; Xu, Lan-Ping; Zhang, Xiao-Hui; Wang, Yu; Liu, Kai-Yan; Chang, Ying-Jun; Huang, Xiao-Jun

2015-12-01

The association of donor characteristics with immune cell composition in allografts remains poorly understood. In this retrospective study, the effects of donor characteristics on immune cell composition in allografts were investigated. The correlations of donor characteristics with the immune cell composition in mixture allografts of granulocyte-colony-stimulating factor-mobilized marrow harvests and peripheral blood harvests of 390 healthy donors (male, 240; female, 150; median age, 40 years old) were analyzed. The median doses of CD3+ T cells, CD4+ T cells, CD8+ T cells, CD3+CD4-CD8- T cells, and monocytes in mixture allografts were 160.57 × 10(6), 89.29 × 10(6), 56.16 × 10(6), 10.87 × 10(6), and 137.94 × 10(6)/kg, respectively. Multivariate analysis showed that younger donor age was associated with a higher dose of CD3+ T cells (p = 0.006), CD3+CD8+ T cells (p < 0.001), CD3+CD4-CD8- T cells (p = 0.004), and monocytes (p = 0.014), as well as a higher ratio of CD3+CD4-CD8- T cells/CD3+ T cells (p < 0.001) in the mixture allografts. A negative association of donor weight with CD3+ T cells (p < 0.001), CD4+ T cells (p = 0.002), CD8+ T cells (p < 0.001), and CD3+CD4-CD8- T cells (p = 0.044) was observed. The count of peripheral blood lymphocyte pre-peripheral blood apheresis was correlated with the yield of CD3+ T cells (p < 0.001) and CD4+ T cells (p = 0.001). The peripheral blood monocyte count before marrow harvest predicted the monocyte dose (p = 0.002). The results suggested that older and overweight donors should not be chosen. The monocyte and lymphocyte counts before harvest could predict the yield of immune cells in allografts. © 2015 AABB.

CD147 as a Novel Prognostic Biomarker for Hepatocellular Carcinoma: A Meta-Analysis.

PubMed

Peng, Fei; Li, Hui; You, Qian; Li, Hongru; Wu, Dongwen; Jiang, Chunxiang; Deng, Guangtong; Li, Yan; Li, Yuyan; Wu, Yi

2017-01-01

We conducted a meta-analysis to investigate the controversial association of CD147 expression with HCC prognosis and clinicopathological characteristics. Eight studies from PubMed (1966-2016), EMBASE (1980-2016), Cochrane Library (1996-2016), Web of Science (1945-2016), China National Knowledge Infrastructure (1982-2016), and Wanfang databases (1988-2016) were considered. The associations between CD147 expression and clinicopathological parameters and overall survival (OS) or DFS/RFS were reassessed using the meta-analysis for odds ratio (OR) or hazard ratio (HR) and 95% confidence interval (CI). CD147 expression was associated with DFS/RFS (HR = 3.26; 95% CI: 1.82-5.83; P < 0.0001) but not with OS (HR = 1.35; 95% CI: 0.56-3.29; P = 0.51). We also delved deeper into the association between median survival time and CD147 expression owing to significant heterogeneity and found significant differences between high and low CD147 expression groups with respect to median survival time. CD147 expression was closely associated with the TNM stage (OR = 0.18; 95% CI: 0.04-0.85; P = 0.03) and venous invasion (OR = 6.29; 95% CI: 1.70-23.20; P = 0.006). In contrast, there was no association between CD147 expression and tumor stage, cirrhosis, differentiation, lymph node metastasis, HBsAg, and serum AFP levels. Thus, CD147 expression is potentially closely related to HCC survival and associated clinicopathological parameters, paving the way for further research.

Cd-induced production of glomalin by arbuscular mycorrhizal fungus (Rhizophagus irregularis) as estimated by monoclonal antibody assay.

PubMed

Malekzadeh, Elham; Aliasgharzad, Nasser; Majidi, Jafar; Aghebati-Maleki, Leili; Abdolalizadeh, Jalal

2016-10-01

Glomalin is a specific fungal glycoprotein produced by arbuscular mycorrhizal (AM) fungi belonging to the Glomerales which could efficiently sequestrate heavy metals. The glomalin has been introduced as a heat shock protein and there are evidences that increasing levels of heavy metals could enhance its production. We examined the influence of Cd concentrations on glomalin production by AM fungus, as well as its contribution to the sequestration of Cd in both pot and in vitro culture conditions. Pot experiment was carried out using pure sand with Trifolium repens L. as host plant, mycorrhized by Rhizophagus irregularis and treated with Cd levels of 0, 15, 30, and 45 μM. In vitro experiment was performed in two-compartment plates containing the transformed carrot roots mycorrhized with the same fungus and treated with Cd levels of 0, 0.001, 0.01, and 0.1 mM. The immunoreactive and Bradford reactive glomalin contents in both experiments increased as so raising Cd concentration. Total Cd sequestrated by hyphal glomalin in both cultures was significantly increased as the levels of Cd increased. The highest contents of Cd sequestration in pot (75.78 μg Cd/mg glomalin) and in vitro (11.44 μg Cd/mg glomalin) cultures were recorded at the uppermost levels of Cd, which significantly differed with other levels. Our results suggested that under Cd-induced stress, stimulated production of glomalin by AM fungus may be a protective mechanism against the toxic effect of Cd.

CD24 can be used to isolate Lgr5+ putative colonic epithelial stem cells in mice

PubMed Central

King, Jeffrey B.; von Furstenberg, Richard J.; Smith, Brian J.; McNaughton, Kirk K.; Galanko, Joseph A.

2012-01-01

A growing body of evidence has implicated CD24, a cell-surface protein, as a marker of colorectal cancer stem cells and target for antitumor therapy, although its presence in normal colonic epithelium has not been fully characterized. Previously, our group showed that CD24-based cell sorting can be used to isolate a fraction of murine small intestinal epithelial cells enriched in actively cycling stem cells. Similarly, we hypothesized that CD24-based isolation of colonic epithelial cells would generate a fraction enriched in actively cycling colonic epithelial stem cells (CESCs). Immunohistochemistry performed on mouse colonic tissue showed CD24 expression in the bottom half of proximal colon crypts and the crypt base in the distal colon. This pattern of distribution was similar to enhanced green fluorescent protein (EGFP) expression in Lgr5-EGFP mice. Areas expressing CD24 contained actively proliferating cells as determined by ethynyl deoxyuridine (EdU) incorporation, with a distinct difference between the proximal colon, where EdU-labeled cells were most frequent in the midcrypt, and the distal colon, where they were primarily at the crypt base. Flow cytometric analyses of single epithelial cells, identified by epithelial cell adhesion molecule (EpCAM) positivity, from mouse colon revealed an actively cycling CD24+ fraction that contained the majority of Lgr5-EGFP+ putative CESCs. Transcript analysis by quantitative RT-PCR confirmed enrichment of active CESC markers [leucine-rich-repeat-containing G protein-coupled receptor 5 (Lgr5), ephrin type B receptor 2 (EphB2), and CD166] in the CD24+EpCAM+ fraction but also showed enrichment of quiescent CESC markers [leucine-rich repeats and immunoglobin domains (Lrig), doublecortin and calmodulin kinase-like 1 (DCAMKL-1), and murine telomerase reverse transcriptase (mTert)]. We conclude that CD24-based sorting in wild-type mice isolates a colonic epithelial fraction highly enriched in actively cycling and quiescent putative CESCs. Furthermore, the presence of CD24 expression in normal colonic epithelium may have important implications for the use of anti-CD24-based colorectal cancer therapies. PMID:22723265

Lack of evidence of rotavirus-dependent molecular mimicry as a trigger of coeliac disease.

