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.

Identification of Multipotent Stem/Progenitor Cells in Murine Sclera

PubMed Central

Tsai, Chia-Ling; Wu, Pei-Chang; Fini, M. Elizabeth; Shi, Songtao

2011-01-01

Purpose. The sclera forms the fibrous outer coat of the eyeball and acts as a supportive framework. The purpose of this study was to examine whether the sclera contains mesenchymal stem/progenitor cells. Method. Scleral tissue from C57BL6/J mice was separated from the retina and choroid and subsequently enzyme digested to release single cells. Proliferation capacity, self-renewal capacity, and ability for multipotent differentiation were analyzed by BrdU labeling, flow cytometry, reverse transcriptase–polymerase chain reaction, immunocytochemistry, and in vivo transplantation. Results. The scleral stem/progenitor cells (SSPCs) possessed clonogenic and high doubling capacities. These cells were positive for the mesenchymal markers Sca-1, CD90.2, CD44, CD105, and CD73 and negative for the hematopoietic markers CD45, CD11b, Flk1, CD34, and CD117. In addition to expressing stem cell genes ABCG2, Six2, Notch1, and Pax6, SSPCs were able to differentiate to adipogenic, chondrogenic, and neurogenic lineages. Conclusions. This study indicates that the sclera contains multipotent mesenchymal stem cells. Further study of SSPCs may help elucidate the cellular and molecular mechanism of scleral diseases such as scleritis and myopia. PMID:21788434

Long-Term Cultured Human Term Placenta-Derived Mesenchymal Stem Cells of Maternal Origin Displays Plasticity

PubMed Central

Sabapathy, Vikram; Ravi, Saranya; Srivastava, Vivi; Srivastava, Alok; Kumar, Sanjay

2012-01-01

Mesenchymal stem cells (MSCs) are an alluring therapeutic resource because of their plasticity, immunoregulatory capacity and ease of availability. Human BM-derived MSCs have limited proliferative capability, consequently, it is challenging to use in tissue engineering and regenerative medicine applications. Hence, placental MSCs of maternal origin, which is one of richest sources of MSCs were chosen to establish long-term culture from the cotyledons of full-term human placenta. Flow analysis established bonafied MSCs phenotypic characteristics, staining positively for CD29, CD73, CD90, CD105 and negatively for CD14, CD34, CD45 markers. Pluripotency of the cultured MSCs was assessed by in vitro differentiation towards not only intralineage cells like adipocytes, osteocytes, chondrocytes, and myotubules cells but also translineage differentiated towards pancreatic progenitor cells, neural cells, and retinal cells displaying plasticity. These cells did not significantly alter cell cycle or apoptosis pattern while maintaining the normal karyotype; they also have limited expression of MHC-II antigens and are Naive for stimulatory factors CD80 and CD 86. Further soft agar assays revealed that placental MSCs do not have the ability to form invasive colonies. Taking together all these characteristics into consideration, it indicates that placental MSCs could serve as good candidates for development and progress of stem-cell based therapeutics. PMID:22550499

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.

Comparative Analysis of Cellular and Growth Factor Composition in Bone Marrow Aspirate Concentrate and Platelet-Rich Plasma.

PubMed

Sugaya, Hisashi; Yoshioka, Tomokazu; Kato, Toshiki; Taniguchi, Yu; Kumagai, Hiroshi; Hyodo, Kojiro; Ohneda, Osamu; Yamazaki, Masashi; Mishima, Hajime

2018-01-01

The purpose of this study was to quantify the stem cell and growth factor (GF) contents in the bone marrow aspirate concentrate (BMAC) and platelet-rich plasma (PRP) prepared from whole blood using a protocol established in our laboratory. We examined 10 patients with osteonecrosis of the femoral head who were treated by autologous BMAC transplantation at our hospital between January 2015 and June 2015. We quantified CD34+ and CD31-CD45-CD90+CD105+ cells in BMAC and PRP by flow cytometry. Additionally, we measured various GFs, that is, basic fibroblast growth factor (b-FGF), platelet-derived growth factor-BB (PDGF-BB), vascular endothelial growth factor (VEGF), transforming growth factor- β 1 (TGF- β 1), and bone morphogenetic protein-2 (BMP-2) in BMAC and PRP using enzyme-linked immunosorbent assays and statistical analyses. CD34+ and CD31-45-90+105+ cells accounted for approximately 1.9% and 0.03% of cells in BMAC and no cells in PRP. The concentration of b-FGF was higher in BMAC than in PRP ( P < 0.001), whereas no significant differences in the levels of PDGF-BB, VEGF, TGF- β 1, and BMP-2 were observed between the two types of sample. BMAC had an average of 1.9% CD34+ and 0.03% CD31-45-90+105+ cells and higher levels of b-FGF than those of PRP.

Endothelium trans differentiated from Wharton's jelly mesenchymal cells promote tissue regeneration: potential role of soluble pro-angiogenic factors.

PubMed

Aguilera, Valeria; Briceño, Luis; Contreras, Hector; Lamperti, Liliana; Sepúlveda, Esperanza; Díaz-Perez, Francisca; León, Marcelo; Veas, Carlos; Maura, Rafael; Toledo, Jorge Roberto; Fernández, Paulina; Covarrubias, Ambart; Zuñiga, Felipe Andrés; Radojkovic, Claudia; Escudero, Carlos; Aguayo, Claudio

2014-01-01

Mesenchymal stem cells have a high capacity for trans-differentiation toward many adult cell types, including endothelial cells. Feto-placental tissue, such as Wharton's jelly is a potential source of mesenchymal stem cells with low immunogenic capacity; make them an excellent source of progenitor cells with a potential use for tissue repair. We evaluated whether administration of endothelial cells derived from mesenchymal stem cells isolated from Wharton's jelly (hWMSCs) can accelerate tissue repair in vivo. Mesenchymal stem cells were isolated from human Wharton's jelly by digestion with collagenase type I. Endothelial trans-differentiation was induced for 14 (hWMSC-End14d) and 30 (hWMSC-End30d) days. Cell phenotyping was performed using mesenchymal (CD90, CD73, CD105) and endothelial (Tie-2, KDR, eNOS, ICAM-1) markers. Endothelial trans-differentiation was demonstrated by the expression of endothelial markers and their ability to synthesize nitric oxide (NO). hWMSCs can be differentiated into adipocytes, osteocytes, chondrocytes and endothelial cells. Moreover, these cells show high expression of CD73, CD90 and CD105 but low expression of endothelial markers prior to differentiation. hWMSCs-End express high levels of endothelial markers at 14 and 30 days of culture, and also they can synthesize NO. Injection of hWMSC-End30d in a mouse model of skin injury significantly accelerated wound healing compared with animals injected with undifferentiated hWMSC or injected with vehicle alone. These effects were also observed in animals that received conditioned media from hWMSC-End30d cultures. These results demonstrate that mesenchymal stem cells isolated from Wharton's jelly can be cultured in vitro and trans-differentiated into endothelial cells. Differentiated hWMSC-End may promote neovascularization and tissue repair in vivo through the secretion of soluble pro-angiogenic factors.

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

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.

Tissue Source and Cell Expansion Condition Influence Phenotypic Changes of Adipose-Derived Stem Cells

PubMed Central

Mangum, Lauren H.; Stone, Randolph; Wrice, Nicole L.; Larson, David A.; Florell, Kyle F.; Christy, Barbara A.; Herzig, Maryanne C.; Cap, Andrew P.

2017-01-01

Stem cells derived from the subcutaneous adipose tissue of debrided burned skin represent an appealing source of adipose-derived stem cells (ASCs) for regenerative medicine. Traditional tissue culture uses fetal bovine serum (FBS), which complicates utilization of ASCs in human medicine. Human platelet lysate (hPL) is one potential xeno-free, alternative supplement for use in ASC culture. In this study, adipogenic and osteogenic differentiation in media supplemented with 10% FBS or 10% hPL was compared in human ASCs derived from abdominoplasty (HAP) or from adipose associated with debrided burned skin (BH). Most (95–99%) cells cultured in FBS were stained positive for CD73, CD90, CD105, and CD142. FBS supplementation was associated with increased triglyceride content and expression of adipogenic genes. Culture in hPL significantly decreased surface staining of CD105 by 31% and 48% and CD142 by 27% and 35% in HAP and BH, respectively (p < 0.05). Culture of BH-ASCs in hPL also increased expression of markers of osteogenesis and increased ALP activity. These data indicate that application of ASCs for wound healing may be influenced by ASC source as well as culture conditions used to expand them. As such, these factors must be taken into consideration before ASCs are used for regenerative purposes. PMID:29138638

Characterization and In Vivo Testing of Mesenchymal Stem Cells Derived from Human Embryonic Stem Cells

PubMed Central

Gruenloh, William; Kambal, Amal; Sondergaard, Claus; McGee, Jeannine; Nacey, Catherine; Kalomoiris, Stefanos; Pepper, Karen; Olson, Scott; Fierro, Fernando

2011-01-01

Mesenchymal stem cells (MSCs) have been shown to contribute to the recovery of tissues through homing to injured areas, especially to hypoxic, apoptotic, or inflamed areas and releasing factors that hasten endogenous repair. In some cases genetic engineering of the MSC is desired, since they are excellent delivery vehicles. We have derived MSCs from the human embryonic stem cell (hESC) line H9 (H9-MSCs). They expressed CD105, CD90, CD73, and CD146, and lacked expression of CD45, CD34, CD14, CD31, and HLA-DR, the hESC pluripotency markers SSEA-4 and Tra-1-81, and the hESC early differentiation marker SSEA-1. Marrow-derived MSCs showed a similar phenotype. H9-MSCs did not form teratoma in our initial studies, whereas the parent H9 line did so robustly. H9-MSCs differentiated into bone, cartilage, and adipocytes in vitro, and displayed increased migration under hypoxic conditions. Finally, using a hindlimb ischemia model, H9-MSCs were shown to home to the hypoxic muscle, but not the contralateral limb, by 48 h after IV injection. In summary, we have defined methods for differentiation of hESCs into MSCs and have defined their characteristics and in vivo migratory properties. PMID:21275830

Analysis for apoptosis and necrosis on adipocytes, stromal vascular fraction, and adipose-derived stem cells in human lipoaspirates after liposuction.

PubMed

Wang, Wei Z; Fang, Xin-Hua; Williams, Shelley J; Stephenson, Linda L; Baynosa, Richard C; Wong, Nancy; Khiabani, Kayvan T; Zamboni, William A

2013-01-01

Adipose-derived stem cells have become the most studied adult stem cells. The authors examined the apoptosis and necrosis rates for adipocyte, stromal vascular fraction, and adipose-derived stem cells in fresh human lipoaspirates. Human lipoaspirate (n = 8) was harvested using a standard liposuction technique. Stromal vascular fraction cells were separated from adipocytes and cultured to obtain purified adipose-derived stem cells. A panel of stem cell markers was used to identify the surface phenotypes of cultured adipose-derived stem cells. Three distinct stem cell subpopulations (CD90/CD45, CD105/CD45, and CD34/CD31) were selected from the stromal vascular fraction. Apoptosis and necrosis were determined by annexin V/propidium iodide assay and analyzed by flow cytometry. The cultured adipose-derived stem cells demonstrated long-term proliferation and differentiation evidenced by cell doubling time and positive staining with oil red O and alkaline phosphatase. Isolated from lipoaspirates, adipocytes exhibited 19.7 ± 3.7 percent apoptosis and 1.1 ± 0.3 percent necrosis; stromal vascular fraction cells revealed 22.0 ± 6.3 percent of apoptosis and 11.2 ± 1.9 percent of necrosis; stromal vascular fraction cells had a higher rate of necrosis than adipocytes (p < 0.05). Among the stromal vascular fraction cells, 51.1 ± 3.7 percent expressed CD90/CD45, 7.5 ± 1.0 percent expressed CD105/CD45, and 26.4 ± 3.8 percent expressed CD34/CD31. CD34/CD31 adipose-derived stem cells had lower rates of apoptosis and necrosis compared with CD105/CD45 adipose-derived stem cells (p < 0.05). Adipose-derived stem cells had a higher rate of apoptosis and necrosis than adipocytes. However, the extent of apoptosis and necrosis was significantly different among adipose-derived stem cell subpopulations.

Tissues from equine cadaver ligaments up to 72 hours of post-mortem: a promising reservoir of stem cells.

PubMed

Shikh Alsook, Mohamad Khir; Gabriel, Annick; Piret, Joëlle; Waroux, Olivier; Tonus, Céline; Connan, Delphine; Baise, Etienne; Antoine, Nadine

2015-12-18

Mesenchymal stem cells (MSCs) harvested from cadaveric tissues represent a promising approach for regenerative medicine. To date, no study has investigated whether viable MSCs could survive in cadaveric tissues from tendon or ligament up to 72 hours of post-mortem. The purpose of the present work was to find out if viable MSCs could survive in cadaveric tissues from adult equine ligaments up to 72 hours of post-mortem, and to assess their ability (i) to remain in an undifferentiated state and (ii) to divide and proliferate in the absence of any specific stimulus. MSCs were isolated from equine cadaver (EC) suspensory ligaments within 48-72 hours of post-mortem. They were evaluated for viability, proliferation, capacity for tri-lineage differentiation, expression of cell surface markers (CD90, CD105, CD73, CD45), pluripotent transcription factor (OCT-4), stage-specific embryonic antigen-1 (SSEA-1), neuron-specific class III beta-tubulin (TUJ-1), and glial fibrillary acidic protein (GFAP). As well, they were characterized by transmission electron microscope (TEM). EC-MSCs were successfully isolated and maintained for 20 passages with high cell viability and proliferation. Phase contrast microscopy revealed that cells with fibroblast-like appearance were predominant in the culture. Differentiation assays proved that EC-MSCs are able to differentiate towards mesodermal lineages (osteogenic, adipogenic, chondrogenic). Flow cytometry analysis demonstrated that EC-MSCs expressed CD90, CD105, and CD73, while being negative for the leukocyte common antigen CD45. Immunofluorescence analysis showed a high percentage of positive cells for OCT-4 and SSEA-1. Surprisingly, in absence of any stimuli, some adherent cells closely resembling neuronal and glial morphology were also observed. Interestingly, our results revealed that approximately 15 % of the cell populations were TUJ-1 positive, whereas GFAP expression was detected in only a few cells. Furthermore, TEM analysis confirmed the stemness of EC-MSCs and identified some cells with a typical neuronal morphology. Our findings raise the prospect that the tissues harvested from equine ligaments up to 72 hours of post-mortem represent an available reservoir of specific stem cells. EC-MSCs could be a promising alternative source for tissue engineering and stem cell therapy in equine medicine.

Characterization and Classification of Mesenchymal Stem Cells in Several Species Using Surface Markers for Cell Therapy Purposes.

PubMed

Ghaneialvar, Hori; Soltani, Leila; Rahmani, Hamid Reza; Lotfi, Abbas Sahebghadam; Soleimani, Masoud

2018-01-01

Mesenchymal stem cells are multipotent cells capable of replicating as undifferentiated cells, and have the potential of differentiating into mesenchymal tissue lineages such as osteocytes, adipocytes and chondrocytes. Such lineages can then be used in cell therapy. The aim of present study was to characterize bone marrow derived mesenchymal stem cells in four different species, including: sheep, goat, human and mouse. Human bone-marrow mesenchymal stem cells were purchased, those of sheep and goat were isolated from fetal bone marrow, and those of mouse were collected by washing bone cavity of femur and tibia with DMEM/F12. Using flow-cytometry, they were characterized by CD surface antigens. Furthermore, cells of third passage were examined for their osteogenic and adipogenic differentiation potential by oil red and alizarin red staining respectively. According to the results, CD markers studied in the four groups of mesenchymal stem cells showed a different expression. Goat and sheep expressed CD44 and CD166, and weakly expressed CD34, CD45, CD105 and CD90. Similarly, human and mouse mesenchymal cells expressed CD44, CD166, CD105 and CD90 whereas the expression of CD34 and CD45 was negative. In conclusion, although all mesenchymal stem cells display plastic adherence and tri-lineage differentiation, not all express the same panel of surface antigens described for human mesenchymal stem cells. Additional panel of CD markers are necessary to characterize regenerative potential and possible application of these stem cells in regenerative medicine and implantology.

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.

Evaluation of Bone Marrow Processing Protocol for Therapeutic Applications via Culture and Characterization of Mesenchymal Stem Cells.

PubMed

Verma, Poonam; Bansal, Himanshu; Agrawal, Anupama; Leon, Jerry; Sundell, I Birgitta; Koka, Prasad S

Human mesenchymal stem cells from bone marrow (hMSCs) have broad therapeutic potential. These cells can be are readily isolated from bone marrow by their property to adhere to tissue culture treated culture wares. However, the proliferation rates and other properties of the cells gradually change during expansion. This study aims to validate the protocol of isolation and differentiation of hMSCs from bone marrow for therapeutic applications. Sixty ml of bone marrow was extracted from 5 patients and MSCs were isolated. These were characterized by Flow Cytometry, CFU assay and were differentiated into bone, fat cells and neurocytes. The cells were having healthy morphology. These were positive for the markers CD105, CD90 and CD73 and negative for CD45, CD34 and HLA-DR. The cells could differentiate into fat, bone and neural cells. MSCs from the bone marrow were isolated and differentiated. These cells were morphologically healthy and passed CFU assay. The cells exhibited differentiation potential into bone, fat and neural tissue. These cells can be used in therapeutic applications.

Mesenchymal stem cell like (MSCl) cells generated from human embryonic stem cells support pluripotent cell growth

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

Varga, Nora; Vereb, Zoltan; Rajnavoelgyi, Eva

2011-10-28

Highlights: Black-Right-Pointing-Pointer MSC like cells were derived from hESC by a simple and reproducible method. Black-Right-Pointing-Pointer Differentiation and immunosuppressive features of MSCl cells were similar to bmMSC. Black-Right-Pointing-Pointer MSCl cells as feeder cells support the undifferentiated growth of hESC. -- Abstract: Mesenchymal stem cell like (MSCl) cells were generated from human embryonic stem cells (hESC) through embryoid body formation, and isolated by adherence to plastic surface. MSCl cell lines could be propagated without changes in morphological or functional characteristics for more than 15 passages. These cells, as well as their fluorescent protein expressing stable derivatives, efficiently supported the growth ofmore » undifferentiated human embryonic stem cells as feeder cells. The MSCl cells did not express the embryonic (Oct4, Nanog, ABCG2, PODXL, or SSEA4), or hematopoietic (CD34, CD45, CD14, CD133, HLA-DR) stem cell markers, while were positive for the characteristic cell surface markers of MSCs (CD44, CD73, CD90, CD105). MSCl cells could be differentiated toward osteogenic, chondrogenic or adipogenic directions and exhibited significant inhibition of mitogen-activated lymphocyte proliferation, and thus presented immunosuppressive features. We suggest that cultured MSCl cells can properly model human MSCs and be applied as efficient feeders in hESC cultures.« less

Simultaneous isolation of vascular endothelial cells and mesenchymal stem cells from the human umbilical cord.

PubMed

Kadam, Sachin S; Tiwari, Shubha; Bhonde, Ramesh R

2009-01-01

The umbilical cord represents the link between mother and fetus during pregnancy. This cord is usually discarded as a biological waste after the child's birth; however, its importance as a "store house" of stem cells has been explored recently. We developed a method of simultaneous isolation of endothelial cells (ECs) from the vein and mesenchymal stem cells from umbilical cord Wharton's jelly of the same cord. The isolation protocol has been simplified, modified, and improvised with respect to choice of enzyme and enzyme mixture, digestion time, cell yield, cell growth, and culture medium. Isolated human umbilical vascular ECs (hUVECs) were positive for von-Willibrand factor, a classical endothelial marker, and could form capillary-like structures when seeded on Matrigel, thus proving their functionality. The isolated human umbilical cord mesenchymal stem cells (hUCMSCs) were found positive for CD44, CD90, CD 73, and CD117 and were found negative for CD33, CD34, CD45, and CD105 surface markers; they were also positive for cytoskeleton markers of smooth muscle actin and vimentin. The hUCMSCs showed multilineage differentiation potential and differentiated into adipogenic, chondrogenic, osteogenic, and neuronal lineages under influence of lineage specific differentiation medium. Thus, isolating endothelial cells as well as mesenchymal cells from the same umbilical cord could lead to complete utilization of the available tissue for the tissue engineering and cell therapy.

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.

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

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

2008-12-01

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. 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. 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. 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.

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.

Human adipose-derived stem cells (ADSC) and human periodontal ligament stem cells (PDLSC) as cellular substrates of a toxicity prediction assay.

PubMed

Corrêa, Natássia Caroline Resende; Kuligovski, Crisciele; Paschoal, Ariane Caroline Campos; Abud, Ana Paula Ressetti; Rebelatto, Carmen Lucia Kuniyoshi; Leite, Lidiane Maria Boldrini; Senegaglia, Alexandra Cristina; Dallagiovanna, Bruno; Aguiar, Alessandra Melo de

2018-02-01

With the increasing need to develop in vitro assays to replace animal use, human stem cell-derived methods are emerging and showing outstanding contributions to the toxicological screening of substances. Adult human stem cells such as adipose-derived stem cells (ADSC) and periodontal ligament stem cells (PDLSC) were used as cell substrates for a cytotoxicity assay and toxicity prediction using the neutral red uptake (NRU) assay. First, primary cell cultures from three independent donors, from each tissue source, were characterized as mesenchymal stem cells (MSC) by plastic adherence and appropriate immunophenotype for MSC markers (positive for CD90, CD73, and CD105 and negative for CD11b, CD34, CD45, HLADR, and CD19). Furthermore, ADSC and PDLSC were able to differentiate into adipocytes and osteoblasts when maintained under the same culture conditions previously established for the NRU assay. NRU assays for three reference test substances were performed. R 2 was higher than 0.85 for all conditions, showing the feasibility to calculate IC 50 values. The IC 50 values were then used to predict the LD 50 of the test substances, which were comparable to previous results and the ICCVAM standard test report. Primary ADSC and PDLSC showed the potential to be considered as additional models for use in cytotoxicity assays. Copyright © 2017 Elsevier Inc. All rights reserved.

Human Adipose-Derived Mesenchymal Stem Cells Cryopreservation and Thawing Decrease α4-Integrin Expression.