PubMed

Ziberna, F; De Lorenzo, G; Schiavon, V; Arnoldi, F; Quaglia, S; De Leo, L; Vatta, S; Martelossi, S; Burrone, O R; Ventura, A; Not, T

2016-12-01

New data suggest the involvement of rotavirus (RV) in triggering autoimmunity in coeliac disease (CD) by molecular mimicry between the human-transglutaminase protein and the dodecapeptide (260-271 aa) of the RV protein VP7 (pVP7). To assess the role of RV in the onset of CD, we measured anti-pVP7 antibodies in the sera of children with CD and of control groups. We analysed serum samples of 118 biopsy-proven CD patients and 46 patients with potential CD; 32 children with other gastrointestinal diseases; 107 no-CD children and 107 blood donors. Using enzyme-linked immunosorbent assay (ELISA) assay, we measured immunoglobulin (Ig)A-IgG antibodies against the synthetic peptides pVP7, the human transglutaminase-derived peptide (476-487 aa) which shows a homology with VP7 protein and a control peptide. The triple-layered RV particles (TLPs) containing the VP7 protein and the double-layered RV-particles (DLPs) lacking the VP7 protein were also used as antigens in ELISA assay. Antibody reactivity to the RV-TLPs was positive in 22 of 118 (18%) CD patients and in both paediatric (17 of 107, 16%) and adult (29 of 107, 27%) control groups, without showing a statistically significant difference among them (P = 0·6, P = 0·1). Biopsy-proven CD patients as well as the adult control group demonstrated a high positive antibody reactivity against both pVP7 (34 of 118, 29% CD patients; 66 of 107, 62% adult controls) and control synthetic peptides (35 of 118, 30% CD patients; 56 of 107, 52% adult controls), suggesting a non-specific response against RV pVP7. We show that children with CD do not have higher immune reactivity to RV, thus questioning the molecular mimicry mechanism as a triggering factor of CD. © 2016 British Society for Immunology.

Maternal serum soluble CD30 is increased in pregnancies complicated with acute Pyelonephritis

PubMed Central

Kusanovic, Juan Pedro; Romero, Roberto; Espinoza, Jimmy; Gotsch, Francesca; Edwin, Samuel; Chaiworapongsa, Tinnakorn; Mittal, Pooja; Soto, Eleazar; Erez, Offer; Mazaki-Tovi, Shali; Than, Nandor Gabor; Friel, Lara; Yoon, Bo Hyun; Mazor, Moshe; Hassan, Sonia

2007-01-01

Objectives Normal pregnancy is characterized by activation of the innate immunity and suppression of the adaptive limb of the immune response. However, pregnant women are more susceptible to the effects of infection and microbial products than non-pregnant women. CD30 is a member of the tumor necrosis factor receptor superfamily and is preferentially expressed by activated T cells producing Th2-type cytokines. Its soluble form (sCD30) is proposed to be an index of Th2 immune response. High serum concentrations of sCD30 have been found in the acute phase of viral infections, such as HIV-1 and hepatitis B. There is, however, conflicting evidence about serum sCD30 concentration in patients with bacterial infections. The objective of this study was to determine whether there are changes in the serum concentration of sCD30 in pregnant women with pyelonephritis. Methods This cross-sectional study included normal pregnant women (N=89) and pregnant women with pyelonephritis (N=41). Maternal serum concentration of sCD30 was measured by a specific and sensitive enzyme-linked immunoassay. Non-parametric tests were used for comparisons. A p value <0.05 was considered statistically significant. Results (1) Pregnant women with pyelonephritis had a significantly higher median serum concentration of sCD30 than those with a normal pregnancy (median: 44.3 U/ml, range: 16–352.5 vs. median: 29.7 U/ml, range: 12.2–313.2, respectively; p<0.001); and (2) No significant differences were found in the median maternal serum concentration of sCD30 between pregnant women with pyelonephritis who had a positive blood culture compared to those with a negative blood culture (median:47.7 U/mL, range: 17.1–118.8 vs. median: 42.6 U/mL, range: 16–352.5, respectively; p=0.86). Conclusions Acute pyelonephritis during pregnancy is associated with a higher maternal serum concentration of sCD30 than normal pregnancy. This finding is novel, and suggests that pregnant women with pyelonephritis may have a complex immune state in which there is activation of some components of what is considered a Th2 immune response. PMID:17853184

Fluorescent carbon dot-gated multifunctional mesoporous silica nanocarriers for redox/enzyme dual-responsive targeted and controlled drug delivery and real-time bioimaging.

PubMed

Wang, Ying; Cui, Yu; Zhao, Yating; He, Bing; Shi, Xiaoli; Di, Donghua; Zhang, Qiang; Wang, Siling

2017-08-01

A distinctive and personalized nanocarrier is described here for controlled and targeted antitumor drug delivery and real-time bioimaging by combining a redox/enzyme dual-responsive disulfide-conjugated carbon dot with mesoporous silica nanoparticles (MSN-SS-CD HA ). The carbon dot with controlling and targeting abilities was prepared through a polymerizing reaction by applying citric acid and HA as starting materials (named CD HA ). The as-prepared MSN-SS-CD HA exhibited not only superior photostability and excellent biocompatibility, but also the ability to target A549 cells with overexpression of CD44 receptors. Upon loading the antitumor drug, doxorubicin (DOX), into the mesoporous channels of MSN nanoparticles, CD HA with a diameter size of 3nm completely blocked the pore entrance of DOX-encapsulated MSN nanoparticles with a pore size of about 3nm, thus preventing the premature leakage of DOX and increasing the antitumor activity until being triggered by specific stimuli in the tumor environment. The results of the cell imaging and cytotoxicity studies demonstrated that the redox/enzyme dual-responsive DOX-encapsulated MSN-SS-CD HA nanoparticles can selectively deliver and control the release of DOX into tumor cells. Ex vivo fluorescence images showed a much stronger fluorescence of MSN-SS-CD HA -DOX in the tumor site than in normal tissues, greatly facilitating the accumulation of DOX in the target tissue. However, its counterpart, MSN-SH-DOX exhibited no or much lower tumor cytotoxicity and drug accumulation in tumor tissue. In addition, MSN-SS-CD was also used as a control to investigate the ability of MSN-SS-CD HA to target A549 cells. The results obtained indicated that MSN-SS-CD HA possessed a higher cellular uptake through the CD44 receptor-mediated endocytosis compared with MSN-SS-CD in the A549 cells. Such specific redox/enzyme dual-responsive targeted nanocarriers are a useful strategy achieving selective controlled and targeted delivery of therapeutic reagents with real-time bioimaging, and may also facilitate the development of drug delivery systems for a number of clinical applications. Copyright © 2017 Elsevier B.V. All rights reserved.

Structural diversification and photocatalytic properties of three Cd(II) coordination polymers decorated with different auxiliary ligands

NASA Astrophysics Data System (ADS)

Yin, Wen-Yu; Zhuang, Guo-Yong; Huang, Zuo-Long; Cheng, Hong-Jian; Zhou, Li; Ma, Man-Hong; Wang, Hao; Tang, Xiao-Yan; Ma, Yun-Sheng; Yuan, Rong-Xin

2016-03-01

Three cadmium coordination polymers, [Cd(bismip)]n (1), {[Cd(bismip)(phen)]·H2O}n (2) and {[Cd2(bismip)2(4,4‧-bipy)]·2H2O}n (3) (H2bismip=5-(1H-benzoimidazol-2-ylsulfanylmethyl)-isophthalic acid, phen=1,10-phenanthroline, 4,4‧-bipy=4,4‧-bipyridine) have been prepared under solvothermal conditions. In 1, the [Cd4(bismip)3] units are jointed by bismip ligands to afford a three-dimensional (3D) architecture. Complex 2 exhibits a 3D supramolecular framework based on the interconnection of 1D chains through hydrogen bonding interactions and π-π packing interactions. 3 is a two-fold interpenetrating 3D architecture with a (4·82)(42·84) Schläfli symbol in which 2D layers are interlinked by 4,4‧-bipy ligands. The diverse structures of compounds 1-3 indicate that the auxiliary ligands have significant effects on the final structures. The photoluminescent properties and photocatalytic properties of these coordination polymers in the solid state were also investigated. Remarkably, 3 shows the wide gap semiconductor nature and exhibit excellent photocatalytic performance.