PubMed

Irioda, Ana Carolina; Cassilha, Rafael; Zocche, Larissa; Francisco, Julio Cesar; Cunha, Ricardo Correa; Ferreira, Priscila Elias; Guarita-Souza, Luiz Cesar; Ferreira, Reginaldo Justino; Mogharbel, Bassam Felipe; Garikipati, Venkata Naga Srikanth; Souza, Daiany; Beltrame, Mirian Perlingeiro; de Carvalho, Katherine Athayde Teixeira

2016-01-01

Aim. The effects of cryopreservation on adipose tissue-derived mesenchymal stem cells are not clearly documented, as there is a growing body of evidence about the importance of adipose-derived mesenchymal stem cells for regenerative therapies. The aim of this study was to analyze human adipose tissue-derived mesenchymal stem cells phenotypic expression (CD34, CD45, CD73, CD90, CD105, and CD49d), colony forming unit ability, viability, and differentiation potential before and after cryopreservation. Materials and Methods. 12 samples of the adipose tissue were collected from a healthy donor using the liposuction technique. The cell isolation was performed by enzymatic digestion and then the cells were cultured up to passage 2. Before and after cryopreservation the immunophenotype, cellular viability analysis by flow cytometer, colony forming units ability, differentiation potential into adipocytes and osteoblasts as demonstrated by Oil Red O and Alizarin Red staining, respectively. Results. The immunophenotypic markers expression was largely preserved, and their multipotency was maintained. However, after cryopreservation, the cells decreased α4-integrin expression (CD49d), cell viability, and number of colony forming units. Conclusions. These findings suggest that ADMSC transplanted after cryopreservation might compromise the retention of transplanted cells in the host tissue. Therefore, further studies are warranted to standardize protocols related to cryopreservation to attain full benefits of stem cell therapy.

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

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

Protective role of hypoxia-inducible factor-1α-dependent CD39 and CD73 in fulminant acute liver failure

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

Tak, Eunyoung

Acute liver failure (ALF) is a severe life-threatening disease which usually arises in patients with-irreversible liver illnesses. Although human ectonucleotide triphosphate diphosphohydrolase-1, E-NTPDase1 (CD39) and ecto-5′-nucleotidase, Ecto5′NTase (CD73) are known to protect tissues from ALF, the expression and function of CD39 and CD73 during ALF are currently not fully investigated. We tested whether CD39 and CD73 are upregulated by hypoxia inducible factor (HIF)-1α, and improve ischemic tolerance to ALF. To test our hypothesis, liver biopsies were obtained and we found that CD39 and CD73 mRNA and proteins from human specimens were dramatically elevated in ALF. We investigated that induction ofmore » CD39 and CD73 in ALF-related with wild type mice. In contrast, deletion of cd39 and cd73 mice has severe ALF. In this study, we concluded that CD39 and CD73 are molecular targets for the development of drugs for ALF patients care. - Highlights: • HIF-1a is stabilized during acute liver failure • Upregulation of CD39 and CD73 following acute liver failure • CD39 and CD73 are transcriptionally induced by HIF-1a • Deletion of Cd39 and CD73 aggravates murine acute liver failure • DMOG treatment induces HIF-1a stabilization, CD39 and CD73 during acute liver failure in WT mice.« less

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.

New insights into the cellular makeup and progenitor potential of palatal connective tissues.

PubMed

Pall, Emoke; Cenariu, Mihai; Kasaj, Adrian; Florea, Adrian; Soancă, Andrada; Roman, Alexandra; Georgiu, Carmen

2017-12-01

The present study investigated the regenerative potential of connective tissues harvested from two palatal areas widely used as donor sites for muco-gingival surgical approaches. Connective tissue grafts (CTGs) were obtained by de-epithelialisation of a free gingival graft (deCTG) and by a split flap approach from a previous donor site (reCTG). Two types of mesenchymal stem cell (MSCs) were isolated and were named de-epithelialised MSCs (deMSCs) and re-entry MSCs (reMSCs). The cells were characterised and cellular functionality was investigated. CTGs were evaluated using immunohistochemical and ultrastructural approaches. No significant differences were observed regarding the frequency of colony-forming unit- fibroblasts, migration potential, and population doubling time between the two cell lines (p > 0.05). Both cell lines showed positivity for CD105, CD73, CD90, and CD44 and negative expression for CD34/45, CD14, CD79a, and HLA-DR. MSCs from both cell lines successfully differentiated into osteogenic, adipogenic, and chondrogenic lineages. Cells expressing antigens characteristic of CD34+ stromal cells (CD34+, αSMA-, CD31-) were traced in both CTGs. Ultrastructural analysis highlighted the presence of putative progenitors, namely fibroblasts,-in the pericapillary regions and in remote regions of the lamina propria- and pericytes-surrounding the capillaries. This study provides supplementary arguments for the use of CTG grafts in clinical practice due to the presence of putative progenitor cell. However, results were inconclusive regarding clinical decision-making to determine optimal harvesting area. Prior harvesting in the donor area did not appear to alter the regenerative capabilities of the connective tissue. © 2017 Wiley Periodicals, Inc.

Differential Expression of Adhesion-Related Proteins and MAPK Pathways Lead to Suitable Osteoblast Differentiation of Human Mesenchymal Stem Cells Subpopulations.

PubMed

Leyva-Leyva, Margarita; López-Díaz, Annia; Barrera, Lourdes; Camacho-Morales, Alberto; Hernandez-Aguilar, Felipe; Carrillo-Casas, Erika M; Arriaga-Pizano, Lourdes; Calderón-Pérez, Jaime; García-Álvarez, Jorge; Orozco-Hoyuela, Gabriel; Piña-Barba, Cristina; Rojas-Martínez, Augusto; Romero-Díaz, Víktor; Lara-Arias, Jorge; Rivera-Bolaños, Nancy; López-Camarillo, César; Moncada-Saucedo, Nidia; Galván-De los Santos, Alejandra; Meza-Urzúa, Fátima; Villarreal-Gómez, Luis; Fuentes-Mera, Lizeth

2015-11-01

Cellular adhesion enables communication between cells and their environment. Adhesion can be achieved throughout focal adhesions and its components influence osteoblast differentiation of human mesenchymal stem cells (hMSCs). Because cell adhesion and osteoblast differentiation are closely related, this article aimed to analyze the expression profiles of adhesion-related proteins during osteoblastic differentiation of two hMSCs subpopulations (CD105(+) and CD105(-)) and propose a strategy for assembling bone grafts based on its adhesion ability. In vitro experiments of osteogenic differentiation in CD105(-) cells showed superior adhesion efficiency and 2-fold increase of α-actinin expression compared with CD105(+) cells at the maturation stage. Interestingly, levels of activated β1-integrin increased in CD105(-) cells during the process. Additionally, the CD105(-) subpopulation showed 3-fold increase of phosphorylated FAK(Y397) compared to CD105(+) cells. Results also indicate that ERK1/2 was activated during CD105(-) bone differentiation and participation of mitogen-activated protein kinase (MAPK)-p38 in CD105(+) differentiation through a focal adhesion kinase (FAK)-independent pathway. In vivo trial demonstrated that grafts containing CD105(-) showed osteocytes embedded in a mineralized matrix, promoted adequate graft integration, increased host vascular infiltration, and efficient intramembranous repairing. In contrast, grafts containing CD105(+) showed deficient endochondral ossification and fibrocartilaginous tissue. Based on the expression of α-actinin, FAKy,(397) and ERK1/2 activation, we define maturation stage as critical for bone graft assembling. By in vitro assays, CD105(-) subpopulation showed superior adhesion efficiency compared to CD105(+) cells. Considering in vitro and in vivo assays, this study suggests that integration of a scaffold with CD105(-) subpopulation at the maturation stage represents an attractive strategy for clinical use in orthopedic bioengineering.

Survival of the Apical Papilla and Its Resident Stem Cells in a Case of Advanced Pulpal Necrosis and Apical Periodontitis.

PubMed

Chrepa, Vanessa; Pitcher, Brandon; Henry, Michael A; Diogenes, Anibal

2017-04-01

Apical papilla represents a source of an enriched mesenchymal stem cell (MSC) population (stem cells of the apical papilla [SCAPs]) that modulates root development and may participate in regenerative endodontic procedures in immature teeth with pulp necrosis. The characteristics and phenotype of this tissue in the presence of inflammation are largely unknown. The purpose of this study was to characterize a human apical papilla sample that was isolated from an immature tooth with pulp necrosis and apical periodontitis. Inflamed periapical tissue that included part of the apical papilla (apical papilla clinical sample [CS]) was collected from an immature mandibular premolar previously diagnosed with pulp necrosis and apical periodontitis during an apexification procedure. Harvested cells from this tissue (SCAP CS) were compared with inflamed periapical progenitor cells (IPAPCs) and normal SCAP (SCAP-RP89) in flow cytometry and quantitative osteogenesis experiments. Part of the issue was further processed for immunohistochemistry and compared with apical papilla and coronal pulp sections from normal immature teeth as well as inflamed periapical tissues from mature teeth. Similar to SCAP-RP89, 96.6% of the SCAP CS coexpressed the MSC markers CD73, CD90, and CD105, whereas only 66.3% of IPAPCs coexpressed all markers. The SCAP CS showed a significantly greater mineralization potential than both SCAP-RP89 and IPAPCs. Finally, immunohistochemical analysis revealed moderate infiltration of cells expressing the inflammatory markers CD45/68 in the apical papilla CS and prominent CD24, CD105, and von Willebrand factor expression. Under inflammatory conditions, human apical papilla was found moderately inflamed with retained SCAP vitality and stemness and increased osteogenic and angiogenesis potential. Copyright © 2016 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

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

Characterisation of synovial fluid and infrapatellar fat pad derived mesenchymal stromal cells: The influence of tissue source and inflammatory stimulus

PubMed Central

Garcia, John; Wright, Karina; Roberts, Sally; Kuiper, Jan Herman; Mangham, Chas; Richardson, James; Mennan, Claire

2016-01-01

The infrapatellar fat pad (FP) and synovial fluid (SF) in the knee serve as reservoirs of mesenchymal stromal cells (MSCs) with potential therapeutic benefit. We determined the influence of the donor on the phenotype of donor matched FP and SF derived MSCs and examined their immunogenic and immunomodulatory properties before and after stimulation with the pro-inflammatory cytokine interferon-gamma (IFN-γ). Both cell populations were positive for MSC markers CD73, CD90 and CD105, and displayed multipotency. FP-MSCs had a significantly faster proliferation rate than SF-MSCs. CD14 positivity was seen in both FP-MSCs and SF-MSCs, and was positively correlated to donor age but only for SF-MSCs. Neither cell population was positive for the co-stimulatory markers CD40, CD80 and CD86, but both demonstrated increased levels of human leukocyte antigen-DR (HLA-DR) following IFN-γ stimulation. HLA-DR production was positively correlated with donor age for FP-MSCs but not SF-MSCs. The immunomodulatory molecule, HLA-G, was constitutively produced by both cell populations, unlike indoleamine 2, 3-dioxygenase which was only produced following IFN-γ stimulation. FP and SF are accessible cell sources which could be utilised in the treatment of cartilage injuries, either by transplantation following ex-vivo expansion or endogenous targeting and mobilisation of cells close to the site of injury. PMID:27073003

Animal serum-free culture conditions for isolation and expansion of multipotent mesenchymal stromal cells from human BM.

PubMed

Müller, I; Kordowich, S; Holzwarth, C; Spano, C; Isensee, G; Staiber, A; Viebahn, S; Gieseke, F; Langer, H; Gawaz, M P; Horwitz, E M; Conte, P; Handgretinger, R; Dominici, M

2006-01-01

Multipotent mesenchymal stromal cells (MSC) have become important tools in regenerative and transplantation medicine. Rapidly increasing numbers of patients are receiving in vitro-expanded MSC. Culture conditions typically include FSC because human serum does not fully support growth of human MSC in vitro (MSC(FCS)). Concerns regarding BSE, other infectious complications and host immune reactions have fueled investigation of alternative culture supplements. As PDGF has long been identified as a growth factor for MSC, we tested media supplementation with platelet lysate for support of MSC proliferation. We found that primary cultures of BM-derived MSC can be established with animal serum-free media containing fresh frozen plasma and platelets (MSC(FFPP)). Moreover, MSC(FFPP) showed vigorous proliferation that was superior to classical culture conditions containing FCS. MSC(FFPP) morphology was equivalent to MSC(FCS), and MSC(FFPP) expressed CD73, CD90, CD105, CD106, CD146 and HLA-ABC while being negative for CD34, CD45 and surface HLA-DR, as expected. In addition to being phenotypically identical, MSC(FFPP) could efficiently differentiate into adipocytes and osteoblasts. In terms of immune regulatory properties, MSC(FFPP) were indistinguishable from MSC(FCS). Proliferation of PBMC induced by IL-2 in combination with OKT-3 or by PHA was inhibited in the presence of MSC(FFPP). Taken together, FCS can be replaced safely by FFPP in cultures of MSC for clinical purposes.

Isolation and characterization of human umbilical cord-derived endothelial colony-forming cells

PubMed Central

Zhang, Hao; Tao, Yanling; Ren, Saisai; Liu, Haihui; Zhou, Hui; Hu, Jiangwei; Tang, Yongyong; Zhang, Bin; Chen, Hu

2017-01-01

Endothelial colony-forming cells (ECFCs) are a population of endothelial progenitor cells (EPCs) that display robust proliferative potential and vessel-forming capability. Previous studies have demonstrated that a limited number of ECFCs may be obtained from adult bone marrow, peripheral blood and umbilical cord (UC) blood. The present study describes an effective method for isolating ECFCs from human UC. The ECFCs derived from human UC displayed the full properties of EPCs. Analysis of the growth kinetics, cell cycle and colony-forming ability of the isolated human UC-ECFCs indicated that the cells demonstrated properties of stem cells, including relative stability and rapid proliferation in vitro. Gene expression of Fms related tyrosine kinase 1, kinase insert domain receptor, vascular endothelial cadherin, cluster of differentiation (CD)31, CD34, epidermal growth factor homology domains-2, von Willebrand factor and endothelial nitric oxide synthase was assessed by reverse transcription-polymerase chain reaction. The cells were positive for CD34, CD31, CD73, CD105 and vascular endothelial growth factor receptor-2, and negative for CD45, CD90 and human leukocyte antigen-antigen D related protein according to flow cytometry. 1,1′-dioctadecyl-3,3,3′,3′-tetra-methyl-indocarbocyanine perchlorate-labeled acetylated low-density lipoprotein and fluorescein isothiocyanate-Ulex europaeus-l were used to verify the identity of the UC-ECFCs. Matrigel was used to investigate tube formation capability. The results demonstrated that the reported technique is a valuable method for isolating human UC-ECFCs, which have potential for use in vascular regeneration. PMID:29067104

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.

Characterization of Amniotic Stem Cells

PubMed Central

Koike, Chika; Zhou, Kaixuan; Takeda, Yuji; Fathy, Moustafa; Okabe, Motonori; Yoshida, Toshiko; Nakamura, Yukio; Kato, Yukio

2014-01-01

Abstract The amnion membrane is developed from embryo-derived cells, and amniotic cells have been shown to exhibit multidifferentiation potential. These cells represent a desirable source for stem cells for a variety of reasons. However, to date very few molecular analyses of amnion-derived cells have been reported, and efficient markers for isolating the stem cells remain unclear. This paper assesses the characterization of amnion-derived cells as stem cells by examining stemness marker expressions for amnion-derived epithelial cells and mesenchymal cells by flow cytometry, immunocytochemistry, and quantitative PCR. Flow cytometry revealed that amnion epithelial cells expressed CD133, CD 271, and TRA-1-60, whereas mecenchymal cells expressed CD44, CD73, CD90, and CD105. Immunohistochemistry showed that both cells expressed the stemness markers Oct3/4, Sox2, Klf4, and SSEA4. Stemness genes' expression in amnion epithelial cells, mesenchymal cells, fibroblast, bone marrow–derived mesenchymal stem cells (MSCs), and induced pluripotent stem cells (iPSCs) was compared by quantitative reverse-transcription polymerase chain reaction (RT-PCR). Amnion-derived epithelial cells and mesenchymal cells expressed Oct3/4, Nanog, and Klf4 more than bone marrow–derived MSCs. The sorted TRA1-60–positive cells expressed Oct3/4, Nanog, and Klf4 more than unsorted cells or TRA1-60–negative cells. TRA1-60 can be a marker for isolating amnion epithelial stem cells. PMID:25068631

CD73-deficient mice have increased antitumor immunity and are resistant to experimental metastasis.

PubMed

Stagg, John; Divisekera, Upulie; Duret, Helene; Sparwasser, Tim; Teng, Michele W L; Darcy, Phillip K; Smyth, Mark J

2011-04-15

CD73 is a cell-surface enzyme that suppresses immune responses by producing extracellular adenosine. In this study, we employed CD73 gene-targeted mice to investigate the role of host-derived CD73 on antitumor immunity and tumor cell metastasis. We found that CD73 ablation significantly suppressed the growth of ovalbumin-expressing MC38 colon cancer, EG7 lymphoma, AT-3 mammary tumors, and B16F10 melanoma. The protective effect of CD73 deficiency on primary tumors was dependent on CD8(+) T cells and associated with an increased frequency of antigen-specific CD8(+) T cells in peripheral blood and tumors and increased antigen-specific IFN-γ production. Replicate studies in bone marrow chimeras established that both hematopoietic and nonhematopoietic expression of CD73 was important to promote tumor immune escape. Using adoptive reconstitution of T regulatory cell (Treg)-depleted DEREG (depletion of regulatory T cells) mice, we demonstrated that part of the protumorigenic effect of Tregs was dependent on their expression of CD73. CD73-deficient mice were also protected against pulmonary metastasis of B16F10 melanoma cells after intravenous injection. Unexpectedly, we found that the prometastatic effect of host-derived CD73 was dependent on CD73 expression on nonhematopoietic cells. CD73 expression on nonhematopoietic cells, most likely endothelial cells, was critical for promoting lung metastasis in a manner independent from immunosuppressive effects. Notably, in vivo blockade of CD73 with a selective inhibitor or anti-CD73 monoclonal antibody significantly reduced tumor growth and metastasis of CD73-negative tumors. Taken together, our findings indicate that CD73 may be targeted at multiple levels to induce anticancer effects including at the level of tumor cells, Tregs, and nonhematopoietic cells. ©2011 AACR.

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.

The Role of the CD39/CD73 Purinergic Pathway in Modulating Arterial Thrombosis in Mice

PubMed Central

Covarrubias, R; Chepurko, E; Reynolds, A; Huttinger, ZM; Huttinger, R; Stanfill, K; Wheeler, DG; Novitskaya, T; Robson, SC; Dwyer, KM; Cowan, PJ; Gumina, RJ

2016-01-01

Objective Circulating blood cells and endothelial cells express Ectonucleoside triphosphate diphosphohydrolase-1 (CD39) and ecto-5’-nucleotidase (CD73). CD39 hydrolyzes extracellular ATP or ADP to AMP. CD73 hydrolyzes AMP to adenosine. The goal of this study was to examine the interplay between CD39 and CD73 cascade in arterial thrombosis. Approach and Results To determine how CD73 activity influences in vivo thrombosis, the time to FeCl3-induced arterial thrombosis was measured in CD73-null mice. In response to 5% FeCl3, but not to 10% FeCl3, there was a significant decrease in the time to thrombosis in CD73-null mice compared to wild-type (WT) mice. In mice overexpressing CD39, ablation of CD73 did not inhibit the prolongation in the time to thrombosis conveyed by CD39 overexpression. However, the CD73 inhibitor α-β-methylene-ADP nullified the prolongation in the time to thrombosis in hC39-Tg/CD73-null mice. To determine if hematopoietic-derived cells or endothelial cell CD39 activity regulates in vivo arterial thrombus, bone marrow transplant studies were conducted. FeCl3-induced arterial thrombosis in chimeric mice revealed a significant prolongation in the time to thrombosis in hCD39-Tg reconstituted WT mice, but not on WT reconstituted hCD39-Tg mice. Monocyte depletion with clodronate-loaded liposomes normalized the time to thrombosis in hCD39-Tg mice compared to hCD39-Tg mice treated with control liposomes, demonstrating that increased CD39 expression on monocytes protects against thrombosis. Conclusions These data demonstrate that ablation of CD73 minimally effects in vivo thrombosis, but increased CD39 expression on hematopoietic-derived cells, especially monocytes, attenuates in vivo arterial thrombosis. PMID:27417582

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

Specific blockade CD73 alters the 'exhausted' phenotype of T cells in head and neck squamous cell carcinoma.

PubMed

Deng, Wei-Wei; Li, Yi-Cun; Ma, Si-Rui; Mao, Liang; Yu, Guang-Tao; Bu, Lin-Lin; Kulkarni, Ashok B; Zhang, Wen-Feng; Sun, Zhi-Jun

2018-04-16

The adenosine-induced immunosuppression hampers the immune response toward tumor cells and facilitates the tumor cells to evade immunosurveillance. CD73, an ecto-5-nucleotidase, is the ectoenzyme dephosphorylating extracellular AMP to adenosine. Here, using immunocompetent transgenic head and neck squamous cell carcinoma (HNSCC) mouse model, immune profiling showed high expression of CD73 on CD4 + and CD8 + T cells was associated with an 'exhausted' phenotype. Further, treatment with anti-CD73 monoclonal antibody (mAb) significantly blunted the tumor growth in the mouse model, and the blockade of CD73 reversed the 'exhausted' phenotype of CD4 + and CD8 + T cells through downregulation of total expression of PD-1 and CTLA-4 on T cells. Whereas the population of CD4 + CD73 hi /CD8 + CD73 hi T cells expressed higher CTLA-4 and PD-1 as compared to untreated controls. In addition, the human tissue microarrays showed the expression of CD73 is upregulated on tumor infiltrating immune cells in patients with primary HNSCC. Moreover, CD73 expression is an independent prognostic factor for poor outcome in our cohort of HNSCC patients. Altogether, these findings highlight the immunoregulatory role of CD73 in the development of HNSCC and we propose that CD73 may prove to be a promising immunotherapeutic target for the treatment of HNSCC. This article is protected by copyright. All rights reserved. © 2018 UICC.

Platelet Lysate: The Better Choice for Jaw Periosteal Cell Mineralization.