Stage 3 immature human natural killer cells found in secondary lymphoid tissue constitutively and selectively express the TH17 cytokine interleukin-22

PubMed Central

Hughes, Tiffany; Becknell, Brian; McClory, Susan; Briercheck, Edward; Freud, Aharon G.; Zhang, Xiaoli; Mao, Hsiaoyin; Nuovo, Gerard; Yu, Jianhua

2009-01-01

Considerable functional heterogeneity within human natural killer (NK) cells has been revealed through the characterization of distinct NK-cell subsets. Accordingly, a small subset of CD56+NKp44+NK cells, termed NK-22 cells, was recently described within secondary lymphoid tissue (SLT) as IL-22− when resting, with a minor fraction of this population becoming IL-22+ when activated. Here we discover that the vast majority of stage 3 immature NK (iNK) cells in SLT constitutively and selectively express IL-22, a TH17 cytokine important for mucosal immunity, whereas earlier and later stages of NK developmental intermediates do not express IL-22. These iNK cells have a surface phenotype of CD34−CD117+CD161+CD94−, largely lack expression of NKp44 and CD56, and do not produce IFN-γ or possess cytolytic activity. In summary, stage 3 iNK cells are highly enriched for IL-22 and IL-26 messenger RNA, and IL-22 protein production, but do not express IL-17A or IL-17F. PMID:19244159

Morpho-functional evaluation of small bowel using wireless motility capsule and video capsule endoscopy in patients with known or suspected Crohn's disease: pilot study.

PubMed

Yung, Diana; Douglas, Sarah; Hobson, Anthony R; Giannakou, Andry; Plevris, John N; Koulaouzidis, Anastasios

2016-04-01

SmartPill(®) (Given Imaging Corp.,Yoqneam,Israel) is an ingestible, non-imaging capsule that records physiological data including contractions and pH throughout the gastrointestinal tract. There are scarce data looking at SmartPill(®) assessment of patients with known/suspected small-bowel Crohn's Disease (CD). This pilot study aims to investigate feasibility and safety of SmartPill(®) to assess gut motility in this group.  Over 1 year, patients with known/suspected CD, referred for small-bowel capsule endoscopy (SBCE), were invited to participate and 12 were recruited (7 female, 5 male, mean age 44.2 ± 16.6 years). They underwent hydrogen breath test to exclude small-bowel bacterial overgrowth, patency capsule (Agile(®)), and provided stool samples for fecal calprotectin (FC). Patients ingested PillCam(®)SB2 and SmartPill(®) 4 hours apart. Using unpublished data, 33 healthy controls also were identified for the study. P < 0.05 was considered statistically significant. Of the 12 patients enrolled, 10 underwent complete Smartpill(®) examination (1 stomach retention, 1 dropout). Pillcam(®) was complete in 10 (1 dropout, 1 stomach retention). Mean fecal calprotectin was 340 ± 307.71 mcg/g. The study group had longer transit times and lower gut motility index than did the controls. The difference in motility appears to be statistically significant (P < 0.05). Longer transit times for SmartPill(®) (not statistically significant) may have been due to different specifications between the capsules. Limitations included transient Smartpill(®) signal loss (5/10 studies). This is the first pilot to attempt combining SBCE and SmartPill(®) to assess small-bowel CD. Data on motility in CD are scarce. Multimodal information can provide a clearer clinical picture. Despite concerns about capsule retention in CD patients, SmartPill(®) seems safe for use if a patency capsule is employed beforehand.

Neem leaf glycoprotein promotes dual generation of central and effector memory CD8(+) T cells against sarcoma antigen vaccine to induce protective anti-tumor immunity.

PubMed

Ghosh, Sarbari; Sarkar, Madhurima; Ghosh, Tithi; Guha, Ipsita; Bhuniya, Avishek; Saha, Akata; Dasgupta, Shayani; Barik, Subhasis; Bose, Anamika; Baral, Rathindranath

2016-03-01

We have previously shown that Neem Leaf Glycoprotein (NLGP) mediates sustained tumor protection by activating host immune response. Now we report that adjuvant help from NLGP predominantly generates CD44(+)CD62L(high)CCR7(high) central memory (TCM; in lymph node) and CD44(+)CD62L(low)CCR7(low) effector memory (TEM; in spleen) CD8(+) T cells of Swiss mice after vaccination with sarcoma antigen (SarAg). Generated TCM and TEM participated either to replenish memory cell pool for sustained disease free states or in rapid tumor eradication respectively. TCM generated after SarAg+NLGP vaccination underwent significant proliferation and IL-2 secretion following SarAg re-stimulation. Furthermore, SarAg+NLGP vaccination helps in greater survival of the memory precursor effector cells at the peak of the effector response and their maintenance as mature memory cells, in comparison to single modality treatment. Such response is corroborated with the reduced phosphorylation of FOXO in the cytosol and increased KLF2 in the nucleus associated with enhanced CD62L, CCR7 expression of lymph node-resident CD8(+) T cells. However, spleen-resident CD8(+) T memory cells show superior efficacy for immediate memory-to-effector cell conversion. The data support in all aspects that SarAg+NLGP demonstrate superiority than SarAg vaccination alone that benefits the host by rapid effector functions whenever required, whereas, central-memory cells are thought to replenish the memory cell pool for ultimate sustained disease free survival till 60 days following post-vaccination tumor inoculation. Copyright © 2016 Elsevier Ltd. All rights reserved.

The Expenditures for Academic Inpatient Care of Inflammatory Bowel Disease Patients Are Almost Double Compared with Average Academic Gastroenterology and Hepatology Cases and Not Fully Recovered by Diagnosis-Related Group (DRG) Proceeds.

PubMed

Baumgart, Daniel C; le Claire, Marie

2016-01-01

Crohn's disease (CD) and ulcerative colitis (UC) challenge economies worldwide. Detailed health economic data of DRG based academic inpatient care for inflammatory bowel disease (IBD) patients in Europe is unavailable. IBD was identified through ICD-10 K50 and K51 code groups. We took an actual costing approach, compared expenditures to G-DRG and non-DRG proceeds and performed detailed cost center and type accounting to identify coverage determinants. Of all 3093 hospitalized cases at our department, 164 were CD and 157 UC inpatients in 2012. On average, they were 44.1 (CD 44.9 UC 43.3 all 58) years old, stayed 10.1 (CD 11.8 UC 8.4 vs. all 8) days, carried 5.8 (CD 6.4 UC 5.2 vs. all 6.8) secondary diagnoses, received 7.4 (CD 7.7 UC 7 vs. all 6.2) procedures, had a higher cost weight (CD 2.8 UC 2.4 vs. all 1.6) and required more intense nursing. Their care was more costly (means: total cost IBD 8477€ CD 9051€ UC 7903€ vs. all 5078€). However, expenditures were not fully recovered by DRG proceeds (means: IBD 7413€, CD 8441€, UC 6384€ vs all 4758€). We discovered substantial disease specific mismatches in cost centers and types and identified the medical ward personnel and materials budgets to be most imbalanced. Non-DRG proceeds were almost double (IBD 16.1% vs. all 8.2%), but did not balance deficits at total coverage analysis, that found medications (antimicrobials, biologics and blood products), medical materials (mostly endoscopy items) to contribute most to the deficit. DRGs challenge sophisticated IBD care.

Increased Intraepithelial Vα24 Invariant NKT Cells in the Celiac Duodenum

PubMed Central

Montalvillo, Enrique; Bernardo, David; Martínez-Abad, Beatriz; Allegretti, Yessica; Fernández-Salazar, Luis; Calvo, Carmen; Chirdo, Fernando G.; Garrote, José A.; Arranz, Eduardo

2015-01-01

Celiac Disease (CD) is an interferon (IFN)γ-mediated duodenal hypersensitivity to wheat gluten occurring in genetically predisposed individuals. Gluten-free diet (GFD) leads to a complete remission of the disease. Vα24-restricted invariant NKT (iNKT) cells are important to maintain immune homeostasis in the gut mucosa because of their unique capacity to rapidly produce large quantities of both T-helper (Th)1 and Th2 cytokines upon stimulation. We studied the presence of these cells in the CD duodenum. Duodenal biopsies were obtained from 45 untreated-CD patients (uCD), 15 Gluten Free Diet-CD patients (GFD-CD), 44 non-inflamed non-CD controls (C-controls) and 15 inflamed non-CD controls (I-controls). Two populations from Spain and Argentina were recruited. Messenger RNA (mRNA) expression of Vα24-Jα18 (invariant TCRα chain of human iNKT cells), IFNγ and intracellular transcription factor Forkhead Box P3 (Foxp3), and flow cytometry intraepithelial lymphocyte (IEL) profile were determined. Both uCD and GFD-CD patients had higher Vα24-Jα18 mRNA levels than non-CD controls (I and C-controls). The expression of Vα24-Jα18 correlated with Marsh score for the severity of mucosal lesion and also with increased mRNA IFNγ levels. uCD and GFD-CD patients had decreased mRNA expression of FoxP3 but increased expression of Vα24-Jα18, which revealed a CD-like molecular profile. Increased numbers of iNKT cells were confirmed by flow cytometry within the intraepithelial lymphocyte compartment of uCD and GFD-CD patients and correlated with Vα24-Jα18 mRNA expression. In conclusion, we have found an increased number of iNKT cells in the duodenum from both uCD and GFD-CD patients, irrespective of the mucosal status. A CD-like molecular profile, defined by an increased mRNA expression of Vα24-Jα18 together with a decreased expression of FoxP3, may represent a pro-inflammatory signature of the CD duodenum. PMID:26529008

Increased Intraepithelial Vα24 Invariant NKT Cells in the Celiac Duodenum.

PubMed

Montalvillo, Enrique; Bernardo, David; Martínez-Abad, Beatriz; Allegretti, Yessica; Fernández-Salazar, Luis; Calvo, Carmen; Chirdo, Fernando G; Garrote, José A; Arranz, Eduardo

2015-10-30

Celiac Disease (CD) is an interferon (IFN)γ-mediated duodenal hypersensitivity to wheat gluten occurring in genetically predisposed individuals. Gluten-free diet (GFD) leads to a complete remission of the disease. Vα24-restricted invariant NKT (iNKT) cells are important to maintain immune homeostasis in the gut mucosa because of their unique capacity to rapidly produce large quantities of both T-helper (Th)1 and Th2 cytokines upon stimulation. We studied the presence of these cells in the CD duodenum. Duodenal biopsies were obtained from 45 untreated-CD patients (uCD), 15 Gluten Free Diet-CD patients (GFD-CD), 44 non-inflamed non-CD controls (C-controls) and 15 inflamed non-CD controls (I-controls). Two populations from Spain and Argentina were recruited. Messenger RNA (mRNA) expression of Vα24-Jα18 (invariant TCRα chain of human iNKT cells), IFNγ and intracellular transcription factor Forkhead Box P3 (Foxp3), and flow cytometry intraepithelial lymphocyte (IEL) profile were determined. Both uCD and GFD-CD patients had higher Vα24-Jα18 mRNA levels than non-CD controls (I and C-controls). The expression of Vα24-Jα18 correlated with Marsh score for the severity of mucosal lesion and also with increased mRNA IFNγ levels. uCD and GFD-CD patients had decreased mRNA expression of FoxP3 but increased expression of Vα24-Jα18, which revealed a CD-like molecular profile. Increased numbers of iNKT cells were confirmed by flow cytometry within the intraepithelial lymphocyte compartment of uCD and GFD-CD patients and correlated with Vα24-Jα18 mRNA expression. In conclusion, we have found an increased number of iNKT cells in the duodenum from both uCD and GFD-CD patients, irrespective of the mucosal status. A CD-like molecular profile, defined by an increased mRNA expression of Vα24-Jα18 together with a decreased expression of FoxP3, may represent a pro-inflammatory signature of the CD duodenum.

Material properties of Cd1-xMgxO alloys synthesized by radio frequency sputtering

NASA Astrophysics Data System (ADS)

Chen, Guibin; Yu, K. M.; Reichertz, L. A.; Walukiewicz, W.

2013-07-01

We have studied structural, electrical, and optical properties of sputter deposited ternary CdMgO alloy thin films with total Mg concentration as high as 44%. We found that only a fraction (50%-60%) of Mg is incorporated as substitutional Mg contributing to the modification of the electronic structures of the alloys. The electrical and optical results of the Cd1-xMgxO alloys are analyzed in terms of a large upward shift of the conduction band edge with increasing Mg concentration. With the increase of the intrinsic bandgap, appropriately doped Cd-rich CdMgO alloys can be potentially useful as transparent conductors for photovoltaics.

ITK Gene Mutation: Effect on Survival of Children with Severe Hemophagocytic Lymphohistiocytosis.

PubMed

Zheng, Fang; Li, Juan; Zha, Hui; Zhang, Jue; Zhang, Zhiquan; Cheng, Fangjun

2016-11-01

Hemophagocytic lymphohistiocytosis (HLH) is characterized by deadly hyperinflammatory syndrome, but data on severe HLH with multi-organ dysfunction in children are scant. The authors report a retrospective study of 8 cases with severe HLH from a pediatric intensive care unit (PICU) over a 1-y period and found that Epstein barr virus (EBV) -infection was the most common etiology. All patients had genetic analysis, which showed that four patients with EBV -infection had one homozygous mutation, c.985+75G>A (at position chr5:156667232) in exon10 of the ITK gene with poor survival rates. ITK + mutation group had higher percentages of CD3 + CD8 + T cells (36.0 ± 8.4 %) than those in ITK - mutation group (28.8 ± 5.5 %), while they had similar levels of CD3 + CD4 + T cells. ITK + mutation group had lower proportion of CD3 - CD19 + B cells (16.3 ± 2.9 %) and CD16 + CD56 + NK cells (8.4 ± 2.6 %) than ITK - mutation group (29.6 ± 5.1 % and 15.9 ± 9.0 % respectively). Most importantly, patients with EBV infection with c.985+75G>A mutation in ITK had lower survival rates than ITK - mutation group which it may be related with cellular immune dysfunction.

Characterization of hepatic progenitors from human fetal liver during second trimester.

PubMed

Rao, Mekala-Subba; Khan, Aleem-Ahmed; Parveen, Nyamath; Habeeb, Mohammed-Aejaz; Habibullah, Chittoor-Mohammed; Pande, Gopal

2008-10-07

To enrich hepatic progenitors using epithelial cell adhesion molecule (EpCAM) as a marker from human fetal liver and investigate the expression of human leukocyte antigen (HLA) and their markers associated with hepatic progenitor cells. EpCAM +ve cells were isolated using magnetic cell sorting (MACS) from human fetuses (n = 10) at 15-25 wk gestation. Expression of markers for hepatic progenitors such as albumin, alpha-fetoprotein (AFP), CD29 (integrin beta1), CD49f (integrin alpha6) and CD90 (Thy 1) was studied by using flow cytometry, immunocytochemistry and RT-PCR; HLA class I (A, B, C) and class II (DR) expression was studied by flow cytometry only. FACS analysis indicated that EpCAM +ve cells were positive for CD29, CD49f, CD90, CD34, HLA class I, albumin and AFP but negative for HLA class II (DR) and CD45. RT PCR showed that EpCAM +ve cells expressed liver epithelial markers (CK18), biliary specific marker (CK19) and hepatic markers (albumin, AFP). On immunocytochemical staining, EpCAM +ve cells were shown positive signals for CK18 and albumin. Our study suggests that these EpCAM +ve cells can be used as hepatic progenitors for cell transplantation with a minimum risk of alloreactivity and these cells may serve as a potential source for enrichment of hepatic progenitor.

Expansion of PD-1-positive effector CD4 T cells in an experimental model of SLE: contribution to the self-organized criticality theory.