PubMed

Wanner, Yvonne; Umrath, Felix; Waidmann, Marc; Reinert, Siegmar; Alexander, Dorothea

2017-01-01

Previously, we demonstrated a high quality of minerals formed by serum-free cultured jaw periosteal cells (JPCs) by Raman spectroscopy but the mineralization extent was not satisfactory. In the present study, we analyzed the proliferation and mineralization potential of human platelet lysate- (hPL-) cultured JPCs in comparison to that of FCS-cultured JPCs. By cell impedance measurements, we detected significantly higher population doubling times of PL-cultured JPCs in comparison to FCS-cultured JPCs. However, this result was not based on lower proliferation activities but on diminished cell sizes which JPCs develop under PL cultivation. The measurements of the metabolic activities clearly showed significantly higher cell proliferation rates under PL culturing. Equivalent levels of the mesenchymal cell markers CD29, CD45, CD73, CD90, and CD105 were detected, but there were significantly increased MSCA-1 levels under PL cultivation. While JPCs only occasionally mineralize under FCS culture conditions, the mineralization potential was significantly stronger under PL cultivation. Moreover, in 4 of 5 analyzed patient cells, the addition of dexamethasone was proved no longer necessary for strong mineralization of PL-cultured JPCs. We conclude that in vitro cultivation of JPCs with platelet lysate is a suitable alternative to FCS culture conditions and a powerful tool for the development of high-quality TE constructs using jaw periosteal cells.

CD4+CD73+ T cells are associated with lower T-cell activation and C reactive protein levels and are depleted in HIV-1 infection regardless of viral suppression

PubMed Central

Schuler, Patrick J.; Macatangay, Bernard J.C.; Saze, Zenichiro; Jackson, Edwin K.; Riddler, Sharon A.; Buchanan, William G.; Hilldorfer, Benedict B.; Mellors, John W.; Whiteside, Theresa L.; Rinaldo, Charles R.

2013-01-01

Background The role of the adenosine (ADO) suppression pathway, specifically CD39-expressing and CD73-expressing CD4+ T cells in HIV-1 infection is unclear. Methods We evaluated the frequency and numbers of CD4+CD39+ and CD4+CD73+ T cells, activated T cells, and plasma C reactive protein (CRP) levels in 36 HIV-1-positive individuals and 10 normal controls (NC). Low-level plasma viremia was evaluated using single copy assay. Mass spectrometry was used to measure hydrolysis of ATP by ectoenzyme-expressing CD4+ T cells, whereas cyclic adenosine monophosphate (cAMP) levels were measured using enzyme immunoassay. Suppression of T-cell function by exogenous ADO and CD4+CD73+ T cells was tested by flow cytometry. Results CD39 and CD73 are expressed in different CD4+ T-cell subsets. CD4+CD73+ T cells do not express CD25 and FOXP3, and their frequency and numbers were lower in HIV-1-positive individuals regardless of virologic suppression (P = 0.005 and P < 0.001, respectively). CD4+CD73+ numbers inversely correlated with CD4+CD38+DR+ (P = 0.002), CD8+CD38+DR+ T-cell frequency (P = 0.05), and plasma CRP levels (P = 0.01). Both subsets are required for hydrolysis of exogenous ATP to ADO and can increase CD4+ T-cell cAMP levels when incubated with exogenous ATP. Low-level viremia did not correlate with activated T-cell frequency. In vitro, ADO suppressed T-cell activation and cytokine expression. CD4+CD73+ T cells suppressed T-cell proliferation only in the presence of exogenous 5′-AMP. Conclusion The ADO-producing CD4+CD73+ subset of T cells is depleted in HIV-1-positive individuals regardless of viral suppression and may play a key role in controlling HIV-1-associated immune activation. PMID:24005375

In vitro expansion and differentiation of rat pancreatic duct-derived stem cells into insulin secreting cells using a dynamicthree-dimensional cell culture system.

PubMed

Chen, X C; Liu, H; Li, H; Cheng, Y; Yang, L; Liu, Y F

2016-06-27

In this study, a dynamic three-dimensional cell culture technology was used to expand and differentiate rat pancreatic duct-derived stem cells (PDSCs) into islet-like cell clusters that can secrete insulin. PDSCs were isolated from rat pancreatic tissues by in situ collagenase digestion and density gradient centrifugation. Using a dynamic three-dimensional culture technique, the cells were expanded and differentiated into functional islet-like cell clusters, which were characterized by morphological and phenotype analyses. After maintaining 1 x 108 isolated rat PDSCs in a dynamic three-dimensional cell culture for 7 days, 1.5 x 109 cells could be harvested. Passaged PDSCs expressed markers of pancreatic endocrine progenitors, including CD29 (86.17%), CD73 (90.73%), CD90 (84.13%), CD105 (78.28%), and Pdx-1. Following 14 additional days of culture in serum-free medium with nicotinamide, keratinocyte growth factor (KGF), and b fibroblast growth factor (FGF), the cells were differentiated into islet-like cell clusters (ICCs). The ICC morphology reflected that of fused cell clusters. During the late stage of differentiation, representative clusters were non-adherent and expressed insulin indicated by dithizone (DTZ)-positive staining. Insulin was detected in the extracellular fluid and cytoplasm of ICCs after 14 days of differentiation. Additionally, insulin levels were significantly higher at this time compared with the levels exhibited by PDSCs before differentiation (P < 0.01). By using a dynamic three-dimensional cell culture system, PDSCs can be expanded in vitro and can differentiate into functional islet-like cell clusters.

Expression of CD105 cancer stem cell marker in three subtypes of renal cell carcinoma.

PubMed

Saeednejad Zanjani, Leili; Madjd, Zahra; Abolhasani, Maryam; Shariftabrizi, Ahmad; Rasti, Arezoo; Asgari, Mojgan

2018-01-01

CD105 is recently described as a cancer stem cell (CSC) marker. The present study was aimed to investigate the expression and prognostic significance of the CSC marker CD105 in different histological subtypes of renal cell carcinoma (RCC). Expression of CD105 was evaluated using immunohistochemistry in RCC samples on tissue microarrays including clear cell RCCs (ccRCCs), papillary, and chromophobe RCCs. The association between CD105 expression and clinicopathological features as well as survival outcomes was determined. In ccRCC, increased tumoral cytoplasmic and endothelial expression of CD105 were significantly associated with advanced stage, renal vein invasion, and microvascular invasion (MVI). In addition, MVI was associated with a worse overall survival (OS). Moreover, in multivariate analysis tumor stage and nuclear grade were independent prognostic factors for OS both in case of tumoral cytoplasmic and endothelial CD105 expression. Additionally, CD105 expression was found to be a predictor of worse OS in univariate analysis. However, in papillary and chromophobe RCC, no significant association was found between CD105 expression and clinicopathological parameters or prognosis. We showed that CD105 expression was associated with more aggressive tumor behavior, more advanced disease, and worse prognosis in ccRCC but not in the other RCC subtypes.

Down-regulation of CD73 on B cells of patients with viremic HIV correlates with B cell activation and disease progression.

PubMed

Kim, Eun-Seong; Ackermann, Christin; Tóth, Ilona; Dierks, Patrick; Eberhard, Johanna M; Wroblewski, Raluca; Scherg, Felix; Geyer, Matthias; Schmidt, Reinhold E; Beisel, Claudia; Bockhorn, Maximilian; Haag, Friedrich; van Lunzen, Jan; Schulze Zur Wiesch, Julian

2017-05-01

Recently, alterations of the T cell expression of the ectonucleotidases, CD39 and CD73, during HIV infection have been described. Here, peripheral ( n = 70) and lymph nodal B cells ( n = 10) of patients with HIV at different stages of disease as well as uninfected individuals were analyzed via multicolor flow cytometry with regard to expression of CD39 and CD73 and differentiation, proliferation, and exhaustion status. Patients with chronic, untreated HIV showed a significantly decreased frequency of CD73-expressing B cells ( P < 0.001) compared with healthy controls. Decreased frequencies of CD39 + CD73 + B cells in patients with HIV correlated with low CD4 + counts ( P < 0.0256) as well as increased proliferation and exhaustion status as determined by Ki-67 and programmed death-1 expression. Down-regulation of CD73 was observed in naive and memory B cells as determined by CD27 and CD21. Neither HIV elite controller patients nor antiretroviral therapy-treated patients had significantly lower CD39 and CD73 expression on B cells compared with healthy controls. Of importance, low CD73 + expression on B cells was associated with modulated in vitro B cell function. Further in vivo studies are warranted to evaluate the in vivo role of phenotypic loss of CD73 in B cell dysregulation in HIV. © Society for Leukocyte Biology.

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

Dental pulp of the third molar: a new source of pluripotent-like stem cells.

PubMed

Atari, Maher; Gil-Recio, Carlos; Fabregat, Marc; García-Fernández, Dani; Barajas, Miguel; Carrasco, Miguel A; Jung, Han-Sung; Alfaro, F Hernández; Casals, Nuria; Prosper, Felipe; Ferrés-Padró, Eduard; Giner, Luis

2012-07-15

Dental pulp is particularly interesting in regenerative medicine because of the accessibility and differentiation potential of the tissue. Dental pulp has an early developmental origin with multi-lineage differentiation potential as a result of its development during childhood and adolescence. However, no study has previously identified the presence of stem cell populations with embryonic-like phenotypes in human dental pulp from the third molar. In the present work, we describe a new population of dental pulp pluripotent-like stem cells (DPPSCs) that were isolated by culture in medium containing LIF, EGF and PDGF. These cells are SSEA4(+), OCT3/4(+), NANOG(+), SOX2(+), LIN28(+), CD13(+), CD105(+), CD34(-), CD45(-), CD90(+), CD29(+), CD73(+), STRO1(+) and CD146(-), and they show genetic stability in vitro based on genomic analysis with a newly described CGH technique. Interestingly, DPPSCs were able to form both embryoid-body-like structures (EBs) in vitro and teratoma-like structures that contained tissues derived from all three embryonic germ layers when injected in nude mice. We examined the capacity of DPPSCs to differentiate in vitro into tissues that have similar characteristics to mesoderm, endoderm and ectoderm layers in both 2D and 3D cultures. We performed a comparative RT-PCR analysis of GATA4, GATA6, MIXL1, NANOG, OCT3/4, SOX1 and SOX2 to determine the degree of similarity between DPPSCs, EBs and human induced pluripotent stem cells (hIPSCs). Our analysis revealed that DPPSCs, hIPSC and EBs have the same gene expression profile. Because DPPSCs can be derived from healthy human molars from patients of different sexes and ages, they represent an easily accessible source of stem cells, which opens a range of new possibilities for regenerative medicine.

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.

Cartilage Protective and Chondrogenic Capacity of WIN-34B, a New Herbal Agent, in the Collagenase-Induced Osteoarthritis Rabbit Model and in Progenitor Cells from Subchondral Bone

PubMed Central

Huh, Jeong-Eun; Park, Yeon-Cheol; Seo, Byung-Kwan; Lee, Jae-Dong; Baek, Yong-Hyeon; Choi, Do-Young; Park, Dong-Suk

2013-01-01

We sought to determine the cartilage repair capacity of WIN-34B in the collagenase-induced osteoarthritis rabbit model and in progenitor cells from subchondral bone. The cartilage protective effect of WIN-34B was measured by clinical and histological scores, cartilage area, and proteoglycan and collagen contents in the collagenase-induced osteoarthritis rabbit model. The efficacy of chondrogenic differentiation of WIN-34B was assessed by expression of CD105, CD73, type II collagen, and aggrecan in vivo and was analyzed by the surface markers of progenitor cells, the mRNA levels of chondrogenic marker genes, and the level of proteoglycan, GAG, and type II collagen in vitro. Oral administration of WIN-34B significantly increased cartilage area, and this was associated with the recovery of proteoglycan and collagen content. Moreover, WIN-34B at 200 mg/kg significantly increased the expression of CD105, CD73, type II collagen, and aggrecan compared to the vehicle group. WIN-34B markedly enhanced the chondrogenic differentiation of CD105 and type II collagen in the progenitor cells from subchondral bone. Also, we confirmed that treatment with WIN-34B strongly increased the number of SH-2(CD105) cells and expression type II collagen in subchondral progenitor cells. Moreover, WIN-34B significantly increased proteoglycan, as measured by alcian blue staining; the mRNA level of type II α1 collagen, cartilage link protein, and aggrecan; and the inhibition of cartilage matrix molecules, such as GAG and type II collagen, in IL-1β-treated progenitor cells. These findings suggest that WIN-34B could be a potential candidate for effective anti-osteoarthritic therapy with cartilage repair as well as cartilage protection via enhancement of chondrogenic differentiation in the collagenase-induced osteoarthritis rabbit model and progenitor cells from subchondral bone. PMID:23983790

CD73 promotes proliferation and migration of human cervical cancer cells independent of its enzyme activity.

PubMed

Gao, Zhao-Wei; Wang, Hui-Ping; Lin, Fang; Wang, Xi; Long, Min; Zhang, Hui-Zhong; Dong, Ke

2017-02-15

CD73 has both enzymatic and non-enzymatic functions in cells. As a nucleotidase, CD73 plays its enzymatic function by catalyzing the hydrolysis of AMP into adenosine and phosphate. In addition to this, accumulating data have shown that CD73 is a key regulatory molecule involved in cancer growth and metastasis, but this non-enzymatic function of CD73 in cervical cancer cells has not been well studied. CD73 was overexpressed by pcDNA-NT5E expression vector transfection in Hela and SiHa cells. Cell's proliferation and migration were evaluated by MTT and scratch healing assay. The CD73 specific antagonist -APCP was used to inhibit CD73 enzymatic activity. And the effect of APCP on CD73 activity was determined by high performance liquid chromatography (HPLC). Expression level was assessed by qRT-PCR and western blotting. In the present study, we used Hela and SiHa cell lines to evaluate the effects of CD73 on cervical cancer cells proliferation and migration, and further explore the potential regulating mechanisms. Our data showed that CD73 overexpression significantly promoted cervical cancer cells proliferation and migration, and this promotive effect was not reverted by blocking CD73 enzymatic activity, both in Hela and SiHa cells. On the other hand, our data also showed that high concentration of adenosine inhibited Hela and SiHa cells proliferation and migration. These results demonstrated that the promotive effect of CD73 on cervical cancer cells proliferation and migration in vitro was independent from its enzymatic activity (i.e. production of adenosine). Furthermore, the expressions of EGFR, VEGF and Akt were significantly increased in CD73 overexpression Hela and SiHa cells. Our data suggested that CD73 might promote proliferation and migration via potentiating EGFR/Akt and VEGF/Akt pathway, which was independent of CD73 enzyme activity. These data provide a novel insight into the regulating function of CD73 in cancer cells and suggest that CD73 may be promising therapeutic target in cervical cancer.

Isolation and characterization of stem cells derived from human third molar tooth germs of young adults: implications in neo-vascularization, osteo-, adipo- and neurogenesis.

PubMed

Yalvac, M E; Ramazanoglu, M; Rizvanov, A A; Sahin, F; Bayrak, O F; Salli, U; Palotás, A; Kose, G T

2010-04-01

A number of studies have reported in the last decade that human tooth germs contain multipotent cells that give rise to dental and peri-odontal structures. The dental pulp, third molars in particular, have been shown to be a significant stem cell source. In this study, we isolated and characterized human tooth germ stem cells (hTGSCs) from third molars and assessed the expression of developmentally important transcription factors, such as oct4, sox2, klf4, nanog and c-myc, to determine their pluri-potency. Flow-cytometry analysis revealed that hTGSCs were positive for CD73, CD90, CD105 and CD166, but negative for CD34, CD45 and CD133, suggesting that these cells are mesenchymal-like stem cells. Under specific culture conditions, hTGSCs differentiated into osteogenic, adipogenic and neurogenic cells, as well as formed tube-like structures in Matrigel assay. hTGSCs showed significant levels of expression of sox2 and c-myc messenger RNA (mRNA), and a very high level of expression of klf4 mRNA when compared with human embryonic stem cells. This study reports for the first time that hTGSCs express developmentally important transcription factors that could render hTGSCs an attractive candidate for future somatic cell re-programming studies to differentiate germs into various tissue types, such as neurons and vascular structures. In addition, these multipotential hTGSCs could be important stem cell sources for autologous transplantation.

Functional significance of CD105-positive cells in papillary renal cell carcinoma.

PubMed

Matak, Damian; Brodaczewska, Klaudia K; Szczylik, Cezary; Koch, Irena; Myszczyszyn, Adam; Lipiec, Monika; Lewicki, Slawomir; Szymanski, Lukasz; Zdanowski, Robert; Czarnecka, Anna M

2017-01-05

CD105 was postulated as a renal cell carcinoma (RCC) stem cell marker, and CD133 as a putative RCC progenitor. Hypoxia, a natural microenvironment that prevails in tumors, was also incorporated into the study, especially in terms of the promotion of hypothetical stem-like cell properties. Within this study, we verify the existence of CD105+ and CD133+ populations in selected papillary subtype RCC (pRCC) cell lines. Both populations were analyzed for correlation with stem-like cell properties, such as stemness gene expression, and sphere and colony formation. For the preliminary analysis, several RCC cell lines were chosen (786-O, SMKT-R2, Caki-2, 796-P, ACHN, RCC6) and the control was human kidney cancer stem cells (HKCSC) and renal cells of embryonic origin (ASE-5063). Four cell lines were chosen for further investigation: Caki-2 (one of the highest numbers of CD105+ cells; primary origin), ACHN (a low number of CD105+ cells; metastatic origin), HKCSC (putative positive control), and ASE-5063 (additional control). In 769-P and RCC6, we could not detect a CD105+ population. Hypoxia variously affects pRCC cell growth, and mainly diminishes the stem-like properties of cells. Furthermore, we could not observe the correlation of CD105 and/or CD133 expression with the enhancement of stem-like properties. Based on this analysis, CD105/CD133 cannot be validated as cancer stem cell markers of pRCC cell lines.

Comparative prognostic relevance of breast intra-tumoral microvessel density evaluated by CD105 and CD146: A pilot study of 42 cases.

PubMed

Martinez, Leandro Marcelo; Labovsky, Vivian; Calcagno, María de Luján; Davies, Kevin Mauro; Rivello, Hernán Garcia; Wernicke, Alejandra; Calvo, Juan Carlos; Chasseing, Norma Alejandra

2016-04-01

Angiogenesis is a key process for metastatic progression. While it has been established that the evaluation of breast tumoral microvessel density by CD105 marker is a potential prognostic parameter, its evaluation by CD146 marker has been poorly studied. The purpose of this study was to compare the prognostic value of intra-tumoral microvessel density assayed by CD105 and CD146 in early breast cancer patients. 42 women with breast infiltrative ductal carcinoma (I and II-stages) were retrospectively reviewed. Intra-tumoral microvessel density was immunohistochemically examined using antibodies anti-CD105 and CD146 in paraffin-embedded tissues, and their association with classical prognostic-markers, metastatic recurrence, metastasis-free survival and overall survival was analyzed. High microvessel density assessed by CD146 was significantly associated with a higher risk of developing metastasis (p=0.0310) and a shorter metastasis-free survival (p=0.0197). In contrast, when we used the CD105-antibody, we did not find any significant association. Finally, CD146 showed to be an independent predictive indicator for metastasis-free survival (p=0.0055). Our data suggest that the intra-tumoral microvessel density evaluated by CD146 may be a more suitable predictor of metastatic development than that evaluated by CD105 in early breast cancer. Copyright © 2016 Elsevier GmbH. All rights reserved.

The Volatile Anesthetic Isoflurane Increases Endothelial Adenosine Generation via Microparticle Ecto-5′-Nucleotidase (CD73) Release

PubMed Central

Kim, Mihwa; Ham, Ahrom; Kim, Katelyn Yu-Mi; Brown, Kevin M.; Lee, H. Thomas

2014-01-01

Endothelial dysfunction is common in acute and chronic organ injury. Isoflurane is a widely used halogenated volatile anesthetic during the perioperative period and protects against endothelial cell death and inflammation. In this study, we tested whether isoflurane induces endothelial ecto-5′-nucleotidase (CD73) and cytoprotective adenosine generation to protect against endothelial cell injury. Clinically relevant concentrations of isoflurane induced CD73 activity and increased adenosine generation in cultured human umbilical vein or mouse glomerular endothelial cells. Surprisingly, isoflurane-mediated induction of endothelial CD73 activity occurred within 1 hr and without synthesizing new CD73. We determined that isoflurane rapidly increased CD73 containing endothelial microparticles into the cell culture media. Indeed, microparticles isolated from isoflurane-treated endothelial cells had significantly higher CD73 activity as well as increased CD73 protein. In vivo, plasma from mice anesthetized with isoflurane had significantly higher endothelial cell-derived CD144+ CD73+ microparticles and had increased microparticle CD73 activity compared to plasma from pentobarbital-anesthetized mice. Supporting a critical role of CD73 in isoflurane-mediated endothelial protection, a selective CD73 inhibitor (APCP) prevented isoflurane-induced protection against human endothelial cell inflammation and apoptosis. In addition, isoflurane activated endothelial cells Rho kinase evidenced by myosin phosphatase target subunit-1 and myosin light chain phosphorylation. Furthermore, isoflurane-induced release of CD73 containing microparticles was significantly attenuated by a selective Rho kinase inhibitor (Y27632). Taken together, we conclude that the volatile anesthetic isoflurane causes Rho kinase-mediated release of endothelial microparticles containing preformed CD73 and increase adenosine generation to protect against endothelial apoptosis and inflammation. PMID:24945528

CD39 is upregulated during activation of mouse and human T cells and attenuates the immune response to Listeria monocytogenes.

PubMed

Raczkowski, Friederike; Rissiek, Anne; Ricklefs, Isabell; Heiss, Kirsten; Schumacher, Valéa; Wundenberg, Kira; Haag, Friedrich; Koch-Nolte, Friedrich; Tolosa, Eva; Mittrücker, Hans-Willi

2018-01-01

The ectoenzymes CD39 and CD73 degrade extracellular ATP to adenosine. ATP is released by stressed or damaged cells and provides pro-inflammatory signals to immune cells through P2 receptors. Adenosine, on the other hand, suppresses immune cells by stimulating P1 receptors. Thus, CD39 and CD73 can shape the quality of immune responses. Here we demonstrate that upregulation of CD39 is a consistent feature of activated conventional CD4+ and CD8+ T cells. Following stimulation in vitro, CD4+ and CD8+ T cells from human blood gained surface expression of CD39 but displayed only low levels of CD73. Activated human T cells from inflamed joints largely presented with a CD39+CD73- phenotype. In line, in spleens of mice with acute Listeria monocytogenes, listeria-specific CD4+ and CD8+ T cells acquired a CD39+CD73- phenotype. To test the function of CD39 in control of bacterial infection, CD39-deficient (CD39-/-) mice were infected with L. monocytogenes. CD39-/- mice showed better initial control of L. monocytogenes, which was associated with enhanced production of inflammatory cytokines. In the late stage of infection, CD39-/- mice accumulated more listeria-specific CD8+ T cells in the spleen than wildtype animals suggesting that CD39 attenuates the CD8+ T-cell response to infection. In conclusion, our results demonstrate that CD39 is upregulated on conventional CD4+ and CD8+ T cells at sites of acute infection and inflammation, and that CD39 dampens responses to bacterial infection.

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

Tetraspanin CD9 and ectonucleotidase CD73 identify an osteochondroprogenitor population with elevated osteogenic properties.

PubMed

Singh, Anju; Lester, Chantel; Drapp, Rebecca; Hu, Dorothy Z; Glimcher, Laurie H; Jones, Dallas

2015-02-01

Cell-based bone regeneration strategies offer promise for traumatic bone injuries, congenital defects, non-union fractures and other skeletal pathologies. Postnatal bone remodeling and fracture healing provide evidence that an osteochondroprogenitor cell is present in adult life that can differentiate to remodel or repair the fractured bone. However, cell-based skeletal repair in the clinic is still in its infancy, mostly due to poor characterization of progenitor cells and lack of knowledge about their in vivo behavior. Here, we took a combined approach of high-throughput screening, flow-based cell sorting and in vivo transplantation to isolate markers that identify osteochondroprogenitor cells. We show that the presence of tetraspanin CD9 enriches for osteochondroprogenitors within CD105(+) mesenchymal cells and that these cells readily form bone upon transplantation. In addition, we have used Thy1.2 and the ectonucleotidase CD73 to identify subsets within the CD9(+) population that lead to endochondral or intramembranous-like bone formation. Utilization of this unique cell surface phenotype to enrich for osteochondroprogenitor cells will allow for further characterization of the molecular mechanisms that regulate their osteogenic properties. © 2015. Published by The Company of Biologists Ltd.

Growth on poly(L-lactic acid) porous scaffold preserves CD73 and CD90 immunophenotype markers of rat bone marrow mesenchymal stromal cells.

PubMed

Zamparelli, Alessandra; Zini, Nicoletta; Cattini, Luca; Spaletta, Giulia; Dallatana, Davide; Bassi, Elena; Barbaro, Fulvio; Iafisco, Michele; Mosca, Salvatore; Parrilli, Annapaola; Fini, Milena; Giardino, Roberto; Sandri, Monica; Sprio, Simone; Tampieri, Anna; Maraldi, Nadir M; Toni, Roberto

2014-10-01

Few data are available on the effect of biomaterials on surface antigens of mammalian bone marrow-derived, adult mesenchymal stromal cells (MSCs). Since poly(L-lactic acid) or PLLA is largely used in tissue engineering of human bones, and we are developing a reverse engineering program to prototype with biomaterials the vascular architecture of bones for their bioartificial reconstruction, both in humans and animal models, we have studied the effect of porous, flat and smooth PLLA scaffolds on the immunophenotype of in vitro grown, rat MSCs in the absence of any coating, co-polymeric enrichment, and differentiation stimuli. Similar to controls on plastic, we show that our PLLA scaffold does not modify the distribution of some surface markers in rat MSCs. In particular, the maintained expression of CD73 and CD90 on two different subpopulations (small and large cells) is consistent with their adhesion to the PLLA scaffold through specialized appendages, and to their prominent content in actin. In addition, our PLLA scaffold favours retention of the intermediate filament desmin, believed a putative marker of undifferentiated state. Finally, it preserves all rat MSCs morphotypes, and allows for their survival, adhesion to the substrate, and replication. Remarkably, a subpopulation of rat MSCs grown on our PLLA scaffold exhibited formation of membrane protrusions of uncertain significance, although in a size range and morphology compatible with either motility blebs or shedding vesicles. In summary, our PLLA scaffold has no detrimental effect on a number of features of rat MSCs, primarily the expression of CD73 and CD90.

Specific gene delivery to liver sinusoidal and artery endothelial cells.

PubMed

Abel, Tobias; El Filali, Ebtisam; Waern, Johan; Schneider, Irene C; Yuan, Qinggong; Münch, Robert C; Hick, Meike; Warnecke, Gregor; Madrahimov, Nodir; Kontermann, Roland E; Schüttrumpf, Jörg; Müller, Ulrike C; Seppen, Jurgen; Ott, Michael; Buchholz, Christian J

2013-09-19

Different types of endothelial cells (EC) fulfill distinct tasks depending on their microenvironment. ECs are therefore difficult to genetically manipulate ex vivo for functional studies or gene therapy. We assessed lentiviral vectors (LVs) targeted to the EC surface marker CD105 for in vivo gene delivery. The mouse CD105-specific vector, mCD105-LV, transduced only CD105-positive cells in primary liver cell cultures. Upon systemic injection, strong reporter gene expression was detected in liver where mCD105-LV specifically transduced liver sinusoidal ECs (LSECs) but not Kupffer cells, which were mainly transduced by nontargeted LVs. Tumor ECs were specifically targeted upon intratumoral vector injection. Delivery of the erythropoietin gene with mCD105-LV resulted in substantially increased erythropoietin and hematocrit levels. The human CD105-specific vector (huCD105-LV) transduced exclusively human LSECs in mice transplanted with human liver ECs. Interestingly, when applied at higher dose and in absence of target cells in the liver, huCD105-LV transduced ECs of a human artery transplanted into the descending mouse aorta. The data demonstrate for the first time targeted gene delivery to specialized ECs upon systemic vector administration. This strategy offers novel options to better understand the physiological functions of ECs and to treat genetic diseases such as those affecting blood factors.

Identification of CD4+ T-cell-derived CD161+ CD39+ and CD39+CD73+ microparticles as new biomarkers for rheumatoid arthritis.

PubMed

Fan, Wenqiang; Wang, Wenxiang; Wu, Jie; Ma, Ling; Guo, Jinyan

2017-02-01

This study aimed to identify CD4 + T-cell-derived microparticles (MPs) and investigate their roles in rheumatoid arthritis (RA). Synovial fluids from 34 RA, 33 osteoarthritis patients and 42 healthy individuals were analyzed by flow cytometry. Human fibroblast-like synoviocytes and peripheral blood mononuclear cells were cultured with or without isolated MPs, chemokines and cytokines were measured by ELISA. CD4 + CD161 + CD39 + and CD4 + CD39 + CD73 + MPs were abundantly present in RA patients, which were positively or negatively correlated with RA features, respectively. Chemokines CCL20, CCL17 and CCL22, and cytokines IL-17 and IL-10 were influenced by these MPs in human fibroblast-like synoviocytes (HFLS) or PMBCs. CD4 + T-cell-derived CD161 + CD39 + and CD39 + CD73 + MPs could serve as new reciprocal biomarkers for RA evaluation.

Platelet Lysate: The Better Choice for Jaw Periosteal Cell Mineralization

PubMed Central

Wanner, Yvonne; Umrath, Felix; Waidmann, Marc; Reinert, Siegmar

2017-01-01

Previously, we demonstrated a high quality of minerals formed by serum-free cultured jaw periosteal cells (JPCs) by Raman spectroscopy but the mineralization extent was not satisfactory. In the present study, we analyzed the proliferation and mineralization potential of human platelet lysate- (hPL-) cultured JPCs in comparison to that of FCS-cultured JPCs. By cell impedance measurements, we detected significantly higher population doubling times of PL-cultured JPCs in comparison to FCS-cultured JPCs. However, this result was not based on lower proliferation activities but on diminished cell sizes which JPCs develop under PL cultivation. The measurements of the metabolic activities clearly showed significantly higher cell proliferation rates under PL culturing. Equivalent levels of the mesenchymal cell markers CD29, CD45, CD73, CD90, and CD105 were detected, but there were significantly increased MSCA-1 levels under PL cultivation. While JPCs only occasionally mineralize under FCS culture conditions, the mineralization potential was significantly stronger under PL cultivation. Moreover, in 4 of 5 analyzed patient cells, the addition of dexamethasone was proved no longer necessary for strong mineralization of PL-cultured JPCs. We conclude that in vitro cultivation of JPCs with platelet lysate is a suitable alternative to FCS culture conditions and a powerful tool for the development of high-quality TE constructs using jaw periosteal cells. PMID:29391870

Periodontal Regeneration Using Periodontal Ligament Stem Cell-Transferred Amnion

PubMed Central

Iwasaki, Kengo; Yokoyama, Naoki; Tanaka, Yuichi; Taki, Atsuko; Honda, Izumi; Kimura, Yasuyuki; Takeda, Masaki; Akazawa, Keiko; Oda, Shigeru; Izumi, Yuichi; Morita, Ikuo

2014-01-01

Periodontal disease is characterized by the destruction of tooth supporting tissues. Regeneration of periodontal tissues using ex vivo expanded cells has been introduced and studied, although appropriate methodology has not yet been established. We developed a novel cell transplant method for periodontal regeneration using periodontal ligament stem cell (PDLSC)-transferred amniotic membrane (PDLSC-amnion). The aim of this study was to investigate the regenerative potential of PDLSC-amnion in a rat periodontal defect model. Cultured PDLSCs were transferred onto amniotic membranes using a glass substrate treated with polyethylene glycol and photolithography. The properties of PDLSCs were investigated by flow cytometry and in vitro differentiation. PDLSC-amnion was transplanted into surgically created periodontal defects in rat maxillary molars. Periodontal regeneration was evaluated by microcomputed tomography (micro-CT) and histological analysis. PDLSCs showed mesenchymal stem cell-like characteristics such as cell surface marker expression (CD90, CD44, CD73, CD105, CD146, and STRO-1) and trilineage differentiation ability (i.e., into osteoblasts, adipocytes, and chondrocytes). PDLSC-amnion exhibited a single layer of PDLSCs on the amniotic membrane and stability of the sheet even with movement and deformation caused by surgical instruments. We observed that the PDLSC-amnion enhanced periodontal tissue regeneration as determined by micro-CT and histology by 4 weeks after transplantation. These data suggest that PDLSC-amnion has therapeutic potential as a novel cell-based regenerative periodontal therapy. PMID:24032400

Periodontal regeneration using periodontal ligament stem cell-transferred amnion.

PubMed

Iwasaki, Kengo; Komaki, Motohiro; Yokoyama, Naoki; Tanaka, Yuichi; Taki, Atsuko; Honda, Izumi; Kimura, Yasuyuki; Takeda, Masaki; Akazawa, Keiko; Oda, Shigeru; Izumi, Yuichi; Morita, Ikuo

2014-02-01

Periodontal disease is characterized by the destruction of tooth supporting tissues. Regeneration of periodontal tissues using ex vivo expanded cells has been introduced and studied, although appropriate methodology has not yet been established. We developed a novel cell transplant method for periodontal regeneration using periodontal ligament stem cell (PDLSC)-transferred amniotic membrane (PDLSC-amnion). The aim of this study was to investigate the regenerative potential of PDLSC-amnion in a rat periodontal defect model. Cultured PDLSCs were transferred onto amniotic membranes using a glass substrate treated with polyethylene glycol and photolithography. The properties of PDLSCs were investigated by flow cytometry and in vitro differentiation. PDLSC-amnion was transplanted into surgically created periodontal defects in rat maxillary molars. Periodontal regeneration was evaluated by microcomputed tomography (micro-CT) and histological analysis. PDLSCs showed mesenchymal stem cell-like characteristics such as cell surface marker expression (CD90, CD44, CD73, CD105, CD146, and STRO-1) and trilineage differentiation ability (i.e., into osteoblasts, adipocytes, and chondrocytes). PDLSC-amnion exhibited a single layer of PDLSCs on the amniotic membrane and stability of the sheet even with movement and deformation caused by surgical instruments. We observed that the PDLSC-amnion enhanced periodontal tissue regeneration as determined by micro-CT and histology by 4 weeks after transplantation. These data suggest that PDLSC-amnion has therapeutic potential as a novel cell-based regenerative periodontal therapy.

Human cadaver multipotent stromal/stem cells isolated from arteries stored in liquid nitrogen for 5 years

PubMed Central

2014-01-01

Introduction Regenerative medicine challenges researchers to find noncontroversial, safe and abundant stem cell sources. In this context, harvesting from asystolic donors could represent an innovative and unlimited reservoir of different stem cells. In this study, cadaveric vascular tissues were established as an alternative source of human cadaver mesenchymal stromal/stem cells (hC-MSCs). We reported the successful cell isolation from postmortem arterial segments stored in a tissue-banking facility for at least 5 years. Methods After thawing, hC-MSCs were isolated with a high efficiency (12 × 106) and characterized with flow cytometry, immunofluorescence, molecular and ultrastructural approaches. Results In early passages, hC-MSCs were clonogenic, highly proliferative and expressed mesenchymal (CD44, CD73, CD90, CD105, HLA-G), stemness (Stro-1, Oct-4, Notch-1), pericyte (CD146, PDGFR-β, NG2) and neuronal (Nestin) markers; hematopoietic and vascular markers were negative. These cells had colony and spheroid-forming abilities, multipotency for their potential to differentiate in multiple mesengenic lineages and immunosuppressive activity to counteract proliferation of phytohemagglutinin-stimulated blood mononuclear cells. Conclusions The efficient procurement of stem cells from cadaveric sources, as postmortem vascular tissues, demonstrates that such cells can survive to prolonged ischemic insult, anoxia, freezing and dehydration injuries, thus paving the way for a scientific revolution where cadaver stromal/stem cells could effectively treat patients demanding cell therapies. PMID:24429026

Development and validation of a procedure to isolate viable bone marrow cells from the vertebrae of cadaveric organ donors for composite organ grafting

PubMed Central

GORANTLA, VIJAY S.; SCHNEEBERGER, STEFAN; MOORE, LINDA R.; DONNENBERG, VERA S.; ZIMMERLIN, LUDOVIC; ANDREW LEE, W. P.; DONNENBERG, ALBERT D.

2014-01-01

Background aims Donor-derived vertebral bone marrow (BM) has been proposed to promote chimerism in solid organ transplantation with cadaveric organs. Reports of successful weaning from immunosuppression in patients receiving directed donor transplants in combination with donor BM or blood cells and novel peri-transplant immunosuppression has renewed interest in implementing similar protocols with cadaveric organs. Methods We performed six pre-clinical full-scale separations to adapt vertebral BM preparations to a good manufacturing practice (GMP) environment. Vertebral bodies L4–T8 were transported to a class 10 000 clean room, cleaned of soft tissue, divided and crushed in a prototype bone grinder. Bone fragments were irrigated with medium containing saline, albumin, DNAse and gentamicin, and strained through stainless steel sieves. Additional cells were eluted after two rounds of agitation using a prototype BM tumbler. Results The majority of recovered cells (70.9 ± 14.1%, mean ± SD) were eluted directly from the crushed bone, whereas 22.3% and 5.9% were eluted after the first and second rounds of tumbling, respectively. Cells were pooled and filtered (500, 200 μm) using a BM collection kit. Larger lumbar vertebrae yielded about 1.6 times the cells of thoracic vertebrae. The average product yielded 5.2 ± 1.2×1010 total cells, 6.2 ± 2.2×108 of which were CD45+ CD34+. Viability was 96.6 ± 1.9% and 99.1 ± 0.8%, respectively. Multicolor flow cytometry revealed distinct populations of CD34+ CD90+ CD117 dim hematopoietic stem cells (15.5 ± 7.5% of the CD34 + cells) and CD45− CD73+ CD105+ mesenchymal stromal cells (0.04 ± 0.04% of the total cells). Conclusions This procedure can be used to prepare clinical-grade cells suitable for use in human allotransplantation in a GMP environment. PMID:21905958

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

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.

NANOG Plays a Hierarchical Role in the Transcription Network Regulating the Pluripotency and Plasticity of Adipose Tissue-Derived Stem Cells

PubMed Central

Pitrone, Maria; Pizzolanti, Giuseppe; Tomasello, Laura; Coppola, Antonina; Morini, Lorenzo; Pantuso, Gianni; Ficarella, Romina; Guarnotta, Valentina; Perrini, Sebastio; Giorgino, Francesco; Giordano, Carla

2017-01-01

The stromal vascular cell fraction (SVF) of visceral and subcutaneous adipose tissue (VAT and SAT) has increasingly come into focus in stem cell research, since these compartments represent a rich source of multipotent adipose-derived stem cells (ASCs). ASCs exhibit a self-renewal potential and differentiation capacity. Our aim was to study the different expression of the embryonic stem cell markers NANOG (homeobox protein NANOG), SOX2 (SRY (sex determining region Y)-box 2) and OCT4 (octamer-binding transcription factor 4) and to evaluate if there exists a hierarchal role in this network in ASCs derived from both SAT and VAT. ASCs were isolated from SAT and VAT biopsies of 72 consenting patients (23 men, 47 women; age 45 ± 10; BMI between 25 ± 5 and 30 ± 5 range) undergoing elective open-abdominal surgery. Sphere-forming capability was evaluated by plating cells in low adhesion plastic. Stem cell markers CD90, CD105, CD29, CD31, CD45 and CD146 were analyzed by flow cytometry, and the stem cell transcription factors NANOG, SOX2 and OCT4 were detected by immunoblotting and real-time PCR. NANOG, SOX2 and OCT4 interplay was explored by gene silencing. ASCs from VAT and SAT confirmed their mesenchymal stem cell (MSC) phenotype expressing the specific MSC markers CD90, CD105, NANOG, SOX2 and OCT4. NANOG silencing induced a significant OCT4 (70 ± 0.05%) and SOX2 (75 ± 0.03%) downregulation, whereas SOX2 silencing did not affect NANOG gene expression. Adipose tissue is an important source of MSC, and siRNA experiments endorse a hierarchical role of NANOG in the complex transcription network that regulates pluripotency. PMID:28545230

Human trabecular meshwork cells exhibit several characteristics of, but are distinct from, adipose-derived mesenchymal stem cells.

PubMed

Morgan, Joshua T; Wood, Joshua A; Walker, Naomi J; Raghunathan, Vijay Krishna; Borjesson, Dori L; Murphy, Christopher J; Russell, Paul

2014-01-01

To support the growing promise of regenerative medicine in glaucoma, we characterized the similarities and differences between human trabecular meshwork (HTM) cells and human mesenchymal stem cells (hMSCs). HTM cells and hMSCs were phenotypically characterized by flow cytometry. Using quantitative polymerase chain reaction, the expression of myoc, angptl7, sox2, pou5f1, and notch1 was determined in both cell types with and without dexamethasone (Dex). Immunosuppressive behavior of HTM cells and hMSCs was determined using T cells activated with phytohemagglutinin. T-cell proliferation was determined using BrdU incorporation and flow cytometry. Multipotency of HTM cells and hMSCs was determined using adipogenic and osteogenic differentiation media as well as aqueous humor (AH). Alpha-smooth muscle actin (αSMA) expression was determined in HTM cells, hMSCs, and HTM tissue. Phenotypically, HTM and hMSCs expressed CD73, CD90, CD105, and CD146 but not CD31, CD34, and CD45 and similar sox2, pou5f1, and notch1 expression. Both cell types suppressed T-cell proliferation. However, HTM cells, but not hMSCs, upregulated myoc and angptl7 in response to Dex. Additionally, HTM cells did not differentiate into adipocytes or osteocytes. Culture of hMSCs in 20%, but not 100%, AH potently induced alkaline phosphatase activity. HTM cells in culture possessed uniformly strong expression of αSMA, which contrasted with the limited expression in hMSCs and spatially discrete expression in HTM tissue. HTM cells possess a number of important similarities with hMSCs but lack multipotency, one of the defining characteristics of stem cells. Further work is needed to explore the molecular mechanisms and functional implications underlying the phenotypic similarities.

Human Trabecular Meshwork Cells Exhibit Several Characteristics of, but Are Distinct from, Adipose-Derived Mesenchymal Stem Cells

PubMed Central

Morgan, Joshua T.; Wood, Joshua A.; Walker, Naomi J.; Raghunathan, Vijay Krishna; Borjesson, Dori L.; Murphy, Christopher J.

2014-01-01

Abstract Purpose: To support the growing promise of regenerative medicine in glaucoma, we characterized the similarities and differences between human trabecular meshwork (HTM) cells and human mesenchymal stem cells (hMSCs). Methods: HTM cells and hMSCs were phenotypically characterized by flow cytometry. Using quantitative polymerase chain reaction, the expression of myoc, angptl7, sox2, pou5f1, and notch1 was determined in both cell types with and without dexamethasone (Dex). Immunosuppressive behavior of HTM cells and hMSCs was determined using T cells activated with phytohemagglutinin. T-cell proliferation was determined using BrdU incorporation and flow cytometry. Multipotency of HTM cells and hMSCs was determined using adipogenic and osteogenic differentiation media as well as aqueous humor (AH). Alpha-smooth muscle actin (αSMA) expression was determined in HTM cells, hMSCs, and HTM tissue. Results: Phenotypically, HTM and hMSCs expressed CD73, CD90, CD105, and CD146 but not CD31, CD34, and CD45 and similar sox2, pou5f1, and notch1 expression. Both cell types suppressed T-cell proliferation. However, HTM cells, but not hMSCs, upregulated myoc and angptl7 in response to Dex. Additionally, HTM cells did not differentiate into adipocytes or osteocytes. Culture of hMSCs in 20%, but not 100%, AH potently induced alkaline phosphatase activity. HTM cells in culture possessed uniformly strong expression of αSMA, which contrasted with the limited expression in hMSCs and spatially discrete expression in HTM tissue. Conclusions: HTM cells possess a number of important similarities with hMSCs but lack multipotency, one of the defining characteristics of stem cells. Further work is needed to explore the molecular mechanisms and functional implications underlying the phenotypic similarities. PMID:24456002

The number of tumor infiltrating T-cell subsets in lymph nodes from patients with Hodgkin lymphoma is associated with the outcome after first line ABVD therapy.

PubMed

Alonso-Álvarez, Sara; Vidriales, Maria Belén; Caballero, Maria Dolores; Blanco, Oscar; Puig, Noemí; Martin, Alejandro; Peñarrubia, Maria Jesús; Zato, Esther; Galende, Josefina; Bárez, Abelardo; Alcoceba, Miguel; Orfão, Alberto; González, Marcos; García-Sanz, Ramón

2017-05-01

Prognostic factors in Hodgkin lymphoma (HL) still fail to accurately identify high-risk patients. Tumor microenvironment in HL is a current focus of research for risk definition but few studies have focused on infiltrating lymphocytes. Here, we analyzed the number of tumor infiltrating lymphocytes by flow cytometry in diagnostic biopsies from 96 HL homogeneously treated patients with ABVD with or without radiotherapy. Most lymph node cells were lymphocytes (90 ± 17), with a median T/B/NK distribution of 74%/26%/0.7%, and CD4 + T-cell predominance. The amount of CD19 + B cells, and NK cells did not show association with disease features. However, high numbers of CD8 + and CD4 + cells were associated with better and poorer outcomes, respectively. Patients with ≥15% cytotoxic CD8 + cells among the total cell population had a longer 10-year freedom from treatment failure (FFTF) (93% vs. 73%, p=.04). In turn, cases with ≥75% of CD4 + infiltrating cells showed a significantly decreased FFTF (73% vs. 96%, p=.021). Consequently, CD4/CD8 ratio ≥5 associated with a poorer 10-year FFTF (69.5% vs. 94%, p=.02). This deleterious effect was particularly prominent in advanced disease (n = 58, p=.01). In multivariate analysis, a CD4/CD8 ratio ≥5 was the only independent variable to predict for treatment failure (HR = 4.5, 95% confidence interval, 1.2-16.8). In conclusion, our study shows that high CD4 + and low CD8 + T-cells infiltrates of tumor specimens associate with poor prognosis in HL patients, and CD4/CD8 ratio might be potentially useful for tailoring therapy.