PubMed

Miyazaki, Yumi; Tsumiyama, Ken; Yamane, Takashi; Ito, Mitsuhiro; Shiozawa, Shunichi

2013-04-18

We have developed a systems biology concept to explain the origin of systemic autoimmunity. From our studies of systemic lupus erythematosus (SLE) we have concluded that this disease is the inevitable consequence of over-stimulating the host's immune system by repeated exposure to antigen to levels that surpass a critical threshold, which we term the system's "self-organized criticality". We observed that overstimulation of CD4 T cells in mice led to the development of autoantibody-inducing CD4 T cells (aiCD4 T) capable of generating various autoantibodies and pathological lesions identical to those observed in SLE. We show here that this is accompanied by the significant expansion of a novel population of effector T cells characterized by expression of programmed death-1 (PD-1)-positive, CD27(low), CD127(low), CCR7(low) and CD44(high)CD62L(low) markers, as well as increased production of IL-2 and IL-6. In addition, repeated immunization caused the expansion of CD8 T cells into fully-matured cytotoxic T lymphocytes (CTL) that express Ly6C(high)CD122(high) effector and memory markers. Thus, overstimulation with antigen leads to the expansion of a novel effector CD4 T cell population that expresses an unusual memory marker, PD-1, and that may contribute to the pathogenesis of SLE.

Nutritional status of children with coeliac disease.

PubMed

Aurangzeb, B; Leach, S T; Lemberg, D A; Day, A S

2010-07-01

The main aim of this study was to assess the nutritional status of children with newly diagnosed Coeliac disease (CD)with comparison to matched controls. A further aim was to assess relationships between presentation patterns and nutrition in childhood CD. The nutritional status of newly diagnosed CD was assessed by anthropometry, Bioelectrical Impedance and serum leptin levels, and contrasted to age and gender matched controls. Twenty-five children with CD (mean age of 8.2 +/- 4.5 years) and 25 control children (mean age 8.1 +/- 4.4.) were enrolled. Thirteen (52%) children with CD had gastrointestinal symptoms with 14 having a family history of CD. At presentation 8.7% were wasted, 4.2% were stunted and 20.8% overweight, although none were obese. Mean height and weight for age, other nutritional parameters and serum leptin did not differ between the groups. Serum leptin correlated with BMI in both groups. Children with CD more commonly present with atypical symptoms than with classical features. Variations in nutrition (under to overnutrition) may be seen at diagnosis, without relationship to the presence of symptoms. Leptin levels were not altered specifically in the setting of CD. Nutritional assessment remains important in the assessment and management of CD in children.

Attrition of memory CD8 T cells during sepsis requires LFA-1.

PubMed

Serbanescu, Mara A; Ramonell, Kimberly M; Hadley, Annette; Margoles, Lindsay M; Mittal, Rohit; Lyons, John D; Liang, Zhe; Coopersmith, Craig M; Ford, Mandy L; McConnell, Kevin W

2016-11-01

CD8 T cell loss and dysfunction have been implicated in the increased susceptibility to opportunistic infections during the later immunosuppressive phase of sepsis, but CD8 T cell activation and attrition in early sepsis remain incompletely understood. With the use of a CLP model, we assessed CD8 T cell activation at 5 consecutive time points and found that activation after sepsis results in a distinct phenotype (CD69 + CD25 int CD62L HI ) independent of cognate antigen recognition and TCR engagement and likely through bystander-mediated cytokine effects. Additionally, we observed that sepsis concurrently results in the preferential depletion of a subset of memory-phenotype CD8 T cells that remain "unactivated" (i.e., fail to up-regulate activation markers) by apoptosis. Unactivated CD44 HI OT-I cells were spared from sepsis-induced attrition, as were memory-phenotype CD8 T cells of mice treated with anti-LFA-1 mAb, 1 h after CLP. Perhaps most importantly, we demonstrate that attrition of memory phenotype cells may have a pathologic significance, as elevated IL-6 levels were associated with decreased numbers of memory-phenotype CD8 T cells in septic mice, and preservation of this subset after administration of anti-LFA-1 mAb conferred improved survival at 7 d. Taken together, these data identify potentially modifiable responses of memory-phenotype CD8 T cells in early sepsis and may be particularly important in the application of immunomodulatory therapies in sepsis. © Society for Leukocyte Biology.

Attrition of memory CD8 T cells during sepsis requires LFA-1

PubMed Central

Serbanescu, Mara A.; Ramonell, Kimberly M.; Hadley, Annette; Margoles, Lindsay M.; Mittal, Rohit; Lyons, John D.; Liang, Zhe; Coopersmith, Craig M.; Ford, Mandy L.; McConnell, Kevin W.

2016-01-01

CD8 T cell loss and dysfunction have been implicated in the increased susceptibility to opportunistic infections during the later immunosuppressive phase of sepsis, but CD8 T cell activation and attrition in early sepsis remain incompletely understood. With the use of a CLP model, we assessed CD8 T cell activation at 5 consecutive time points and found that activation after sepsis results in a distinct phenotype (CD69+CD25intCD62LHI) independent of cognate antigen recognition and TCR engagement and likely through bystander-mediated cytokine effects. Additionally, we observed that sepsis concurrently results in the preferential depletion of a subset of memory-phenotype CD8 T cells that remain “unactivated” (i.e., fail to up-regulate activation markers) by apoptosis. Unactivated CD44HI OT-I cells were spared from sepsis-induced attrition, as were memory-phenotype CD8 T cells of mice treated with anti-LFA-1 mAb, 1 h after CLP. Perhaps most importantly, we demonstrate that attrition of memory phenotype cells may have a pathologic significance, as elevated IL-6 levels were associated with decreased numbers of memory-phenotype CD8 T cells in septic mice, and preservation of this subset after administration of anti-LFA-1 mAb conferred improved survival at 7 d. Taken together, these data identify potentially modifiable responses of memory-phenotype CD8 T cells in early sepsis and may be particularly important in the application of immunomodulatory therapies in sepsis. PMID:27286793

A three-dimensional coordination polymer based on 1,2,3-triazole-4,5-dicarboxylic acid (H{sub 3}tda): ([Cd{sub 12}(tda){sub 8}(H{sub 2}O){sub 11}] · (H{sub 2}O)6.25){sub n}

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhou, Xin-Hui, E-mail: iamxhzhou@njupt.edu.cn; Chen, Qiang

The title coordination polymer ([Cd{sub 12}(tda){sub 8}(H{sub 2}O){sub 11}] · (H{sub 2}O){sub 6.25}){sub n} (H{sub 3}tda = 1,2,3-triazole-4,5-dicarboxylic acid), has been hydrothermally synthesized and structurally characterized by single-crystal X-ray diffraction. Complex crystallizes in orthorhombic sp. gr. Pmn2{sub 1} with Z = 4. The Cd{sub 2} unit doublebridged by one carboxylate oxygen atom and two neighboring nitrogen atoms from the tda{sup 3–} ligands are linked by the tda{sup 3–}ligands to lead to the 2D (4,4) network in the ac plane. The almost coplanar Cd{sub 2}(μ{sub 5}-tda){sub 2} unit comprised of two Cd ions double-bridged by two tda{sup 3–} ligands through themore » neighboring nitrogen atoms is connected with the other four Cd{sub 2}(μ{sub 5}-tda){sub 2} units form the undulating 2D network in the ac plane. The (4,4) networks and undulating 2D networks are alternatively connected along the b axis by the tda{sup 3–} ligands coordinating to the Cd ions to form the 3D framework.« less

Multilayer polymer light-emitting diodes by blade coating method

NASA Astrophysics Data System (ADS)

Tseng, Shin-Rong; Meng, Hsin-Fei; Lee, Kuan-Chen; Horng, Sheng-Fu

2008-10-01

Multilayer polymer light-emitting diodes fabricated by blade coating are presented. Multilayer of polymers can be easily deposited by blade coating on a hot plate. The multilayer structure is confirmed by the total thickness and the cross section view in the scanning electron microscope. The film thickness variation is only 3.3% in 10cm scale and the film roughness is about 0.3nm in the micron scale. The efficiency of single layer poly(para-phenylene vinylene) copolymer Super Yellow and poly(9,9-dioctylfluorene) (PFO, deep blue) devices are 9 and 1.7cd/A, respectively, by blade coating. The efficiency of the PFO device is raised to 2.9cd/A with a 2-(4-tert-butylphenyl)-5-(4-biphenylyl)-1,3,4-oxadiazole (PBD) hole-blocking layer and to 2.3cd/A with a poly[(9,9-dioctylfluorenyl-2,7-diyl)-co-(4,4'-(N-(4-sec-butylphenyl))diphenylamine)] elec-tron-blocking layer added by blade coating.