Identification and validation of multiple cell surface markers of clinical-grade adipose-derived mesenchymal stromal cells as novel release criteria for good manufacturing practice-compliant production.

PubMed

Camilleri, Emily T; Gustafson, Michael P; Dudakovic, Amel; Riester, Scott M; Garces, Catalina Galeano; Paradise, Christopher R; Takai, Hideki; Karperien, Marcel; Cool, Simon; Sampen, Hee-Jeong Im; Larson, A Noelle; Qu, Wenchun; Smith, Jay; Dietz, Allan B; van Wijnen, Andre J

2016-08-11

Clinical translation of mesenchymal stromal cells (MSCs) necessitates basic characterization of the cell product since variability in biological source and processing of MSCs may impact therapeutic outcomes. Although expression of classical cell surface markers (e.g., CD90, CD73, CD105, and CD44) is used to define MSCs, identification of functionally relevant cell surface markers would provide more robust release criteria and options for quality control. In addition, cell surface expression may distinguish between MSCs from different sources, including bone marrow-derived MSCs and clinical-grade adipose-derived MSCs (AMSCs) grown in human platelet lysate (hPL). In this work we utilized quantitative PCR, flow cytometry, and RNA-sequencing to characterize AMSCs grown in hPL and validated non-classical markers in 15 clinical-grade donors. We characterized the surface marker transcriptome of AMSCs, validated the expression of classical markers, and identified nine non-classical markers (i.e., CD36, CD163, CD271, CD200, CD273, CD274, CD146, CD248, and CD140B) that may potentially discriminate AMSCs from other cell types. More importantly, these markers exhibit variability in cell surface expression among different cell isolates from a diverse cohort of donors, including freshly prepared, previously frozen, or proliferative state AMSCs and may be informative when manufacturing cells. Our study establishes that clinical-grade AMSCs expanded in hPL represent a homogeneous cell culture population according to classical markers,. Additionally, we validated new biomarkers for further AMSC characterization that may provide novel information guiding the development of new release criteria. Use of Autologous Bone Marrow Aspirate Concentrate in Painful Knee Osteoarthritis (BMAC): Clinicaltrials.gov NCT01931007 . Registered August 26, 2013. MSC for Occlusive Disease of the Kidney: Clinicaltrials.gov NCT01840540 . Registered April 23, 2013. Mesenchymal Stem Cell Therapy in Multiple System Atrophy: Clinicaltrials.gov NCT02315027 . Registered October 31, 2014. Efficacy and Safety of Adult Human Mesenchymal Stem Cells to Treat Steroid Refractory Acute Graft Versus Host Disease. Clinicaltrials.gov NCT00366145 . Registered August 17, 2006. A Dose-escalation Safety Trial for Intrathecal Autologous Mesenchymal Stem Cell Therapy in Amyotrophic Lateral Sclerosis. Clinicaltrials.gov NCT01609283 . Registered May 18, 2012.

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

Deletion of CD73 in mice leads to aortic valve dysfunction.

PubMed

Zukowska, P; Kutryb-Zajac, B; Jasztal, A; Toczek, M; Zabielska, M; Borkowski, T; Khalpey, Z; Smolenski, R T; Slominska, E M

2017-06-01

Aortic stenosis is known to involve inflammation and thrombosis. Changes in activity of extracellular enzyme - ecto-5'-nucleotidase (referred also as CD73) can alter inflammatory and thrombotic responses. This study aimed to evaluate the effect of CD73 deletion in mice on development of aortic valve dysfunction and to compare it to the effect of high-fat diet. Four groups of mice (normal-diet Wild Type (WT), high-fat diet WT, normal diet CD73-/-, high-fat diet CD73-/-) were maintained for 15weeks followed by echocardiographic analysis of aortic valve function, measurement of aortic surface activities of nucleotide catabolism enzymes as well as alkaline phosphatase activity, mineral composition and histology of aortic valve leaflets. CD73-/- knock out led to an increase in peak aortic flow (1.06±0.26m/s) compared to WT (0.79±0.26m/s) indicating obstruction. Highest values of peak aortic flow (1.26±0.31m/s) were observed in high-fat diet CD73-/- mice. Histological analysis showed morphological changes in CD73-/- including thickening and accumulation of dark deposits, proved to be melanin. Concentrations of Ca 2+ , Mg 2+ and PO 4 3- in valve leaflets were elevated in CD73-/- mice. Alkaline phosphatase (ALP) activity was enhanced after ATP treatment and reduced after adenosine treatment in aortas incubated in osteogenic medium. AMP hydrolysis in CD73-/- was below 10% of WT. Activity of ecto-adenosine deaminase (eADA), responsible for adenosine deamination, in the CD73-/- was 40% lower when compared to WT. Deletion of CD73 in mice leads to aortic valve dysfunction similar to that induced by high-fat diet suggesting important role of this surface protein in maintaining heart valve integrity. Copyright © 2017 Elsevier B.V. All rights reserved.

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.

Osteogenic differentiation of human dental papilla mesenchymal cells

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

Ikeda, Etsuko; Hirose, Motohiro; Kotobuki, Noriko

We isolated dental papilla from impacted human molar and proliferated adherent fibroblastic cells after collagenase treatment of the papilla. The cells were negative for hematopoietic markers but positive for CD29, CD44, CD90, CD105, and CD166. When the cells were further cultured in the presence of {beta}-glycerophosphate, ascorbic acid, and dexamethasone for 14 days, mineralized areas together with osteogenic differentiation evidenced by high alkaline phosphatase activity and osteocalcin contents were observed. The differentiation was confirmed at both protein and gene expression levels. The cells can also be cryopreserved and, after thawing, could show in vivo bone-forming capability. These results indicate thatmore » mesenchymal type cells localize in dental papilla and that the cells can be culture expanded/utilized for bone tissue engineering.« less

[Differentiation of bone marrow derived from mesenchymal stem cells into cardiomyocyte-like cells induced by co-culture with rat myocardial cells].

PubMed

Zhang, Rong-Li; Jiang, Er-Lie; Wang, Mei; Zhou, Zheng; Zhai, Wen-Jing; Zhai, Wei-Hua; Wang, Hua; Wang, Zhi-Yong; Bao, Yu-Shi; DU, Hong; Han, Ming-Zhe

2008-10-01

The study was purposed to investigate the differentiation ability of mesenchymal stem cells (MSCs) into myocardial cells in vitro. Rat bone marrow-derived MSCs were labeled and co-cultured with neonatal rat cardiomyocytes (CM) for 5 - 7 days. The expression of cell surface antigens was detected by flow cytometry, and the expression of muscle-specific marker myosin and troponin T in labeled cells was detected by immunofluorescence. The results showed that in vitro cultured MSCs expressed CD90, CD44, CD105, CD54, not expressed CD34, CD45, CD31. After co-cultured with neonatal rat CM, labeled MSCs differentiated into cardiomyocyte-like cells expressing myosin and troponin T. It is concluded that MSCs can differentiate into cardiomyocyte-like cells when co-cultured with neonatal myocardial cells in vitro. In co-culture of two kind of cells in ratio of four to one showed obvious efficacy differentiating MSCs into CMs.

Generation of immunosuppressive mesenchymal stem cells in allogeneic human serum.

PubMed

Le Blanc, Katarina; Samuelsson, Håkan; Lönnies, Lena; Sundin, Mikael; Ringdén, Olle

2007-10-27

Mesenchymal stem cells (MSC) may be used to treat acute graft-versus-host disease and for tissue repair. In vitro expansion of MSC has been achieved in the presence of fetal calf serum (FCS). For safety and regulatory reasons, we explored if FCS could be replaced by human blood group AB serum. Proliferation and fold increase of MSC was higher in the presence of AB-serum, compared to FCS. Similar to cells generated in FCS media, MSC from AB-serum media were more than 95% positive for CD90, CD105 and human leukocyte antigen (HLA) class I, and negative for hematopoietic and endothelial markers CD14, CD31, CD34, CD45, and CD80. HLA class II expression was higher in MSC generated in AB-serum, but decreased with higher passage numbers. MSC generated in AB-serum suppressed lymphocyte proliferation in mixed lymphocyte cultures and after stimulation with phytohemagglutinin. MSC expanded in AB-serum and FCS have similar in vitro properties.

Endoglin (CD105) expression on microvessel endothelial cells in juvenile nasopharyngeal angiofibroma: tissue microarray analysis and association with prognostic significance.

PubMed

Wang, Jing-Jing; Sun, Xi-Cai; Hu, Li; Liu, Zhuo-Fu; Yu, Hua-Peng; Li, Han; Wang, Shu-Yi; Wang, De-Hui

2013-12-01

The purpose of this study was to examine endoglin (CD105) expression on microvessel endothelial cells (ECs) in juvenile nasopharyngeal angiofibroma (JNA) and its relationship with recurrence. Immunohistochemistry was performed to detect CD105 expression in a tissue microarray from 70 patients with JNA. Correlation between CD105 expression on microvessel ECs and clinicopathological features, as well as tumor recurrence, were analyzed. Immunohistochemistry revealed CD105 expression on ECs but not in stroma of patients with JNA. Chi-square analysis indicated CD105-based microvessel density (MVD) was correlated with JNA recurrence (p = .013). Univariate and multivariate analyses determined that MVD was a significant predictor of time to recurrence (p = .009). The CD105-based MVD was better for predicting disease recurrence (AUROC: 0.673; p = .036) than other clinicopathological features. MVD is a useful predictor for poor prognosis of patients with JNA after curative resection. Angiogenesis, which may play an important role in the occurrence and development of JNA, is therefore a potential therapeutic target for JNA. Copyright © 2013 Wiley Periodicals, Inc., A Wiley Company.

[Long-term expansion of multipotent mesenchymal stromal cells under reduced oxygen tension].

PubMed

Rylova, Iu V; Buravkova, L B

2013-01-01

We have shown that the decrease in oxygen tension in the culture medium of multipotent mesenchymal stromal cells (MMSCs) results in a short-term reduction in the proportion of CD73(+)-cells in the population, without effecting the number of cells expressing other constitutive surface markers (CD90 and CD105). In this case, the heterogeneity of the cell population declined: large spread cells disappeared. The proliferative activity of MMSCs significantly increased and remained stable in conditions in which the oxygen content was close to the tissue oxygen levels (5% O2). At lower oxygen concentration, proliferative activity of the cells gradually reduced from passages 3-4. The increase in proliferative activity was not accompanied by increased expression of telomerase gene indicateding the alsance of cell transformation. However, genome-wide analysis of MMSC gene expression level revealed changes in expression of cyclins (CCND2 and PCNA), regulatory subunit cyclin-dependent kinase (CKS2) and an inhibitor of cyclin-dependent kinase (CDKN2C), regulating the cell cycle, which is obviously facilitated the increase in the proliferative capacity of cells at lower oxygen tension.

Production and Breeding of Transgenic Cloned Pigs Expressing Human CD73.

PubMed

Lee, Seung-Chan; Lee, Haesun; Oh, Keon Bong; Hwang, In-Sul; Yang, Hyeon; Park, Mi-Ryung; Ock, Sun-A; Woo, Jae-Seok; Im, Gi-Sun; Hwang, Seongsoo

2017-06-01

One of the reasons to causing blood coagulation in the tissue of xenografted organs was known to incompatibility of the blood coagulation and anti-coagulation regulatory system between TG pigs and primates. Thus, overexpression of human CD73 (hCD73) in the pig endothelial cells is considered as a method to reduce coagulopathy after pig-to-non-human-primate xenotransplantation. This study was performed to produce and breed transgenic pigs expressing hCD73 for the studies immune rejection responses and could provide a successful application of xenotransplantation. The transgenic cells were constructed an hCD73 expression vector under control porcine Icam2 promoter (pIcam2-hCD73) and established donor cell lines expressing hCD73. The numbers of transferred reconstructed embryos were 127 ± 18.9. The pregnancy and delivery rate of surrogates were 8/18 (44%) and 3/18 (16%). The total number of delivered cloned pigs were 10 (2 alive, 7 mummy, and 1 died after birth). Among them, three live hCD73-pigs were successfully delivered by Caesarean section, but one was dead after birth. The two hCD73 TG cloned pigs had normal reproductive ability. They mated with wild type (WT) MGH (Massachusetts General Hospital) female sows and produced totally 16 piglets. Among them, 5 piglets were identified as hCD73 TG pigs. In conclusion, we successfully generated the hCD73 transgenic cloned pigs and produced their litters by natural mating. It can be possible to use a mate for the production of multiple transgenic pigs such as α-1,3-galactosyltransferase knock-out /hCD46 for xenotransplantation.

Effective elimination of liver cancer stem-like cells by CD90 antibody targeted thermosensitive magnetoliposomes

PubMed Central

Yang, Rui; An, Li Y.; Miao, Qin F.; Li, Feng M.; Han, Yong; Wang, Hui X.; Liu, Dang P.; Chen, Rong; Tang, Sha Q.

2016-01-01

Aim To investigate the use of thermosensitive magnetoliposomes (TMs) loaded with magnetic iron oxide (Fe3O4) and the anti-cancer stem cell marker CD90 (CD90@TMs) to target and kill CD90+ liver cancer stem cells (LCSCs). Methods The hepatocellular carcinoma cell line Huh7 was used to separate CD90+ LCSCs by magnetic-activated cell sorting. CD90@TMs was characterized and their ability to target CD90+ LCSCs was determined. Experiments were used to investigate whether CD90@TMs combined with magnetic hyperthermia could effectively eliminate CD90+ LCSCs. Results The present study demonstrated that CD90+ LCSCs with stem cells properties were successfully isolated. We also successfully prepared CD90@TMs that was almost spherical and uniform with an average diameter of 130±4.6 nm and determined that magnetic iron oxide could be incorporated and retained a superparamagnetic response. CD90@TMs showed good targeting and increased inhibition of CD90+ LCSCs in vitro and in vivo compared to TMs. Conclusion CD90@TMs can be used for controlled and targeted delivery of anticancer drugs, which may offer a promising alternative for HCC therapy. PMID:27145285

A small population of resident limb bud mesenchymal cells express few MSC-associated markers, but the expression of these markers is increased immediately after cell culture.

PubMed

Marín-Llera, Jessica Cristina; Chimal-Monroy, Jesús

2018-05-01

Skeletal progenitors are derived from resident limb bud mesenchymal cells of the vertebrate embryos. However, it remains poorly understood if they represent stem cells, progenitors, or multipotent mesenchymal stromal cells (MSC). Derived-MSC of different adult tissues under in vitro experimental conditions can differentiate into the same cellular lineages that are present in the limb. Here, comparing non-cultured versus cultured mesenchymal limb bud cells, we determined the expression of MSC-associated markers, the in vitro differentiation capacity and their gene expression profile. Results showed that in freshly isolated limb bud mesenchymal cells, the proportion of cells expressing Sca1, CD44, CD105, CD90, and CD73 is very low and a low expression of lineage-specific genes was observed. However, recently seeded limb bud mesenchymal cells acquired Sca1 and CD44 markers and the expression of the key differentiation genes Runx2 and Sox9, while Scx and Pparg genes decreased. Also, their chondrogenic differentiation capacity decreased through cellular passages while the osteogenic increased. Our findings suggest that the modification of the cell adhesion process through the in vitro method changed the limb mesenchymal cell immunophenotype leading to the expression and maintenance of common MSC-associated markers. These findings could have a significant impact on MSC study and isolation strategy because they could explain common variations observed in the MSC immunophenotype in different tissues. © 2018 International Federation for Cell Biology.

Mesenchymal stem cells derived from bone marrow of diabetic patients portrait unique markers influenced by the diabetic microenvironment.

PubMed

Phadnis, Smruti M; Ghaskadbi, Surendra M; Hardikar, Anandwardhan A; Bhonde, Ramesh R

2009-01-01

Cellular microenvironment is known to play a critical role in the maintenance of human bone marrow-derived mesenchymal stem cells (BM-MSCs). It was uncertain whether BM-MSCs obtained from a 'diabetic milieu' (dBM-MSCs) offer the same regenerative potential as those obtained from healthy (non-diabetic) individuals (hBM-MSCs). To investigate the effect of diabetic microenvironment on human BM-MSCs, we isolated and characterized these cells from diabetic patients (dBM-MSCs). We found that dBM-MSCs expressed mesenchymal markers such as vimentin, smooth muscle actin, nestin, fibronectin, CD29, CD44, CD73, CD90, and CD105. These cells also exhibited multilineage differentiation potential, as evident from the generation of adipocytes, osteocytes, and chondrocytes when exposed to lineage specific differentiation media. Although the cells were similar to hBM-MSCs, 6% (3/54) of dBM-MSCs expressed proinsulin/C-peptide. Emanating from the diabetic microenvironmental milieu, we analyzed whether in vitro reprogramming could afford the maturation of the islet-like clusters (ICAs) derived from dBM-MSCs. Upon mimicking the diabetic hyperglycemic niche and the supplementation of fetal pancreatic extract, to differentiate dBM-MSCs into pancreatic lineage in vitro, we observed rapid differentiation and maturation of dBM-MSCs into islet-like cell aggregates. Thus, our study demonstrated that diabetic hyperglycemic microenvironmental milieu plays a major role in inducing the differentiation of human BM-MSCs in vivo and in vitro.

Expression and structural features of endoglin (CD105), a transforming growth factor beta1 and beta3 binding protein, in human melanoma.

PubMed Central

Altomonte, M.; Montagner, R.; Fonsatti, E.; Colizzi, F.; Cattarossi, I.; Brasoveanu, L. I.; Nicotra, M. R.; Cattelan, A.; Natali, P. G.; Maio, M.

1996-01-01

Human endoglin (CD105) is a member of the transforming growth factor beta (TGF-beta) receptor family that binds TGF-beta1 and -beta3, but not TGF-beta2, on human endothelial cells. Immunohistochemical analyses demonstrated that CD105 is expressed on normal and neoplastic cells of the melanocytic lineage. The anti-CD105 MAb, MAEND3, stained 50, 25 and 34% of intradermal naevi, primary and metastatic melanomas investigated, respectively, and nine out of 12 melanoma cell lines. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed that CD105 expressed by melanoma cells consists of a homodimeric protein with an apparent molecular weight of 180 and 95 kDa under non-reducing and reducing conditions. Cross-linking of 125I-labelled TGF-beta1 to melanoma cells, Mel 97, by disuccinimidyl suberate (DSS) demonstrated that CD105 expressed on pigmented cells binds TGF-beta1; the pattern of binding of TGF-beta1 to melanoma cells was found to be similar to that of human umbilical vein endothelial cells. The addition of exogenous, bioactive TGF-beta1 significantly (P<0.05) inhibited the growth of CD105-positive melanoma cells, Mel 97, but did not affect that of CD105-negative melanoma cells, F0-1. These data, altogether, demonstrate that CD105 is expressed on pigmented cells and might play a functionally relevant role in the biology of human melanoma cells by regulating their sensitivity to TGF-betas. Images Figure 1 Figure 3 Figure 4 PMID:8932339

[Expression, crystallization and crystallographic study of the 1st IgV domain of human CD96].

PubMed

Jiang, Wenjing; Zhang, Shuijun; Yan, Jinghua; Guo, Ning

2013-05-01

CD96 (Tactile) is an adhesion receptor expressed mainly on activated T cells, NK cells. As a family member of the immunoglobulin-like cell receptor, CD96 consists of three immunoglobulin-like domains (V1, V2/C and C) in the extracellular region. Recent studies have shown that the 1st IgV domain of CD96 (CD96V1) plays an essential role in cell adhesion and NK cell-mediated killing. In this study, the 1st IgV domain of human CD96 (hCD96V1) was cloned and expressed in Escherichia coli (BL21). The soluble protein was obtained by refolding of the hCD96V1 inclusion bodies. From analytical ultracentrifugation, we could predict that CD96 V1 maily exists as dimer with approximate molecular weight of 26.9 kDa. The protein was then successfully crystallized using the sitting-drop vapour-diffusion method. The crystals diffracted to 1.9 angstrom resolution and belonged to space group P21, with unit-cell parameters a = 35.1, b = 69.5, c = 49.6A, alpha=gamma=90 degrees, beta=105.4 degrees.

Odontoblastic differentiation of dental pulp stem cells from healthy and carious teeth on an original PCL-based 3D scaffold.

PubMed

Louvrier, A; Euvrard, E; Nicod, L; Rolin, G; Gindraux, F; Pazart, L; Houdayer, C; Risold, P Y; Meyer, F; Meyer, C

2018-05-01

To isolate and characterize dental pulp stem cells (DPSCs) obtained from carious and healthy mature teeth extracted when conservative treatment was not possible or for orthodontic reasons; to evaluate the ability of DPSCs to colonize, proliferate and differentiate into functional odontoblast-like cells when cultured onto a polycaprolactone cone made by jet-spraying and prototyped into a design similar to a gutta-percha cone. DPSCs were obtained from nine carious and 12 healthy mature teeth. Then cells were characterized by flow cytometry and submitted to multidifferentiation to confirm their multipotency. These DPSCs were then cultured on a polycaprolactone cone in an odontoblastic differentiation medium. Cell proliferation, colonization of the biomaterial and functional differentiation of cells were histologically assessed. For the characterization, a t-Student test was used to compare the two groups. In all cell cultures, characterization highlighted a mesenchymal stem cell phenotype (CD105+, CD90+, CD73+, CD11b-, CD34-, CD45-, HLA-DR-). No significant differences were found between cultures obtained from carious and healthy mature teeth. DPSCs from both origins were able to differentiate into osteocytes, adipocytes and chondrocytes. Cell colonization was observed both on the surface and in the thickness of polycaprolactone cones as well as a mineralized pericellular matrix deposit composed of type I collagen, alkaline phosphatase, osteocalcin and dentin sialophosphoprotein. DPSCs were isolated from both carious and healthy mature teeth. They were able to colonize and proliferate within a polycaprolactone cone and could be differentiated into functional odontoblast-like cells. © 2017 International Endodontic Journal. Published by John Wiley & Sons Ltd.