[Expression of c-MPL in leukemic stem cells from acute myeloid leukemia patients].

PubMed

Yu, Pei; Qiu, Shao-Wei; Rao, Qing; Lin, Dong; Xing, Hai-Yan; Tang, Ke-Jing; Tian, Zheng; Wang, Min; Wang, Jian-Xiang

2012-10-01

This study was aimed to investigate the expression of c-MPL in acute myeloid leukemia (AML) and the correlation of the c-MPL expression with CD34 and CD38, so as to define the expression of c-MPL in leukemic stem cells. The expression levels of CD34, CD38 and c-MPL were detected by flow cytometry in bone marrow cells from 29 newly diagnosed AML patients. The relationship of c-MPL positive cell ratio with clinical parameters and correlation of c-MPL with CD34 and CD38 expression in AML patients were analyzed. The results showed that expression level of c-MPL in AML patients was significantly higher than that of normal controls (P < 0.05), and the expression level of c-MPL did not correlate with age, sex, white blood cell count, AML1-ETO fusion gene and remission after chemotherapy, but the expression of c-MPL in M2 and M5 patients was higher than that of normal control (P < 0.05). Expression of c-MPL in CD34 positive AML patients was obviously higher than that in CD34 negative AML patients (P < 0.01). c-MPL was significantly higher expressed in CD34(+) cells than that in CD34(-) cells (P < 0.001), while c-MPL expression was not significantly different between CD34(+)CD38(-) and CD34(+)CD38(-) cell groups. Positive correlation between c-MPL and CD34 expression was observed (r = 0.380, P = 0.042). It is concluded that expression of c-MPL is higher in AML patients, and positively correlates with the expression level of CD34. The c-MPL expresses in leukemic stem cells.

Role of bone marrow-derived CD11c+ dendritic cells in systolic overload-induced left ventricular inflammation, fibrosis and hypertrophy.

PubMed

Wang, Huan; Kwak, Dongmin; Fassett, John; Liu, Xiaohong; Yao, Wu; Weng, Xinyu; Xu, Xin; Xu, Yawei; Bache, Robert J; Mueller, Daniel L; Chen, Yingjie

2017-05-01

Inflammatory responses play an important role in the development of left ventricular (LV) hypertrophy and dysfunction. Recent studies demonstrated that increased T-cell infiltration and T-cell activation contribute to LV hypertrophy and dysfunction. Dendritic cells (DCs) are professional antigen-presenting cells that orchestrate immune responses, especially by modulating T-cell function. In this study, we investigated the role of bone marrow-derived CD11c + DCs in transverse aortic constriction (TAC)-induced LV fibrosis and hypertrophy in mice. We observed that TAC increased the number of CD11c + cells and the percentage of CD11c + MHCII + (major histocompatibility complex class II molecule positive) DCs in the LV, spleen and peripheral blood in mice. Using bone marrow chimeras and an inducible CD11c + DC ablation model, we found that depletion of bone marrow-derived CD11c + DCs significantly attenuated LV fibrosis and hypertrophy in mice exposed to 24 weeks of moderate TAC. CD11c + DC ablation significantly reduced TAC-induced myocardial inflammation as indicated by reduced myocardial CD45 + cells, CD11b + cells, CD8 + T cells and activated effector CD8 + CD44 + T cells in LV tissues. Moreover, pulsing of autologous DCs with LV homogenates from TAC mice promoted T-cell proliferation. These data indicate that bone marrow-derived CD11c + DCs play a maladaptive role in hemodynamic overload-induced cardiac inflammation, hypertrophy and fibrosis through the presentation of cardiac self-antigens to T cells.

Immunophenotype of infiltrating cells in protocol renal allograft biopsies from tacrolimus-versus cyclosporine-treated patients.

PubMed

Serón, Daniel; O'Valle, Francisco; Moreso, Francesc; Gomà, Montse; Hueso, Miguel; Grinyó, Josep M; Garcia del Moral, Raimundo

2007-03-15

The prevalence of subclinical rejection is lower in patients receiving tacrolimus than in patients treated with cyclosporine. However, it is not known whether this difference is related to the modulation of a specific cell immunophenotype. We perform a two case-one control study in patients treated with tacrolimus (n=44) or cyclosporine (n=22) with a protocol biopsy performed at 4 to 6 months. Immunophenotype of infiltrating cells was evaluated with monoclonal antibodies directed against CD45 (all leukocytes), CD3 (T lymphocytes), CD68 (monocytes/macrophages), and CD20 (B lymphocytes) and expressed as interstitial positive cells/mm(2). The number of interstitial CD45 (290+/-209 vs. 696+/-560; P<0.01), CD3 (121+/-84 vs. 208+/-104; P<0.01), and CD68 (155+/-232 vs. 242+/-280; P<0.05) but not CD20 (137+/-119 vs. 197+/-154) positive cells was lower in tacrolimus-treated patients. T lymphocytes and macrophages interstitial infiltration was reduced in tacrolimus treated patients evaluated with protocol biopsies in comparison to cyclosporine-treated patients.

Manganese-induced cadmium stress tolerance in rice seedlings: Coordinated action of antioxidant defense, glyoxalase system and nutrient homeostasis.

PubMed

Rahman, Anisur; Nahar, Kamrun; Hasanuzzaman, Mirza; Fujita, Masayuki

The accumulation of cadmium (Cd) alters different physiological and biochemical attributes that affect plant growth and yield. In our study, we investigated the regulatory role of supplemental manganese (Mn) on hydroponically grown rice (Oryza sativa L. cv. BRRI dhan29) seedlings under Cd-stress conditions. Exposure of 14-d-old seedlings to 0.3mM CdCl 2 for three days caused growth inhibition, chlorosis, nutrient imbalance, and higher Cd accumulation. Higher Cd uptake caused oxidative stress through lipid peroxidation, loss of plasma membrane integrity, and overproduction of reactive oxygen species (ROS) and methylglyoxal (MG). The exogenous application of 0.3mM MnSO 4 to Cd-treated seedlings partly recovered Cd-induced water loss, chlorosis, growth inhibition, and nutrient imbalance by reducing Cd uptake and its further translocation to the upper part of the plant. Supplemental Mn also reduced Cd-induced oxidative damage and lipid peroxidation by improved antioxidant defense and glyoxalase systems through enhancing ROS and MG detoxification, respectively. Copyright © 2016 Académie des sciences. Published by Elsevier SAS. All rights reserved.

Effect of CdTe Back Surface Field on the Efficiency Enhancement of a CGS Based Thin Film Solar Cell

NASA Astrophysics Data System (ADS)

Khattak, Yousaf Hameed; Baig, Faisal; Marí, Bernabé; Beg, Saira; Gillani, Syed Rizwan; Ahmed, Tanveer

2018-05-01

Numerical analysis of the proposed solar cell is based on cadmium telluride (CdTe) and copper gallium sulfide (CuGaS2), also known as CGS, is proposed in this research work. Performance of a CdTe/CGS/CdS/ZnO cell is analyzed in Solar Cell Capacitance Simulator (SCAPS) software, by changing the physical parameters like doping density of acceptor, doping density of donor, absorber thickness and buffer thickness. The cell structure is in the same order as the CGS/CdS/ZnO with CdTe used for the back surface field layer. Power conversion efficiency of the CGS/CdS/ZnO solar cell without CdTe is 10.578% (with FF = 83.70%, V oc = 0.82 V, J sc = 15.40 mA/cm2) and conversion efficiency of CdTe/CGS/CdS/ZnO is 28.20% (with FF = 77.66%, V oc = 1.22 V, J sc = 29.63 mA/cm3). The overall investigation and simulation results from the modeling of a proposed device in SCAPS is very useful for the understanding of the fundamentals of photovoltaic devices and gives feedback to engineers and designers for the fabrication of CdTe/CGS based solar cells.

Isolation and identification of tumor-initiating cell properties in human gallbladder cancer cell lines using the marker cluster of differentiation 133.

PubMed

Yu, Jiwei; Tang, Zhaohui; Gong, Wei; Zhang, Mingdi; Quan, Zhiwei

2017-12-01

The present study aimed to isolate and identify the properties of the cluster of differentiation (CD)133 + subset in human gallbladder cancer cells. The CD133 + and CD133 - subpopulations of the GBC-SD cell line were separated using immunomagnetic separation, and the biological features of the two subpopulations were analyzed in vitro and in vivo . In particular, the present study aimed to determine whether the two subpopulations were resistant to anti-tumor reagents and to identify the underlying molecular mechanisms involved. Following cell sorting of GBC-SD cells using immunomagnetic beads, 90.2±2% of cells were identified as CD133 + . Immunofluorescence confirmed that CD133 was expressed at higher levels in the Cd133 + group compared with the CD133 - group. The proliferation of the CD133 + group was significantly increased compared with the CD133 - group in vitro and in vivo . Following treatment with fluorouracil or gemcitabine, cells in the CD133 + group exhibited a decreased sensitivity to these drugs. The number of transmembrane cells was significantly increased in the CD133 + group compared with the CD133 - group. In addition, the expression levels of ATP binding cassette subfamily G member 2, CD44, C-X-C motif chemokine receptor 4 (CXCR4), phosphorylated-protein kinase B (Akt) and CD133 in the CD133 + group were significantly increased, compared with those in the CD133 - group. In CD133 + GBC-SD cells, stromal cell-derived factor 1α (SDF-1α) or treatment with AMD3100, an inhibitor of CXCR4, promotes or suppresses the SDF-1α/CXCR4 axis, respectively, resulting in increased or decreased CD133 expression through the Akt signaling pathway. Inhibition of the Akt signaling pathway resulted in decreased CD133 expression in GBC-SD cells. Immunomagnetic beads were successfully used for isolation of the CD133 + subset from GBC-SD cells. Furthermore, the CD133 + subset revealed an increased potential for tumor formation, cell proliferation, invasion and resistance to chemotherapeutic agents with expression of stem cell-associated genes. Therefore, in GBC-SD cells, the CXCR4/Akt/CD133 signaling pathways may be activated.

Early elevation of soluble CD14 may help identify trauma patients at high risk for infection.

PubMed

Carrillo, E H; Gordon, L; Goode, E; Davis, E; Polk, H C

2001-05-01

Elevated levels of soluble CD14 (sCD14) have been implicated in both gram-positive and gram-negative sepsis, and it has been associated with high mortality in trauma patients who become infected. Eleven healthy volunteers and 25 adult trauma patients with multiple injuries and a mean Injury Severity Score of 32 participated. Whole blood was obtained at intervals. Immunohistochemistry was used to quantify membrane CD14 (mCD14), by flow cytometry and plasma levels of sCD14 by enzyme-linked immunosorbent assay. Analysis of variance and Student's T test with Mann-Whitney posttest were used to determine significance at p < 0.05. On posttrauma day 1, sCD14 was significantly different in the plasma of infected patients compared with normal controls (7.16 +/- 1.87 microg/mL vs. 4.4 +/- 0.92 microg/mL, p < 0.01), but not significantly different from noninfected patients. The percentage of monocytes expressing mCD14 in trauma patients did not differentiate them from normal controls; however, mCD14 receptor density did demonstrate significance in septic trauma patients (n = 15) versus normal controls on posttrauma day 3 (p = 0.0065). On the basis of our data, mCD14 did not differentiate infected and noninfected trauma patients, although trauma in general reduced mCD14 and elevated sCD14. Interestingly, 100% of patients who exceeded plasma levels of 8 microg/mL of sCD14 on day 1 after injury developed infections. Therefore, early high expressers of sCD14 may be at higher risk for infectious complications after trauma.

Task-shifting of CD4 T cell count monitoring by the touchscreen-based Muse™ Auto CD4/CD4% single-platform system for CD4 T cell numeration: Implication for decentralization in resource-constrained settings.

PubMed

Kouabosso, André; Mossoro-Kpinde, Christian Diamant; Bouassa, Ralph-Sydney Mboumba; Longo, Jean De Dieu; Mbeko Simaleko, Marcel; Grésenguet, Gérard; Bélec, Laurent

2018-04-01

The accuracy of CD4 T cell monitoring by the recently developed flow cytometry-based CD4 T cell counting Muse™ Auto CD4/CD4% Assay analyzer (EMD Millipore Corporation, Merck Life Sciences, KGaA, Darmstadt, Germany) was evaluated in trained lay providers against laboratory technicians. After 2 days of training on the Muse™ Auto CD4/CD4% analyzer, EDTA-blood samples from 6 HIV-positive and 4 HIV-negative individuals were used for CD4 T cell counting in triplicate in parallel by 12 trained lay providers as compared to 10 lab technicians. Mean number of CD4 T cells in absolute number was 829 ± 380 cells/μl by lay providers and 794 ± 409 cells/μl by technicians (P > 0.05); and in percentage 36.2 ± 14.8%CD4 by lay providers and 36.1 ± 15.0%CD4 by laboratory technician (P > 0.05). The unweighted linear regression and Passing-Bablok regression analyses on CD4 T cell results expressed in absolute count revealed moderate correlation between CD4 T cell counts obtained by lay providers and lab technicians. The mean absolute bias measured by Bland-Altman analysis between CD4 T cell/μl obtained by lay providers and lab technicians was -3.41 cells/μl. Intra-assay coefficient of variance (CV) of Muse™ Auto CD4/CD4% in absolute number was 10.1% by lay providers and 8.5% by lab technicians (P > 0.05), and in percentage 5.5% by lay providers and 4.4% by lab technicians (P > 0.05). The inter-assay CV of Muse™ Auto CD4/CD4% in absolute number was 13.4% by lay providers and 10.3% by lab technicians (P > 0.05), and in percentage 7.8% by lay providers and 6.9% by lab technicians (P > 0.05). The study demonstrates the feasibility of CD4 T cell counting using the alternative flow cytometer Muse™ Auto CD4/CD4% analyzer by trained lay providers and therefore the practical possibility of decentralization CD4 T cell counting to health community centers. Copyright © 2018. Published by Elsevier B.V.

Over-expression of CD8+ T-cell activation is associated with decreased CD4+ cells in patients seeking treatment of Alcohol Use Disorder.

PubMed

Zuluaga, Paola; Sanvisens, Arantza; Martínez-Cáceres, Eva; Teniente, Aina; Tor, Jordi; Muga, Robert

2017-11-01

Harmful alcohol consumption may have an impact on the adaptive immune system through an imbalance in T cell subpopulations and changes in cell activation. We aimed to analyze profiles of CD4 and CD8T cell activation in patients with alcohol use disorder (AUD). We used a cross-sectional study with patients seeking treatment of the disorder. Blood samples for immunophenotyping were obtained at admission. Profiles of T cell activation were defined: (I) CD38 + /HLA-DR + , (II) CD38 + /HLA-DR - , (III) CD38 - /HLA-DR + , (IV) CD38 - /HLA-DR - and compared with healthy controls. We calculated a CD8 + T cell activation indicator (AI) that was defined as the quotient of non-activated cells (CD38 - /HLA-DR - ) and activated cells (CD38 + /HLA-DR + ). 60 patients were eligible (83%M); median age was 49 years [IQR: 44-54] and alcohol consumption was 145g/day [IQR: 90-205]. Mean±SD of CD38 + /HLA-DR - was 50.3±50.6 cells/μL in patients and 33.5±24.5 cells/μL in controls (p=0.03), for the CD38 - /HLA-DR + it was 61±62.2 cells/μL in patients and 21.2±17.3 cells/μL in controls (p<0.001) and for the CD38 + /HLA-DR + it was 20.2±15.6 cells/μL in patients and 10.8±10.3 cells/μL in controls (p<0.001). In patients, an inverse correlation was observed between absolute number and percentage of CD4 + T cells, and the percentage of CD38 + /HLA-DR + CD8 + T cells (r=0.37, p=0.003; r=0.2, p=0.086, respectively). Patients with AUD have an increased expression of immune activation with respect to healthy individuals. This excess of activated CD8 + T cells correlates with the absolute CD4 + T cells. Copyright © 2017 Elsevier B.V. All rights reserved.