Ex vivo identification and characterization of a population of CD13(high) CD105(+) CD45(-) mesenchymal stem cells in human bone marrow.

PubMed

Muñiz, Carmen; Teodosio, Cristina; Mayado, Andrea; Amaral, Ana Teresa; Matarraz, Sergio; Bárcena, Paloma; Sanchez, Maria Luz; Alvarez-Twose, Iván; Diez-Campelo, María; García-Montero, Andrés C; Blanco, Juan F; Del Cañizo, Maria Consuelo; del Pino Montes, Javier; Orfao, Alberto

2015-09-07

Mesenchymal stem cells (MSCs) are multipotent cells capable of self-renewal and multilineage differentiation. Their multipotential capacity and immunomodulatory properties have led to an increasing interest in their biological properties and therapeutic applications. Currently, the definition of MSCs relies on a combination of phenotypic, morphological and functional characteristics which are typically evaluated upon in vitro expansion, a process that may ultimately lead to modulation of the immunophenotypic, functional and/or genetic features of these cells. Therefore, at present there is great interest in providing markers and phenotypes for direct in vivo and ex vivo identification and isolation of MSCs. Multiparameter flow cytometry immunophenotypic studies were performed on 65 bone marrow (BM) samples for characterization of CD13(high) CD105(+) CD45(-) cells. Isolation and expansion of these cells was performed in a subset of samples in parallel to the expansion of MSCs from mononuclear cells following currently established procedures. The protein expression profile of these cells was further assessed on (paired) primary and in vitro expanded BM MSCs, and their adipogenic, chondrogenic and osteogenic differentiation potential was also determined. Our results show that the CD13(high) CD105(+) CD45(-) immunophenotype defines a minor subset of cells that are systematically present ex vivo in normal/reactive BM (n = 65) and that display immunophenotypic features, plastic adherence ability, and osteogenic, adipogenic and chondrogenic differentiation capacities fully compatible with those of MSCs. In addition, we also show that in vitro expansion of these cells modulates their immunophenotypic characteristics, including changes in the expression of markers currently used for the definition of MSCs, such as CD105, CD146 and HLA-DR. BM MSCs can be identified ex vivo in normal/reactive BM, based on a robust CD13(high) CD105(+) and CD45(-) immunophenotypic profile. Furthermore, in vitro expansion of these cells is associated with significant changes in the immunophenotypic profile of MSCs.

Circulating Mesenchymal Stem Cells Microparticles in Patients with Cerebrovascular Disease

PubMed Central

Cho, Yeon Hee; Kang, Ho Young; Hyung, Na Kyum; Kim, Donghee; Lee, Ji Hyun; Nam, Ji Yoon; Bang, Oh Young

2012-01-01

Preclinical and clinical studies have shown that the application of CD105+ mesenchymal stem cells (MSCs) is feasible and may lead to recovery after stroke. In addition, circulating microparticles are reportedly functional in various disease conditions. We tested the levels of circulating CD105+ microparticles in patients with acute ischemic stroke. The expression of CD105 (a surface marker of MSCs) and CXCR4 (a CXC chemokine receptor for MSC homing) on circulating microparticles was evaluated by flow cytometry of samples from 111 patients and 50 healthy subjects. The percentage of apoptotic CD105 microparticles was determined based on annexin V (AV) expression. The relationship between serum levels of CD105+/AV− microparticles, stromal cells derived factor-1α (SDF-1α), and the extensiveness of cerebral infarcts was also evaluated. CD105+/AV− microparticles were higher in stroke patients than control subjects. Correlation analysis showed that the levels of CD105+/AV− microparticles increased as the baseline stroke severity increased. Multivariate testing showed that the initial severity of stroke was independently associated with circulating CD105+/AV− microparticles (OR, 1.103 for 1 point increase in the NIHSS score on admission; 95% CI, 1.032–1.178) after adjusting for other variables. The levels of CD105+/CXCR4+/AV− microparticles were also increased in patients with severe disability (r = 0.192, p = 0.046 for NIHSS score on admission), but were decreased with time after stroke onset (r = −0.204, p = 0.036). Risk factor profiles were not associated with the levels of circulating microparticles or SDF-1α. In conclusion, our data showed that stroke triggers the mobilization of MSC-derived microparticles, especially in patients with extensive ischemic stroke. PMID:22615882

A novel small-molecule compound targeting CD147 inhibits the motility and invasion of hepatocellular carcinoma cells

PubMed Central

Peng, Jian-long; Wang, Shi-jie; Geng, Jie-jie; Liu, Ji-de; Feng, Fei; Song, Fei; Li, Ling; Zhu, Ping; Jiang, Jian-li; Chen, Zhi-nan

2016-01-01

CD147, a type I transmembrane glycoprotein, is highly expressed in various cancer types and plays important roles in tumor progression, especially by promoting the motility and invasion of hepatocellular carcinoma (HCC) cells. These crucial roles make CD147 an attractive target for therapeutic intervention in HCC, but no small-molecule inhibitors of CD147 have been developed to date. To identify a candidate inhibitor, we used a pharmacophore model derived from the structure of CD147 to virtually screen over 300,000 compounds. The 100 highest-ranked compounds were subjected to biological assays, and the most potent one, dubbed AC-73 (ID number: AN-465/42834501), was studied further. We confirmed that AC-73 targeted CD147 and further demonstrated it can specifically disrupt CD147 dimerization. Moreover, molecular docking and mutagenesis experiments showed that the possible binding sites of AC-73 on CD147 included Glu64 and Glu73 in the N-terminal IgC2 domain, which two residues are located in the dimer interface of CD147. Functional assays revealed that AC-73 inhibited the motility and invasion of typical HCC cells, but not HCC cells that lacked the CD147 gene, demonstrating on-target action. Further, AC-73 reduced HCC metastasis by suppressing matrix metalloproteinase (MMP)-2 via down-regulation of the CD147/ERK1/2/signal transducer and activator of transcription 3 (STAT3) signaling pathway. Finally, AC-73 attenuated progression in an orthotopic nude mouse model of liver metastasis, suggesting that AC-73 or its derivatives have potential for use in HCC intervention. We conclude that the novel small-molecule inhibitor AC-73 inhibits HCC mobility and invasion, probably by disrupting CD147 dimerization and thereby mainly suppressing the CD147/ERK1/2/STAT3/MMP-2 pathways, which are crucial for cancer progression. PMID:26882566

A novel small-molecule compound targeting CD147 inhibits the motility and invasion of hepatocellular carcinoma cells.

PubMed

Fu, Zhi-guang; Wang, Li; Cui, Hong-yong; Peng, Jian-long; Wang, Shi-jie; Geng, Jie-jie; Liu, Ji-de; Feng, Fei; Song, Fei; Li, Ling; Zhu, Ping; Jiang, Jian-li; Chen, Zhi-nan

2016-02-23

CD147, a type I transmembrane glycoprotein, is highly expressed in various cancer types and plays important roles in tumor progression, especially by promoting the motility and invasion of hepatocellular carcinoma (HCC) cells. These crucial roles make CD147 an attractive target for therapeutic intervention in HCC, but no small-molecule inhibitors of CD147 have been developed to date. To identify a candidate inhibitor, we used a pharmacophore model derived from the structure of CD147 to virtually screen over 300,000 compounds. The 100 highest-ranked compounds were subjected to biological assays, and the most potent one, dubbed AC-73 (ID number: AN-465/42834501), was studied further. We confirmed that AC-73 targeted CD147 and further demonstrated it can specifically disrupt CD147 dimerization. Moreover, molecular docking and mutagenesis experiments showed that the possible binding sites of AC-73 on CD147 included Glu64 and Glu73 in the N-terminal IgC2 domain, which two residues are located in the dimer interface of CD147. Functional assays revealed that AC-73 inhibited the motility and invasion of typical HCC cells, but not HCC cells that lacked the CD147 gene, demonstrating on-target action. Further, AC-73 reduced HCC metastasis by suppressing matrix metalloproteinase (MMP)-2 via down-regulation of the CD147/ERK1/2/signal transducer and activator of transcription 3 (STAT3) signaling pathway. Finally, AC-73 attenuated progression in an orthotopic nude mouse model of liver metastasis, suggesting that AC-73 or its derivatives have potential for use in HCC intervention. We conclude that the novel small-molecule inhibitor AC-73 inhibits HCC mobility and invasion, probably by disrupting CD147 dimerization and thereby mainly suppressing the CD147/ERK1/2/STAT3/MMP-2 pathways, which are crucial for cancer progression.

Flow cytometric characterization of culture expanded multipotent mesenchymal stromal cells (MSCs) from horse adipose tissue: towards the definition of minimal stemness criteria.

PubMed

Pascucci, L; Curina, G; Mercati, F; Marini, C; Dall'Aglio, C; Paternesi, B; Ceccarelli, P

2011-12-15

In the last decades, multipotent mesenchymal progenitor cells have been isolated from many adult tissues of different species. The International Society for Cellular Therapy (ISCT) has recently established that multipotent mesenchymal stromal cells (MSCs) is the currently recommended designation. In this study, we used flow cytometry to evaluate the expression of several molecules related to stemness (CD90, CD44, CD73 and STRO-1) in undifferentiated, early-passaged MSCs isolated from adipose tissue of four donor horses (AdMSCs). The four populations unanimously expressed high levels of CD90 and CD44. On the contrary, they were unexpectedly negative to CD73. A small percentage of the cells, finally, showed the expression of STRO-1. This last result might be due to the existence of a small subpopulation of STRO-1+ cells or to a poor cross-reactivity of the antibody. A remarkable donor-to-donor consistency and reproducibility of these findings was demonstrated. The data presented herein support the idea that equine AdMSCs may be easily isolated and selected by adherence to tissue culture plastic and exhibit a surface profile characterized by some peculiar differences in comparison to those described in other species. Continued characterization of these cells will help to clarify several aspects of their biology and may ultimately enable the isolation of specific, purified subpopulations. Copyright © 2011 Elsevier B.V. All rights reserved.

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.

Differentiation of human umbilical cord mesenchymal stem cells into dermal fibroblasts in vitro

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

Han, Yanfu; Chai, Jiake, E-mail: cjk304@126.com; Sun, Tianjun

2011-10-07

Highlights: {yields} Mesenchymal stem cells (MSCs) are potential seed cells for tissue-engineered skin. {yields} Tissue-derived umbilical cord MSCs (UCMSCs) can readily be isolated in vitro. {yields} We induce UCMSCs to differentiate into dermal fibroblasts via conditioned medium. {yields} Collagen type I and collagen type III mRNA level was higher in differentiated cells. {yields} UCMSCs-derived fibroblast-like cells strongly express fibroblast-specific protein. -- Abstract: Tissue-derived umbilical cord mesenchymal stem cells (UCMSCs) can be readily obtained, avoid ethical or moral constraints, and show excellent pluripotency and proliferation potential. UCMSCs are considered to be a promising source of stem cells in regenerative medicine. Inmore » this study, we collected newborn umbilical cord tissue under sterile conditions and isolated UCMSCs through a tissue attachment method. UCMSC cell surface markers were examined using flow cytometry. On the third passage, UCMSCs were induced to differentiate into dermal fibroblasts in conditioned induction media. The induction results were detected using immunofluorescence with a fibroblast-specific monoclonal antibody and real time PCR for type I and type III collagen. UCMSCs exhibited a fibroblast-like morphology and reached 90% confluency 14 to 18 days after primary culture. Cultured UCMSCs showed strong positive staining for CD73, CD29, CD44, CD105, and HLA-I, but not CD34, CD45, CD31, or HLA-DR. After differentiation, immunostaining for collagen type I, type III, fibroblast-specific protein, vimentin, and desmin were all strongly positive in induced cells, and staining was weak or negative in non-induced cells; total transcript production of collagen type I and collagen type III mRNA was higher in induced cells than in non-induced cells. These results demonstrate that UCMSCs can be induced to differentiate into fibroblasts with conditioned induction media and, in turn, could be used as seed cells for tissue-engineered dermis.« less

Morphology, cell viability, karyotype, expression of surface markers and plasticity of three human primary cell line cultures before and after the cryostorage in LN2 and GN2.

PubMed

Del Pino, Alberto; Ligero, Gertrudis; López, María B; Navarro, Héctor; Carrillo, Jose A; Pantoll, Siobhan C; Díaz de la Guardia, Rafael

2015-02-01

Primary cell line cultures from human skin biopsies, adipose tissue and tumor tissue are valuable samples for research and therapy. In this regard, their derivation, culture, storage, transport and thawing are important steps to be studied. Towards this end, we wanted to establish the derivation, and identify the culture characteristics and the loss of viability of three human primary cell line cultures (human adult dermal fibroblasts (hADFs), human adult mesenchymal stem cells (hMSCs), and primary culture of tumor cells from lung adenocarcinoma (PCTCLA)). Compared to fresh hADFs, hMSCs and PCTCLA, thawed cells stored in a cryogenic Dewar tanks with liquid nitrogen (LN2), displayed 98.20% ± 0.99, 95.40% ± 1.41 and 93.31% ± 3.83 of cell viability, respectively. Thawed cells stored in a Dry Vapor Shipper container with gas phase (GN2), for 20 days, in addition displayed 4.61% ± 2.78, 3.70% ± 4.09 and 9.13% ± 3.51 of average loss of cells viability, respectively, showing strong correlation between the loss of viability in hADFs and the number of post-freezing days in the Dry Vapor Shipper. No significant changes in morphological characteristics or in the expression of surface markers (being hADFs, hMSCs and PCTCLA characterized by positive markers CD73+; CD90+; CD105+; and negative markers CD14-; CD20-; CD34-; and CD45-; n=2) were found. Chromosome abnormalities in the karyotype were not found. In addition, under the right conditions hMSCs were differentiated into adipogenic, osteogenic and chondrogenic lineages in vitro. In this paper, we have shown the characteristics of three human primary cell line cultures when they are stored in LN2 and GN2. Copyright © 2014 Elsevier Inc. All rights reserved.

Pluripotent Nontumorigenic Adipose Tissue‐Derived Muse Cells have Immunomodulatory Capacity Mediated by Transforming Growth Factor‐β1

PubMed Central

Gimeno, María L.; Fuertes, Florencia; Barcala Tabarrozzi, Andres E.; Attorressi, Alejandra I.; Cucchiani, Rodolfo; Corrales, Luis; Oliveira, Talita C.; Sogayar, Mari C.; Labriola, Leticia; Dewey, Ricardo A.

2016-01-01

Abstract Adult mesenchymal stromal cell‐based interventions have shown promising results in a broad range of diseases. However, their use has faced limited effectiveness owing to the low survival rates and susceptibility to environmental stress on transplantation. We describe the cellular and molecular characteristics of multilineage‐differentiating stress‐enduring (Muse) cells derived from adipose tissue (AT), a subpopulation of pluripotent stem cells isolated from human lipoaspirates. Muse‐AT cells were efficiently obtained using a simple, fast, and affordable procedure, avoiding cell sorting and genetic manipulation methods. Muse‐AT cells isolated under severe cellular stress, expressed pluripotency stem cell markers and spontaneously differentiated into the three germ lineages. Muse‐AT cells grown as spheroids have a limited proliferation rate, a diameter of ∼15 µm, and ultrastructural organization similar to that of embryonic stem cells. Muse‐AT cells evidenced high stage‐specific embryonic antigen‐3 (SSEA‐3) expression (∼60% of cells) after 7–10 days growing in suspension and did not form teratomas when injected into immunodeficient mice. SSEA‐3+‐Muse‐AT cells expressed CD105, CD29, CD73, human leukocyte antigen (HLA) class I, CD44, and CD90 and low levels of HLA class II, CD45, and CD34. Using lipopolysaccharide‐stimulated macrophages and antigen‐challenged T‐cell assays, we have shown that Muse‐AT cells have anti‐inflammatory activities downregulating the secretion of proinflammatory cytokines, such as interferon‐γ and tumor necrosis factor‐α. Muse‐AT cells spontaneously gained transforming growth factor‐β1 expression that, in a phosphorylated SMAD2‐dependent manner, might prove pivotal in their observed immunoregulatory activity through decreased expression of T‐box transcription factor in T cells. Collectively, the present study has demonstrated the feasibility and efficiency of obtaining Muse‐AT cells that can potentially be harnessed as immunoregulators to treat immune‐related disorders. Stem Cells Translational Medicine 2017;6:161–173 PMID:28170177

CD73 Protein as a Source of Extracellular Precursors for Sustained NAD+ Biosynthesis in FK866-treated Tumor Cells*

PubMed Central

Grozio, Alessia; Sociali, Giovanna; Sturla, Laura; Caffa, Irene; Soncini, Debora; Salis, Annalisa; Raffaelli, Nadia; De Flora, Antonio; Nencioni, Alessio; Bruzzone, Santina

2013-01-01

NAD+ is mainly synthesized in human cells via the “salvage” pathways starting from nicotinamide, nicotinic acid, or nicotinamide riboside (NR). The inhibition with FK866 of the enzyme nicotinamide phosphoribosyltransferase (NAMPT), catalyzing the first reaction in the “salvage” pathway from nicotinamide, showed potent antitumor activity in several preclinical models of solid and hematologic cancers. In the clinical studies performed with FK866, however, no tumor remission was observed. Here we demonstrate that low micromolar concentrations of extracellular NAD+ or NAD+ precursors, nicotinamide mononucleotide (NMN) and NR, can reverse the FK866-induced cell death, this representing a plausible explanation for the failure of NAMPT inhibition as an anti-cancer therapy. NMN is a substrate of both ectoenzymes CD38 and CD73, with generation of NAM and NR, respectively. In this study, we investigated the roles of CD38 and CD73 in providing ectocellular NAD+ precursors for NAD+ biosynthesis and in modulating cell susceptibility to FK866. By specifically silencing or overexpressing CD38 and CD73, we demonstrated that endogenous CD73 enables, whereas CD38 impairs, the conversion of extracellular NMN to NR as a precursor for intracellular NAD+ biosynthesis in human cells. Moreover, cell viability in FK866-treated cells supplemented with extracellular NMN was strongly reduced in tumor cells, upon pharmacological inhibition or specific down-regulation of CD73. Thus, our study suggests that genetic or pharmacologic interventions interfering with CD73 activity may prove useful to increase cancer cell sensitivity to NAMPT inhibitors. PMID:23880765

Cannabidiol Modulates the Immunophenotype and Inhibits the Activation of the Inflammasome in Human Gingival Mesenchymal Stem Cells

PubMed Central

Libro, Rosaliana; Scionti, Domenico; Diomede, Francesca; Marchisio, Marco; Grassi, Gianpaolo; Pollastro, Federica; Piattelli, Adriano; Bramanti, Placido; Mazzon, Emanuela; Trubiani, Oriana

2016-01-01

Human Gingival Mesenchymal Stem Cells (hGMSCs) are multipotential cells that can expand and differentiate in culture under specific and standardized conditions. In the present study, we have investigated whether in vitro pre-treatment of hGMSCs with Cannabidiol (CBD) can influence their expression profile, improving the therapeutic potential of this cell culture. Following CBD treatment (5 μM) for 24 h, gene expression analysis through Next Generation Sequencing (NGS) has revealed several genes differentially expressed between CBD-treated hGMSCs (CBD-hGMSCs) and control cells (CTR-hGMSCs) that were linked to inflammation and apoptosis. In particular, we have demonstrated that CBD treatment in hGMSCs prevented the activation of the NALP3-inflammasome pathway by suppressing the levels of NALP3, CASP1, and IL18, and in parallel, inhibited apoptosis, as demonstrated by the suppression of Bax. CBD treatment was also able to modulate the expression of the well-known mesenchymal stem cell markers (CD13, CD29, CD73, CD44, CD90, and CD166), and other surface antigens. Specifically, CBD led to the downregulation of genes codifying for antigens involved in the activation of the immune system (CD109, CD151, CD40, CD46, CD59, CD68, CD81, CD82, CD99), while it led to the upregulation of those implicated in the inhibition of the immune responses (CD47, CD55, CD276). In conclusion, the present study will provide a new simple and reproducible method for preconditioning hGMSCs with CBD, before transplantation, as an interesting strategy for improving the hGMSCs molecular phenotype, reducing the risk of immune or inflammatory reactions in the host, and in parallel, for increasing their survival and thus, their long-term therapeutic efficacy. PMID:27932991

Platinum desensitization in patients with carboplatin hypersensitivity: A single-institution retrospective study.

PubMed

Altwerger, Gary; Gressel, Gregory M; English, Diana P; Nelson, Wendelin K; Carusillo, Nina; Silasi, Dan-Arin; Azodi, Masoud; Santin, Alessandro; Schwartz, Peter E; Ratner, Elena S

2017-01-01

The carboplatin desensitization (CD) protocol presented here allows patients with either a positive skin test or a prior hypersensitivity reaction (HSR) to safely, rapidly and effectively continue with carboplatin infusions. Newly described factors can identify patients at risk for developing adverse events during CD. A retrospective review was performed on patients with gynecologic cancer who underwent CD between 2005 and 2014. The CD protocol uses a four-step dilution process over 3.5h. 129 patients underwent CD and completed a total of 788cycles. The desensitization protocol prevented HSRs in 96% (753 out of 788) of these cycles. Patients achieved an average of 6.1cycles (SD±4.55, range 0-23) with CD. The CD protocol allowed 73% (94 of 129) of the patients to undergo carboplatin infusion without reaction. Patients with moderate to life-threatening HSRs (grade 2 through 4) were 10.5years younger at initial CD than patients with grades 0 or 1 HSRs (52.3 vs. 63, P = 0.0307). One patient death occurred during her thirteenth desensitization cycle. The HSR in this case was complicated by pre-exisiting pulmonary hypertension. This is the largest study of its kind showing a safe, effective and rapid (3.5h) CD protocol. The majority of patients with a history of either carboplatin hypersensitivity reaction or a positive skin test completed the CD protocol without HSRs. Age was identified as a risk factor for HSR severity during CD. Age can be employed along with pre-load dependent cardiac conditions as a way to help risk stratify patients undergoing CD. Copyright © 2016 Elsevier Inc. All rights reserved.

1α,25-dihydroxyvitamin D3 acts via transforming growth factor-β to up-regulate expression of immunosuppressive CD73 on human CD4+ Foxp3- T cells.

PubMed

Mann, Elizabeth H; Chambers, Emma S; Chen, Yin-Huai; Richards, David F; Hawrylowicz, Catherine M

2015-11-01

Vitamin D deficiency is associated with increased incidence and severity of various immune-mediated diseases. Active vitamin D (1α,25-dihydroxyvitamin D3; 1,25(OH)2 D3) up-regulates CD4(+) T-cell expression of the purine ectonucleotidase CD39, a molecule that is associated with the generation of anti-inflammatory adenosine. Here we aimed to investigate the direct impact of 1,25(OH)2 D3 on expression of the downstream ecto-5'-nucleotidase CD73 by human CD4 T cells, and components of the transforming growth factor-β (TGF-β) pathway, which have been implicated in the modulation of CD73 by murine T cells. At 10(-8) to 10(-7) m, 1,25(OH)2 D3 significantly increased expression of CD73 on peripheral human CD4(+) T cells. Although 1,25(OH)2 D3 did not affect the mRNA expression of latent TGF-β1 , 1,25(OH)2 D3 did up-regulate expression of TGF-β-associated molecules [latency-associated peptide (LAP), glycophorin A repetitions predominant (GARP), GP96, neuropilin-1, thrombospondin-1 and αv integrin] which is likely to have contributed to the observed enhancement in TGF-β bioactivity. CD73 was highly co-expressed with LAP and GARP following 1,25(OH)2 D3 treatment, but unexpectedly, each of these cell surface molecules was expressed primarily on CD4(+) Foxp3(-) T cells, rather than CD4(+) Foxp3(+) T cells. Notably, neutralization of TGF-β significantly impaired 1,25(OH)2 D3-mediated induction of CD73. Collectively, we show that 1,25(OH)2 D3 enhances expression of CD73 on CD4(+) Foxp3(-) T cells in a process that is at least partially TGF-β-dependent. These data reveal an additional contributing mechanism by which vitamin D may be protective in immune-mediated disease. © 2015 John Wiley & Sons Ltd.

CD73-Generated Adenosine Is Critical for Immune Regulation during Toxoplasma gondii Infection

PubMed Central

Mahamed, Deeqa A.; Toussaint, Leon E.

2014-01-01

As an obligate intracellular pathogen, the apicomplexan parasite Toxoplasma gondii evades immune system-mediated clearance by undergoing stage differentiation to persist indefinitely in susceptible hosts. Previously, we found that mice deficient in the ectoenzyme CD73, which generates adenosine in the extracellular matrix, were resistant to chronic toxoplasmosis after oral infection with T. gondii. Resistance in CD73 knockout mice was due to a delay in parasite differentiation in the central nervous system (CNS). To further clarify the role of CD73 and extracellular adenosine in T. gondii pathogenesis, we infected wild-type (WT) and CD73−/− mice with T. gondii cysts systemically by the intraperitoneal (i.p.) route. In contrast to oral infection, i.p. infected CD73−/− mice were highly susceptible to immune-mediated pathology, with significantly increased infiltration of neutrophils and T cells into the peritoneal cavity. Administration of the broad-spectrum adenosine receptor agonist 5′-N-ethylcarboxamidoadenosine (NECA) protected CD73−/− mice against T. gondii-induced immunopathology, suggesting that the absence of CD73-generated adenosine led to the increased susceptibility in these mice. Peritoneal exudate cells from infected CD73−/− mice produced higher levels of the inflammatory mediators nitric oxide, tumor necrosis factor alpha (TNF-α), and interleukin-1β (IL-1β), without enhanced parasite killing or clearance. Bone marrow chimeras established that CD73 expression in both hematopoietic and nonhematopoietic compartments contributes to limiting T. gondii-induced immunopathology. In addition, mice deficient in the adenosine receptor A2A were more susceptible to immunopathology during intraperitoneal infection with T. gondii than WT mice. Thus, extracellular adenosine is a key immune regulator that limits collateral tissue damage due to an intracellular pathogen and promotes host survival. PMID:25452548

Elucidation of the EP defect in Diamond-Blackfan anemia by characterization and prospective isolation of human EPs.

PubMed

Iskander, Deena; Psaila, Bethan; Gerrard, Gareth; Chaidos, Aristeidis; En Foong, Hui; Harrington, Yvonne; Karnik, Leena C; Roberts, Irene; de la Fuente, Josu; Karadimitris, Anastasios

2015-04-16

Diamond-Blackfan anemia (DBA) is a disorder characterized by a selective defect in erythropoiesis. Delineation of the precise defect is hampered by a lack of markers that define cells giving rise to erythroid burst- and erythroid colony-forming unit (BFU-E and CFU-E) colonies, the clonogenic assays that quantify early and late erythroid progenitor (EEP and LEP) potential, respectively. By combining flow cytometry, cell-sorting, and single-cell clonogenic assays, we identified Lin(-)CD34(+)CD38(+)CD45RA(-)CD123(-)CD71(+)CD41a(-)CD105(-)CD36(-) bone marrow cells as EEP giving rise to BFU-E, and Lin(-)CD34(+/-)CD38(+)CD45RA(-)CD123(-)CD71(+)CD41a(-)CD105(+)CD36(+) cells as LEP giving rise to CFU-E, in a hierarchical fashion. We then applied these definitions to DBA and identified that, compared with controls, frequency, and clonogenicity of DBA, EEP and LEP are significantly decreased in transfusion-dependent but restored in corticosteroid-responsive patients. Thus, both quantitative and qualitative defects in erythroid progenitor (EP) contribute to defective erythropoiesis in DBA. Prospective isolation of defined EPs will facilitate more incisive study of normal and aberrant erythropoiesis. © 2015 by The American Society of Hematology.

Characterization of CD22 Expression in Acute Lymphoblastic Leukemia

PubMed Central

Shah, Nirali N.; Stetler-Stevenson, Maryalice; Yuan, Constance M.; Richards, Kelly; Delbrook, Cindy; Kreitman, Robert J.; Pastan, Ira; Wayne, Alan S.

2015-01-01

Background CD22 is a B-lineage differentiation antigen that has emerged as a leading therapeutic target in acute lymphoblastic leukemia (ALL). Procedure Properties of CD22 expression relevant to therapeutic targeting were characterized in primary samples obtained from children and young adults with relapsed and chemotherapy refractory B-precursor (pre-B) ALL. Results CD22 expression was demonstrated in all subjects (n=163) with detection on at least 90% of blasts in 155 cases. Median antigen site density of surface CD22 was 3,470 sites/cell (range 349 – 19,653, n=160). Blasts from patients with known 11q23 (MLL) rearrangement had lower site density (median 1,590 sites/cell, range 349-3,624, n=20 versus 3,853 sites/cell, range 451-19,653, n=140; p=<0.0001) and 6 of 21 cases had sub-populations of blasts lacking CD22 expression (22% – 82% CD22+). CD22 expression was maintained in serial studies of 73 subjects, including those treated with anti-CD22 targeted therapy. The levels of soluble CD22 in blood and marrow by ELISA were low and not expected to influence the pharmacokinetics of anti-CD22 directed agents. Conclusions These characteristics make CD22 an excellent potential therapeutic target in patients with relapsed and chemotherapy-refractory ALL, although cases with MLL rearrangement require close study to exclude the presence of a CD22-negative blast population. PMID:25728039

Efficient expansion of mesenchymal stromal cells in a disposable fixed bed culture system.

PubMed

Mizukami, Amanda; Orellana, Maristela D; Caruso, Sâmia R; de Lima Prata, Karen; Covas, Dimas T; Swiech, Kamilla

2013-01-01

The need for efficient and reliable technologies for clinical-scale expansion of mesenchymal stromal cells (MSC) has led to the use of disposable bioreactors and culture systems. Here, we evaluate the expansion of cord blood-derived MSC in a disposable fixed bed culture system. Starting from an initial cell density of 6.0 × 10(7) cells, after 7 days of culture, it was possible to produce of 4.2(±0.8) × 10(8) cells, which represents a fold increase of 7.0 (±1.4). After enzymatic retrieval from Fibra-Cell disks, the cells were able to maintain their potential for differentiation into adipocytes and osteocytes and were positive for many markers common to MSC (CD73, CD90, and CD105). The results obtained in this study demonstrate that MSC can be efficiently expanded in the culture system. This novel approach presents several advantages over the current expansion systems, based on culture flasks or microcarrier-based spinner flasks and represents a key element for MSC cellular therapy according to GMP compliant clinical-scale production system. Copyright © 2013 American Institute of Chemical Engineers.

Isolation, Characterization, and Differentiation of Dental Pulp Stem Cells in Ferrets.

PubMed

Homayounfar, Negar; Verma, Prashant; Nosrat, Ali; El Ayachi, Ikbale; Yu, Zongdong; Romberg, Elaine; Huang, George T-J; Fouad, Ashraf F

2016-03-01

The ferret canine tooth has been introduced as a suitable model for studying dental pulp regeneration. The aim of this study was to isolate and characterize ferret dental pulp stem cells (fDPSCs) and their differentiation potential. Dental pulp stem cells were isolated from freshly extracted ferret canine teeth. The cells were examined for the expression of stem cell markers STRO-1, CD90, CD105, and CD146. The osteo/odontogenic and adipogenic differentiation potential of fDPSCs was evaluated. Osteogenic and odontogenic marker genes were evaluated using quantitative real-time polymerase chain reaction (qRT-PCR) on days 1, 4, and 8 after osteo/odontogenic induction of fDPSCs including dentin sialophosphoprotein (DSPP), dentin matrix protein-1, osteopontin, and alkaline phosphatase. Human dental pulp cells were used as the control. The results were analyzed using 3-way analysis of variance. fDPSCs were positive for STRO1, CD90, and CD105 and negative for CD146 markers with immunohistochemistry. fDPSCs showed strong osteogenic and weak adipogenic potential. The overall expression of DSPP was not significantly different between fDPSCs and human dental pulp cells. The expression of DSPP in osteo/odontogenic media was significantly higher in fDPSCs on day 4 (P < .01). The overall expression of dentin matrix protein-1, osteopontin, and alkaline phosphatase was significantly higher in fDPSCs (P = .0005). fDPSCs were positive for several markers of dental pulp stem cells resembling human DPSCs and appeared to show a stronger potential to differentiate to osteoblastic rather than odontoblastic lineage. Copyright © 2016 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

BCL2 expression in CD105 positive neoangiogenic cells and tumor progression in angioimmunoblastic T-cell lymphoma.

PubMed

Ratajczak, Philippe; Leboeuf, Christophe; Wang, Li; Brière, Josette; Loisel-Ferreira, Irmine; Thiéblemont, Catherine; Zhao, Wei-Li; Janin, Anne

2012-06-01

The angiogenic microenvironment has been known to be a component of angioimmunoblastic T-cell lymphoma since its initial characterization. We have shown that angioimmunoblastic T-cell lymphoma endothelial cells produce vascular endothelial growth factor-A (VEGFA), and participate in lymphoma progression. In squamous cell carcinoma, endothelial BCL2 expression induces a crosstalk with tumor cells through VEGFA, a major mediator of tumoral angiogenesis. In the present study, we analyzed BCL2 and VEGFA in 30 angioimmunoblastic T-cell lymphomas, using triple immunofluorescence to identify protein coexpression in well-characterized lymphoma cells and microenvironment neoangiogenic endothelial cells. Using quantitative real-time PCR, we assessed mRNA expression levels in laser-microdissected endothelial and lymphoma cells. In lymphoma cells, as in endothelial cells, BCL2 and VEGFA proteins were coexpressed. BCL2 was expressed only in neoangiogenic CD34(+)CD105(+) endothelial cells. In laser-microdissected cells, mRNA studies showed a significant relationship between BCL2 and VEGFA levels in CD34(+) endothelial cells, but not in CD3(+)CD10(+)lymphoma cells, or in CD34(+) endothelial cells from lymph node hyperplasia. Further study showed that, in AITL, BCL2 mRNA levels in CD34(+)CD105(+) neoangiogenic endothelial cells also correlated with microvessel density, International Prognostic Index, Ann Arbor stage, bone marrow involvement and elevated LDH. BCL2 expression by CD105(+) neoangiogenic endothelial cells is related to tumor progression in angioimmunoblastic T-cell lymphoma.

Plasma Soluble CD163 Level Independently Predicts All-Cause Mortality in HIV-1-Infected Individuals.

PubMed

Knudsen, Troels Bygum; Ertner, Gideon; Petersen, Janne; Møller, Holger Jon; Moestrup, Søren K; Eugen-Olsen, Jesper; Kronborg, Gitte; Benfield, Thomas

2016-10-15

CD163, a monocyte- and macrophage-specific scavenger receptor, is shed as soluble CD163 (sCD163) during the proinflammatory response. Here, we assessed the association between plasma sCD163 levels and progression to AIDS and all-cause mortality among individuals infected with human immunodeficiency virus type 1 (HIV). Plasma sCD163 levels were measured in 933 HIV-infected individuals. Hazard ratios (HRs) with 95% confidence intervals (CIs) associated with mortality were computed by Cox proportional hazards regression. At baseline, 86% were receiving antiretroviral treatment, 73% had plasma a HIV RNA level of <50 copies/mL, and the median CD4(+) T-cell count was 503 cells/µL. During 10.5 years of follow-up, 167 (17.9%) died. Plasma sCD163 levels were higher in nonsurvivors than in survivors (4.92 mg/L [interquartile range {IQR}, 3.29-8.65 mg/L] vs 3.16 mg/L [IQR, 2.16-4.64 mg/L]; P = .0001). The cumulative incidence of death increased with increasing plasma sCD163 levels, corresponding to a 6% or 35% increased risk of death for each milligram per liter or quartile increase, respectively, in baseline plasma sCD163 level (adjusted HR, 1.06 [95% CI, 1.03-1.09] and 1.35 [95% CI, 1.13-1.63], respectively). Plasma sCD163 was an independent marker of all-cause mortality in a cohort of HIV-infected individuals, suggesting that monocyte/macrophage activation may play a role in HIV pathogenesis and be a target of intervention. © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

Umbilical cord tissue-derived mesenchymal stromal cells maintain immunomodulatory and angiogenic potencies after cryopreservation and subsequent thawing.

PubMed

Bárcia, Rita N; Santos, Jorge M; Teixeira, Mariana; Filipe, Mariana; Pereira, Ana Rita S; Ministro, Augusto; Água-Doce, Ana; Carvalheiro, Manuela; Gaspar, Maria Manuela; Miranda, Joana P; Graça, Luis; Simões, Sandra; Santos, Susana Constantino Rosa; Cruz, Pedro; Cruz, Helder

2017-03-01

The effect of cryopreservation on mesenchymal stromal cell (MSC) therapeutic properties has become highly controversial. However, data thus far have indiscriminately involved the assessment of different types of MSCs with distinct production processes. This study assumed that MSC-based products are affected differently depending on the tissue source and manufacturing process and analyzed the effect of cryopreservation on a specific population of umbilical cord tissue-derived MSCs (UC-MSCs), UCX ® . Cell phenotype was assessed by flow cytometry through the evaluation of the expression of relevant surface markers such as CD14, CD19, CD31, CD34, CD44, CD45, CD90, CD105, CD146, CD200, CD273, CD274 and HLA-DR. Immunomodulatory activity was analyzed in vitro through the ability to inhibit activated T cells and in vivo by the ability to reverse the signs of inflammation in an adjuvant-induced arthritis (AIA) model. Angiogenic potential was evaluated in vitro using a human umbilical vein endothelial cell-based angiogenesis assay, and in vivo using a mouse model for hindlimb ischemia. Phenotype and immunomodulatory and angiogenic potencies of this specific UC-MSC population were not impaired by cryopreservation and subsequent thawing, both in vitro and in vivo. This study suggests that potency impairment related to cryopreservation in a given tissue source can be avoided by the production process. The results have positive implications for the development of advanced-therapy medicinal products. Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

High-resolution molecular validation of self-renewal and spontaneous differentiation in adipose-tissue derived human mesenchymal stem cells cultured in human platelet lysate

PubMed Central

Dudakovic, Amel Dudakovic; Camilleri, Emily; Riester, Scott M.; Lewallen, Eric A.; Kvasha, Sergiy; Chen, Xiaoyue; Radel, Darcie J.; Anderson, Jarett M.; Nair, Asha A.; Evans, Jared M.; Krych, Aaron J.; Smith, Jay; Deyle, David R.; Stein, Janet L.; Stein, Gary S.; Im, Hee-Jeong; Cool, Simon M.; Westendorf, Jennifer J.; Kakar, Sanjeev; Dietz, Allan B.; van Wijnen, Andre J.

2014-01-01

Improving the effectiveness of adipose-tissue derived human mesenchymal stromal/stem cells (AMSCs) for skeletal therapies requires a detailed characterization of mechanisms supporting cell proliferation and multi-potency. We investigated the molecular phenotype of AMSCs that were either actively proliferating in platelet lysate or in a basal non-proliferative state. Flow cytometry combined with high-throughput RNA sequencing (RNASeq) and RT-qPCR analyses validate that AMSCs express classic mesenchymal cell surface markers (e.g., CD44, CD73/NT5E, CD90/THY1 and CD105/ENG). Expression of CD90 is selectively elevated at confluence. Self-renewing AMSCs express a standard cell cycle program that successively mediates DNA replication, chromatin packaging, cyto-architectural enlargement and mitotic division. Confluent AMSCs preferentially express genes involved in extracellular matrix (ECM) formation and cellular communication. For example, cell cycle-related biomarkers (e.g., cyclins E2 and B2, transcription factor E2F1) and histone-related genes (e.g., H4, HINFP, NPAT) are elevated in proliferating AMSCs, while ECM genes are strongly upregulated (>10 fold) in quiescent AMSCs. AMSCs also express pluripotency genes (e.g., POU5F1, NANOG, KLF4) and early mesenchymal markers (e.g., NES, ACTA2) consistent with their multipotent phenotype. Strikingly, AMSCs modulate expression of WNT signaling components and switch production of WNT ligands (from WNT5A/WNT5B/WNT7B to WNT2/WNT2B), while up-regulating WNT-related genes (WISP2, SFRP2 and SFRP4). Furthermore, post-proliferative AMSCs spontaneously express fibroblastic, osteogenic, chondrogenic and adipogenic biomarkers when maintained in confluent cultures. Our findings validate the biological properties of self-renewing and multi-potent AMSCs by providing high-resolution quality control data that support their clinical versatility. PMID:24905804

Gene Expression Profiling of Liver Cancer Stem Cells by RNA-Sequencing

PubMed Central

Lam, Chi Tat; Ng, Michael N. P.; Yu, Wan Ching; Lau, Joyce; Wan, Timothy; Wang, Xiaoqi; Yan, Zhixiang; Liu, Hang; Fan, Sheung Tat

2012-01-01

Background Accumulating evidence supports that tumor growth and cancer relapse are driven by cancer stem cells. Our previous work has demonstrated the existence of CD90+ liver cancer stem cells (CSCs) in hepatocellular carcinoma (HCC). Nevertheless, the characteristics of these cells are still poorly understood. In this study, we employed a more sensitive RNA-sequencing (RNA-Seq) to compare the gene expression profiling of CD90+ cells sorted from tumor (CD90+CSCs) with parallel non-tumorous liver tissues (CD90+NTSCs) and elucidate the roles of putative target genes in hepatocarcinogenesis. Methodology/Principal Findings CD90+ cells were sorted respectively from tumor and adjacent non-tumorous human liver tissues using fluorescence-activated cell sorting. The amplified RNAs of CD90+ cells from 3 HCC patients were subjected to RNA-Seq analysis. A differential gene expression profile was established between CD90+CSCs and CD90+NTSCs, and validated by quantitative real-time PCR (qRT-PCR) on the same set of amplified RNAs, and further confirmed in an independent cohort of 12 HCC patients. Five hundred genes were differentially expressed (119 up-regulated and 381 down-regulated genes) between CD90+CSCs and CD90+NTSCs. Gene ontology analysis indicated that the over-expressed genes in CD90+CSCs were associated with inflammation, drug resistance and lipid metabolism. Among the differentially expressed genes, glypican-3 (GPC3), a member of glypican family, was markedly elevated in CD90+CSCs compared to CD90+NTSCs. Immunohistochemistry demonstrated that GPC3 was highly expressed in forty-two human liver tumor tissues but absent in adjacent non-tumorous liver tissues. Flow cytometry indicated that GPC3 was highly expressed in liver CD90+CSCs and mature cancer cells in liver cancer cell lines and human liver tumor tissues. Furthermore, GPC3 expression was positively correlated with the number of CD90+CSCs in liver tumor tissues. Conclusions/Significance The identified genes, such as GPC3 that are distinctly expressed in liver CD90+CSCs, may be promising gene candidates for HCC therapy without inducing damages to normal liver stem cells. PMID:22606345

The volatile anesthetic isoflurane induces ecto-5′-nucleotidase (CD73) to protect against renal ischemia and reperfusion injury

PubMed Central

Kim, Mihwa; Ham, Ahrom; Kim, Joo Yun; Brown, Kevin M.; D’Agati, Vivette D.; Lee, H. Thomas

2013-01-01

The volatile anesthetic isoflurane protects against renal ischemia and reperfusion injury by releasing renal tubular TGF-β1. Since adenosine is a powerful cytoprotective molecule, we tested whether TGF-β1 generated by isoflurane induces renal tubular ecto-5′-nucleotidase (CD73) and adenosine to protect against renal ischemia and reperfusion injury. Isoflurane induced new CD73 synthesis and increased adenosine generation in cultured kidney proximal tubule cells and in mouse kidney. Moreover, a TGF-β1 neutralizing antibody prevented isoflurane-mediated induction of CD73 activity. Mice anesthetized with isoflurane after renal ischemia and reperfusion had significantly reduced plasma creatinine and decreased renal tubular necrosis, neutrophil infiltration and apoptosis compared to pentobarbital-anesthetized mice. Isoflurane failed to protect against renal ischemia and reperfusion injury in CD73 deficient mice, in mice pretreated with a selective CD73 inhibitor or mice treated with an adenosine receptor antagonist. The TGF-β1 neutralizing antibody or the CD73 inhibitor attenuated isoflurane-mediated protection against HK-2 cell apoptosis. Thus, isoflurane causes TGF-β1-dependent induction of renal tubular CD73 and adenosine generation to protect against renal ischemia and reperfusion injury. Modulation of this pathway may have important therapeutic implications to reduce morbidity and mortality arising from ischemic acute kidney injury. PMID:23423261

Immunohistochemical Comparison of the Expression of CD34 and CD105 in Odontogenic Keratocyst and Dentigerous Cyst

PubMed Central

Jamshidi, Shokoofeh; Zargaran, Massoumeh; Roshanaei, Ghodratollah; Hadadi, Fatemeh; Dehghani| Nazhvani, Ali

2017-01-01

Statement of the Problem: Odontogenic keratocyst (OKC) is a developmental odontogenic cyst with specific histopathological features, high recurrence rate, and aggressive clinical behavior. Angiogenesis might be considered as an important factor for the growth, expansion, and distribution of this lesion. Purpose: The aim of the present study was to determine the mean vascular densities (MVD) of OKCs and dentigerous cysts to evaluate their relationship with the biologic behavior of these lesions. Materials and Method: In this cross-sectional analytical study, angiogenesis was assessed in OKC and dentigerous cyst by measuring the MVD. Immunohistochemistry was carried out using CD34 and CD105. The results were analyzed with independent samples t-test. The data were analyzed, setting p value at 0.05. Results: The MVDs with the use of CD34 and CD105 markers were significantly higher in OKC compared to dentigerous cyst (p< 0.05). In addition, MVDs obtained by CD105 in dentigerous cysts and OKC were significantly less than those based on CD34 (p< 0.05). Conclusion: Based on the results of the present study, it can be suggested that angiogenesis might be one of the possible mechanisms involved in higher aggressive biologic behavior and greater recurrence rate of OKC compared to dentigerous cysts. PMID:28280759

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.