Vitamin D increases programmed death receptor-1 expression in Crohn’s disease

PubMed Central

Bendix, Mia; Greisen, Stinne; Dige, Anders; Hvas, Christian L.; Bak, Nina; Jørgensen, Søren P.; Dahlerup, Jens F.; Deleuran, Bent; Agnholt, Jørgen

2017-01-01

Background: Vitamin D modulates inflammation in Crohns disease (CD). Programmed death (PD)-1 receptor contributes to the maintenance of immune tolerance. Vitamin D might modulate PD-1 signalling in CD. Aim: To investigate PD-1 expression on T cell subsets in CD patients treated with vitamin D or placebo. Methods: We included 40 CD patients who received 1200 IU vitamin D3 for 26 weeks or placebo and eight healthy controls. Peripheral blood mononuclear cells (PBMCs) and plasma were isolated at baseline and week 26. The expressions of PD-1, PD-L1, and surface activation markers were analysed by flow cytometry. Soluble PD-1 plasma levels were measured by ELISA. Results: PD-1 expression upon T cell stimulation was increased in CD4+CD25+int T cells in vitamin D treated CD patients from 19% (range 10 39%) to 29% (11 79%)(p = 0.03) compared with placebo-treated patients. Vitamin D treatment, but not placebo, decreased the expression of the T cell activation marker CD69 from 42% (31 62%) to 33% (19 - 54%)(p = 0.01). Soluble PD-1 levels were not influenced by vitamin D treatment. Conclusions: Vitamin D treatment increases CD4+CD25+int T cells ability to up-regulate PD-1 in response to activation and reduces the CD69 expression in CD patients. PMID:28412753

Synergetic effects of DA-6/GA₃ with EDTA on plant growth, extraction and detoxification of Cd by Lolium perenne.

PubMed

He, Shanying; Wu, Qiuling; He, Zhenli

2014-12-01

Research is needed to improve efficiency of phytoextraction of heavy metals from contaminated soils. A pot experiment was carried out to study the effects of plant growth regulators (PGRs) (diethyl aminoethyl hexanoate (C18H33NO8, DA-6) and gibberellic acid 3 (C19H22O6, GA3)) and/or EDTA on Cd extraction, subcellular distribution and chemical forms in Lolium perenne. The addition of EDTA or PGRs significantly enhanced Cd extraction efficiency (P<0.05), with the decreasing order of: 1 μM DA-6>10 μM DA-6>10 μM GA3>2.5 mmol kg(-1) EDTA>other treatments of PGR alone. PGRs+EDTA resulted in a further increase in Cd extraction efficiency, with EDTA+1 μM DA-6 being the most efficient. At the subcellular level, about 44-57% of Cd was soluble fraction, 18-44% in cell walls, and 12-25% in cellular organelles fraction. Chemical speciation analysis showed that 40-54% of Cd was NaCl extractable, 7-23% HAc extractable, followed by other fractions. EDTA increased the proportions of Cd in soluble and cellular organelles fraction, as well as the metal migration in shoot; therefore, the toxicity to plant increased and plant growth was inhibited. Conversely, PGRs fixed more Cd in cell walls and reduced Cd migration in shoot; thus, metal toxicity was reduced. In addition, PGRs promoted plant biomass growth significantly (P<0.05), with 1 μM DA-6 being the most effective. A combination of DA-6/GA3 with EDTA can alleviate the adverse effect of EDTA on plant growth, and the treatment of EDTA+1 μM DA-6 appears to be optimal for improving the remediation efficiency of L. perenne for Cd contaminated soil. Copyright © 2014 Elsevier Ltd. All rights reserved.

miR-375 induces human decidua basalis-derived stromal cells to become insulin-producing cells.

PubMed

Shaer, Anahita; Azarpira, Negar; Vahdati, Akbar; Karimi, Mohammad Hosein; Shariati, Mehrdad

2014-09-01

This paper focuses on the development of renewable sources of isletreplacement tissue for the treatment of type I diabetes mellitus. Placental tissue-derived mesenchymal stem cells (MSCs) are a promising source for regenerative medicine due to their plasticity and easy availability. They have the potential to differentiate into insulin-producing cells. miR-375 is a micro RNA that is expressed in the pancreas and involved in islet development. Human placental decidua basalis MSCs (PDB-MSCs) were cultured from full-term human placenta. The immunophenotype of the isolated cells was checked for CD90, CD105, CD44, CD133 and CD34 markers. The MSCs (P3) were chemically transfected with hsa-miR-375. Total RNA was extracted 4 and 6 days after transfection. The expressions of insulin, NGN3, GLUT2, PAX4, PAX6, KIR6.2, NKX6.1, PDX1, and glucagon genes were evaluated using real-time qPCR. On day 6, we tested the potency of the clusters in response to the high glucose challenge and assessed the presence of insulin and NGN3 proteins via immunocytochemistry. Flow cytometry analysis confirmed that more than 90% of the cells were positive for CD90, CD105 and CD44 and negative for CD133 and CD34. Morphological changes were followed from day 2. Cell clusters formed during day 6. Insulin-producing clusters showed a deep red color with DTZ. The expression of pancreatic-specific transcription factors increased remarkably during the four days after transfection and significantly increased on day 7. The clusters were positive for insulin and NGN3 proteins, and C-peptide and insulin secretion increased in response to changes in the glucose concentration (2.8 mM and 16.7 mM). In conclusion, the MSCs could be programmed into functional insulin-producing cells by transfection of miR-375.

Mass Cytometry Identifies Distinct Lung CD4+ T Cell Patterns in Löfgren’s Syndrome and Non-Löfgren’s Syndrome Sarcoidosis

PubMed Central

Kaiser, Ylva; Lakshmikanth, Tadepally; Chen, Yang; Mikes, Jaromir; Eklund, Anders; Brodin, Petter; Achour, Adnane; Grunewald, Johan

2017-01-01

Sarcoidosis is a granulomatous disorder of unknown etiology, characterized by accumulation of activated CD4+ T cells in the lungs. Disease phenotypes Löfgren’s syndrome (LS) and “non-LS” differ in terms of clinical manifestations, genetic background, HLA association, and prognosis, but the underlying inflammatory mechanisms largely remain unknown. Bronchoalveolar lavage fluid cells from four HLA-DRB1*03+ LS and four HLA-DRB1*03− non-LS patients were analyzed by mass cytometry, using a panel of 33 unique markers. Differentially regulated CD4+ T cell populations were identified using the Citrus algorithm, and t-stochastic neighborhood embedding was applied for dimensionality reduction and single-cell data visualization. We identified 19 individual CD4+ T cell clusters differing significantly in abundance between LS and non-LS patients. Seven clusters more frequent in LS patients were characterized by significantly higher expression of regulatory receptors CTLA-4, PD-1, and ICOS, along with low expression of adhesion marker CD44. In contrast, 12 clusters primarily found in non-LS displayed elevated expression of activation and effector markers HLA-DR, CD127, CD39, as well as CD44. Hierarchical clustering further indicated functional heterogeneity and diverse origins of T cell receptor Vα2.3/Vβ22-restricted cells in LS. Finally, a near-complete overlap of CD8 and Ki-67 expression suggested larger influence of CD8+ T cell activity on sarcoid inflammation than previously appreciated. In this study, we provide detailed characterization of pulmonary T cells and immunological parameters that define separate disease pathways in LS and non-LS. With direct association to clinical parameters, such as granuloma persistence, resolution, or chronic inflammation, these results provide a valuable foundation for further exploration and potential clinical application. PMID:28955342