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.

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

Characterization of CD22 expression in acute lymphoblastic leukemia.

PubMed

Shah, Nirali N; Stevenson, Maryalice Stetler; Yuan, Constance M; Richards, Kelly; Delbrook, Cindy; Kreitman, Robert J; Pastan, Ira; Wayne, Alan S

2015-06-01

CD22 is a B-lineage differentiation antigen that has emerged as a leading therapeutic target in acute lymphoblastic leukemia (ALL). Properties of CD22 expression relevant to therapeutic targeting were characterized in primary samples obtained from children and young adults with relapsed and chemotherapy refractory B-precursor (pre-B) ALL. CD22 expression was demonstrated in all subjects (n = 163) with detection on at least 90% of blasts in 155 cases. Median antigen site density of surface CD22 was 3,470 sites/cell (range 349-19,653, n = 160). Blasts from patients with known 11q23 (MLL) rearrangement had lower site density (median 1,590 sites/cell, range 349-3,624, n = 20 versus 3,853 sites/cell, range 451-19,653, n = 140; P = <0.0001) and 6 of 21 cases had sub-populations of blasts lacking CD22 expression (22%-82% CD22 +). CD22 expression was maintained in serial studies of 73 subjects, including those treated with anti-CD22 targeted therapy. The levels of soluble CD22 in blood and marrow by ELISA were low and not expected to influence the pharmacokinetics of anti-CD22 directed agents. These characteristics make CD22 an excellent potential therapeutic target in patients with relapsed and chemotherapy-refractory ALL, although cases with MLL rearrangement require close study to exclude the presence of a CD22-negative blast population. Published 2015. This article is a U.S. Government work and is in the public domain in the USA.

Endochondral ossification is required for haematopoietic stem-cell niche formation.

PubMed

Chan, Charles K F; Chen, Ching-Cheng; Luppen, Cynthia A; Kim, Jae-Beom; DeBoer, Anthony T; Wei, Kevin; Helms, Jill A; Kuo, Calvin J; Kraft, Daniel L; Weissman, Irving L

2009-01-22

Little is known about the formation of niches, local micro-environments required for stem-cell maintenance. Here we develop an in vivo assay for adult haematopoietic stem-cell (HSC) niche formation. With this assay, we identified a population of progenitor cells with surface markers CD45(-)Tie2(-)alpha(V)(+)CD105(+)Thy1.1(-) (CD105(+)Thy1(-)) that, when sorted from 15.5 days post-coitum fetal bones and transplanted under the adult mouse kidney capsule, could recruit host-derived blood vessels, produce donor-derived ectopic bones through a cartilage intermediate and generate a marrow cavity populated by host-derived long-term reconstituting HSC (LT-HSC). In contrast, CD45(-)Tie2(-)alpha(V)(+)CD105(+)Thy1(+) (CD105(+)Thy1(+)) fetal bone progenitors form bone that does not contain a marrow cavity. Suppressing expression of factors involved in endochondral ossification, such as osterix and vascular endothelial growth factor (VEGF), inhibited niche generation. CD105(+)Thy1(-) progenitor populations derived from regions of the fetal mandible or calvaria that do not undergo endochondral ossification formed only bone without marrow in our assay. Collectively, our data implicate endochondral ossification, bone formation that proceeds through a cartilage intermediate, as a requirement for adult HSC niche formation.

Endoglin (CD105) expression differentiates between aseptic loosening and periprosthetic joint infection after total joint arthroplasty.

PubMed

Jansen, Philipp; Mumme, Torsten; Randau, Thomas; Gravius, Sascha; Hermanns-Sachweh, Benita

2014-01-01

The differentiation between aseptic loosening and periprosthetic joint infection (PJI) after total joint arthroplasty is essential for successful therapy. A better understanding of pathogenesis of aseptic loosening and PJI may help to prevent or treat these complications. Previous investigations revealed an increased vascularization in the periprosthetic membrane in cases of PJI via PET signals. Based on these findings our hypothesis was that PJI is associated with an increased neovascularization in the periprosthetic membrane. Tissue samples from periprosthetic membranes of the bone-implant interface were investigated histologically for inflammation, wear particles, vascularization and fibrosis. To identify vascular structures antibodies against CD 31, CD 34, factor VIII and CD 105 (endoglin) were applied for immunohistochemical investigations. According to a consensus classification of Morawietz the tissue samples were divided into four types: type I (wear particle induced type, n = 11), type II (infectious type, n = 7), type III (combined type, n = 7) and type IV (indeterminate type, n = 7). Patients with PJI (type II) showed a pronounced infiltration of neutrophil granulocytes in the periprosthetic membrane and an enhanced neovascularization indicated by positive immunoreaction with antibodies against CD 105 (endoglin). Tissue samples classified as type I, type III and type IV showed significantly less immune reaction for CD 105. In cases of aseptic loosening and PJI vascularization is found in different expression in periprosthetic membranes. However, in aseptic loosening, there is nearly no neovascularization with CD 105-positive immune reaction. Therefore, endoglin (CD 105) expression allows for differentiation between aseptic loosening and PJI.

Targeting Microvascular Pericytes in Angiogenic Vessels of Prostate Cancer

DTIC Science & Technology

2005-04-01

endothelial cells (BEC) were identified using a cocktail of rat antibodies against mouse endoglin ( CD 105), PECAM-1 (CD3 1), and VEGF receptor-2 (flk...immunohistochemistry, and imaging angiogenic tufts composed of endothelial cells positive for endoglin ( CD 105), PECAM- I (CD3 1), and VEGF receptor...TYPE AND DATES COVERED (Leave blank) - April 2005 Annual (1 Apr 2004 - 31 Mar 2005) 4. TITLE AND SUBTITLE 5. FUNDING NUMBERS Targeting Microvascular

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.

Adipose-derived mesenchymal stromal cells from aged patients with coronary artery disease keep mesenchymal stromal cell properties but exhibit characteristics of aging and have impaired angiogenic potential.

PubMed

Efimenko, Anastasia; Dzhoyashvili, Nina; Kalinina, Natalia; Kochegura, Tatiana; Akchurin, Renat; Tkachuk, Vsevolod; Parfyonova, Yelena

2014-01-01

Tissue regeneration is impaired in aged individuals. Adipose-derived mesenchymal stromal cells (ADSCs), a promising source for cell therapy, were shown to secrete various angiogenic factors and improve vascularization of ischemic tissues. We analyzed how patient age affected the angiogenic properties of ADSCs. ADSCs were isolated from subcutaneous fat tissue of patients with coronary artery disease (CAD; n = 64, 43-77 years old) and without CAD (n = 31, 2-82 years old). ADSC phenotype characterized by flow cytometry was CD90(+)/CD73(+)/CD105(+)/CD45(-)/CD31(-) for all samples, and these cells were capable of adipogenic and osteogenic differentiation. ADSCs from aged patients had shorter telomeres (quantitative reverse transcription polymerase chain reaction) and a tendency to attenuated telomerase activity. ADSC-conditioned media (ADSC-CM) stimulated capillary-like tube formation by endothelial cells (EA.hy926), and this effect significantly decreased with the age of patients both with and without CAD. Angiogenic factors (vascular endothelial growth factor, placental growth factor, hepatocyte growth factor, angiopoetin-1, and angiogenin) in ADSC-CM measured by enzyme-linked immunosorbent assay significantly decreased with patient age, whereas levels of antiangiogenic factors thrombospondin-1 and endostatin did not. Expression of angiogenic factors in ADSCs did not change with patient age (real-time polymerase chain reaction); however, gene expression of factors related to extracellular proteolysis (urokinase and its receptor, plasminogen activator inhibitor-1) and urokinase-type plasminogen activator receptor surface expression increased in ADSCs from aged patients with CAD. ADSCs from aged patients both with and without CAD acquire aging characteristics, and their angiogenic potential declines because of decreasing proangiogenic factor secretion. This could restrict the effectiveness of autologous cell therapy with ADSCs in aged patients.

Transplantation of human placenta-derived mesenchymal stem cells in a silk fibroin/hydroxyapatite scaffold improves bone repair in rabbits.

PubMed

Jin, Jun; Wang, Jun; Huang, Jian; Huang, Fang; Fu, Jianhong; Yang, Xinjing; Miao, Zongning

2014-11-01

The main requirements for successful tissue engineering of the bone are non-immunogenic cells with osteogenic potential and a porous biodegradable scaffold. The purpose of this study is to evaluate the potential of a silk fibroin/hydroxyapatite (SF/HA) porous material as a delivery vehicle for human placenta-derived mesenchymal stem cells (PMSCs) in a rabbit radius defect model. In this study, we randomly assigned 16 healthy adult New Zealand rabbits into two groups, subjected to transplantation with either SF/HA and PMSCs (experimental group) or SF/HA alone (control group). To evaluate fracture healing, we assessed the extent of graft absorption, the quantity of newly formed bone, and re-canalization of the cavitas medullaris using radiographic and histological tools. We performed flow cytometric analysis to characterize PMSCs, and found that while they express CD90, CD105 and CD73, they stain negative for HLA-DR and the hematopoietic cell surface markers CD34 and CD45. When PMSCs were exposed to osteogenic induction medium, they secreted calcium crystals that were identified by von Kossa staining. Furthermore, when seeded on the surface of SF/HA scaffold, they actively secreted extracellular matrix components. Here, we show, through radiographic and histological analyses, that fracture healing in the experimental group is significantly improved over the control group. This strongly suggests that transplantation of human PMSCs grown in an SF/HA scaffold into injured radius segmental bone in rabbits, can markedly enhance tissue repair. Our finding provides evidence supporting the utility of human placenta as a potential source of stem cells for bone tissue engineering. Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Effect of culture medium on propagation and phenotype of corneal stroma-derived stem cells.

PubMed

Sidney, Laura E; Branch, Matthew J; Dua, Harminder S; Hopkinson, Andrew

2015-12-01

The limbal area of the corneal stroma has been identified as a source of mesenchymal-like stem cells, which have potential for exploitation as a cell therapy. However, the optimal culture conditions are disputed and few direct media comparisons have been performed. In this report, we evaluated several media types to identify the optimal for inducing an in vitro stem cell phenotype. Primary human corneal stroma-derived stem cells (CSSCs) were extracted from corneoscleral rims. Culture in seven different media types was compared: Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS); M199 with 20% FBS; DMEM-F12 with 20% serum replacement, basic fibroblast growth factor and leukemia inhibitory factor (SCM); endothelial growth medium (EGM); semi-solid MethoCult; serum-free keratinocyte medium (K-SFM); and StemPro-34. Effects on proliferation, morphology, protein and messenger RNA expression were evaluated. All media supported proliferation of CSSCs with the exception of K-SFM and StemPro-34. Morphology differed between media: DMEM produced large cells, whereas EGM produced very small cells. Culture in M199 produced a typical mesenchymal stromal cell phenotype with high expression of CD105, CD90 and CD73 but not CD34. Culture in SCM produced a phenotype more reminiscent of a progenitor cell type with expression of CD34, ABCG2, SSEA-4 and PAX6. Culture medium can significantly influence CSSC phenotype. SCM produced a cell phenotype closest to that of a pluripotent stem cell, and we consider it to be the most appropriate for development as a clinical-grade medium for the production of CSSC phenotypes suitable for cell therapy. Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Murine Mesenchymal Stem Cell Commitment to Differentiation is Regulated by Mitochondrial Dynamics

PubMed Central

Forni, Maria Fernanda; Peloggia, Julia; Trudeau, Kyle; Shirihai, Orian; Kowaltowski, Alicia J.

2015-01-01

Mouse skin mesenchymal stem cells (msMSCs) are dermis CD105+CD90+CD73+CD29+CD34− mesodermal precursors which, after in vitro induction, undergo chondro, adipo and osteogenesis. Extensive metabolic reconfiguration has been found to occur during differentiation, and the bioenergetic status of a cell is known to be dependent on the quality and abundance of the mitochondrial population, which may be regulated by fusion and fission. However, little is known regarding the impact of mitochondrial dynamics on the differentiation process. We addressed this knowledge gap by isolating MSCs from Swiss female mice, inducing these cells to differentiate into osteo, chondro and adipocytes and measuring changes in mass, morphology, dynamics and bioenergetics. Mitochondrial biogenesis was increased in adipogenesis, as evaluated through confocal microscopy, citrate synthase activity and mtDNA content. The early steps of adipo and osteogenesis involved mitochondrial elongation, as well as increased expression of mitochondrial fusion proteins Mfn1 and 2. Chondrogenesis involved a fragmented mitochondrial phenotype, increased expression of fission proteins Drp1, Fis1 and 2 and enhanced mitophagy. These events were accompanied by profound bioenergetic alterations during the commitment period. Moreover, knockdown of Mfn2 in adipo and osteogenesis and the overexpression of a dominant negative form of Drp1 during chondrogenesis resulted in a loss of differentiation ability. Overall, we find that mitochondrial morphology and its regulating processes of fission/fusion are modulated early on during commitment, leading to alterations in the bioenergetic profile that are important for differentiation. We thus propose a central role for mitochondrial dynamics in the maintenance/commitment of mesenchymal stem cells. PMID:26638184

Compared to the amniotic membrane, Wharton's jelly may be a more suitable source of mesenchymal stem cells for cardiovascular tissue engineering and clinical regeneration.

PubMed

Pu, Lei; Meng, Mingyao; Wu, Jian; Zhang, Jing; Hou, Zongliu; Gao, Hui; Xu, Hui; Liu, Boyu; Tang, Weiwei; Jiang, Lihong; Li, Yaxiong

2017-03-21

The success of developing cardiovascular tissue engineering (CTE) grafts greatly needs a readily available cell substitute for endothelial and interstitial cells. Perinatal annexes have been proposed as a valuable source of mesenchymal stem cells (MSCs) for tissue engineering and regenerative medicine. The objective of the present study is to evaluate the potential of human Wharton's jelly MSCs (WJ-MSCs) and amniotic membrane MSCs (AM-MSCs) as a seeding cell in CTE and cardiovascular regenerative medicine. WJ-MSCs/AM-MSCs were isolated and characterized in vitro according to their morphology, proliferation, self-renewal, phenotype, and multipotency. More importantly, the characteristics of hemocompatibility, extracellular matrix deposition, and gene expression and viability of both MSCs were investigated. Fibroblast-like human WJ-MSCs and AM-MSCs were successfully isolated and positively expressed the characteristic markers CD73, CD90, and CD105 but were negative for CD34, CD45, and HLA-DR. Both MSCs shared trilineage differentiation toward the adipogenic, osteogenic, and chondrogenic lineages. The proliferative and self-renewal capacity of WJ-MSCs was significantly higher than that of AM-MSCs (P < 0.001). WJ-MSCs provided comparable properties of antiplatelet adhesion and did not activate the coagulation cascade to endothelial cells. However, aggregated platelets were visualized on the surface of AM-MSCs-derived cell sheets and the intrinsic pathway was activated. Furthermore, WJ-MSCs have superior properties of collagen deposition and higher viability than AM-MSCs during cell sheet formation. This study highlights that WJ-MSCs could act as a functional substitute of endothelial and interstitial cells, which could serve as an appealing and practical single-cell source for CTE and regenerative therapy.

Adipose-derived mesenchymal stem cells from liposuction and resected fat are feasible sources for regenerative medicine.

PubMed

Schneider, Sandra; Unger, Marina; van Griensven, Martijn; Balmayor, Elizabeth R

2017-05-19

The use of mesenchymal stem cells (MSCs) in research and in regenerative medicine has progressed. Bone marrow as a source has drawbacks because of subsequent morbidities. An easily accessible and valuable source is adipose tissue. This type of tissue contains a high number of MSCs, and obtaining higher quantities of tissue is more feasible. Fat tissue can be harvested using different methods such as liposuction and resection. First, a detailed isolation protocol with complete characterization is described. This also includes highlighting problems and pitfalls. Furthermore, some comparisons of these different harvesting methods exist. However, the later characterization of the cells is conducted poorly in most cases. We performed an in-depth characterization over five passages including an investigation of the effect of freezing and thawing. Characterization was performed using flow cytometry with CD markers, metabolic activity with Alamar Blue, growth potential in between passages, and cytoskeleton staining. Our results show that the cells isolated with distinct isolation methods (solid versus liposuction "liquid") have the same MSC potential. However, the percentage of cells positive for the markers CD73, CD90, and CD105 is initially quite low. The cells isolated from the liquid fat tissue grow faster at higher passages, and significantly more cells display MSC markers. In summary, we show a simple and efficient method to isolate adipose-derived mesenchymal stem cells from different preparations. Liposuctions and resection can be used, whereas liposuction has more growth potential at higher passages.

Cementogenic potential of multipotential mesenchymal stem cells purified from the human periodontal ligament.

PubMed

Torii, Daisuke; Konishi, Kiyoshi; Watanabe, Nobuyuki; Goto, Shinichi; Tsutsui, Takeki

2015-01-01

The periodontal ligament (PDL) consists of a group of specialized connective tissue fibers embedded in the alveolar bone and cementum that are believed to contain progenitors for mineralized tissue-forming cell lineages. These progenitors may contribute to regenerative cell therapy or tissue engineering methods aimed at recovery of tissue formation and functions lost in periodontal degenerative changes. Some reports using immortal clonal cell lines of cementoblasts, which are cells containing mineralized tissue-forming cell lineages, have shown that their phenotypic alteration and gene expression are associated with mineralization. Immortal, multipotential PDL-derived cell lines may be useful biological tools for evaluating differentiation-inducing agents. In this study, we confirmed the gene expression and mineralization potential of primary and immortal human PDL cells and characterized their immunophenotype. Following incubation with mineralization induction medium containing β-glycerophosphate, ascorbic acid, and dexamethasone, normal human PDL (Pel) cells and an immortal derivative line (Pelt) cells showed higher levels of mineralization compared with cells grown in normal growth medium. Both cell types were positive for putative surface antigens of mesenchymal cells (CD44, CD73, CD90, and CD105). They were also positive for stage-specific embryonic antigen-3, a marker of multipotential stem cells. Furthermore, PDL cells expressed cementum attachment protein and cementum protein 1 when cultured with recombinant human bone morphogenetic protein-2 or -7. The results suggest that normal and immortal human PDL cells contain multipotential mesenchymal stem cells with cementogenic potential.

Mesenchymal stromal cells (MSC) from JAK2+ myeloproliferative neoplasms differ from normal MSC and contribute to the maintenance of neoplastic hematopoiesis

PubMed Central

Sánchez-Abarca, Luis Ignacio; Rosón-Burgo, Beatriz; Redondo, Alba; Rico, Ana; Preciado, Silvia; Ortega, Rebeca; Rodríguez, Concepción; Muntión, Sandra; Hernández-Hernández, Ángel; De Las Rivas, Javier; González, Marcos; González Porras, José Ramón; del Cañizo, Consuelo; Sánchez-Guijo, Fermín

2017-01-01

There is evidence of continuous bidirectional cross-talk between malignant cells and bone marrow-derived mesenchymal stromal cells (BM-MSC), which favors the emergence and progression of myeloproliferative neoplastic (MPN) diseases. In the current work we have compared the function and gene expression profile of BM-MSC from healthy donors (HD-MSC) and patients with MPN (JAK2V617F), showing no differences in the morphology, proliferation and differentiation capacity between both groups. However, BM-MSC from MPN expressed higher mean fluorescence intensity (MIF) of CD73, CD44 and CD90, whereas CD105 was lower when compared to controls. Gene expression profile of BM-MSC showed a total of 169 genes that were differentially expressed in BM-MSC from MPN patients compared to HD-MSC. In addition, we studied the ability of BM-MSC to support the growth and survival of hematopoietic stem/progenitor cells (HSPC), showing a significant increase in the number of CFU-GM colonies when MPN-HSPC were co-cultured with MPN-MSC. Furthermore, MPN-MSC showed alteration in the expression of genes associated to the maintenance of hematopoiesis, with an overexpression of SPP1 and NF-kB, and a downregulation of ANGPT1 and THPO. Our results suggest that BM-MSC from JAK2+ patients differ from their normal counterparts and favor the maintenance of malignant clonal hematopoietic cells. PMID:28796790

Isolation and Characterisation of Mesenchymal Stem Cells from Rat Bone Marrow and the Endosteal Niche: A Comparative Study

PubMed Central

Yusop, Norhayati; Battersby, Paul; Alraies, Amr; Moseley, Ryan

2018-01-01

Within bone, mesenchymal stromal cells (MSCs) exist within the bone marrow stroma (BM-MSC) and the endosteal niche, as cells lining compact bone (CB-MSCs). This study isolated and characterised heterogeneous MSC populations from each niche and subsequently investigated the effects of extensive cell expansion, analysing population doublings (PDs)/cellular senescence, colony-forming efficiencies (CFEs), MSC cell marker expression, and osteogenic/adipogenic differentiation. CB-MSCs and BM-MSCs demonstrated similar morphologies and PDs, reaching 100 PDs. Both populations exhibited consistent telomere lengths (12–17 kb), minimal senescence, and positive telomerase expression. CB-MSCs (PD15) had significantly lower CFEs than PD50. CB-MSCs and BM-MSCs both expressed MSC (CD73/CD90/CD105); embryonic (Nanog) and osteogenic markers (Runx2, osteocalcin) but no hematopoietic markers (CD45). CB-MSCs (PD15) strongly expressed Oct4 and p16INK4A. At early PDs, CB-MSCs possessed a strong osteogenic potency and low potency for adipogenesis, whilst BM-MSCs possessed greater overall bipotentiality for osteogenesis and adipogenesis. At PD50, CB-MSCs demonstrated reduced potency for both osteogenesis and adipogenesis, compared to BM-MSCs at equivalent PDs. This study demonstrates similarities in proliferative and mesenchymal cell characteristics between CB-MSCs and BM-MSCs, but contrasting multipotentiality. Such findings support further comparisons of human CB-MSCs and BM-MSCs, facilitating selection of optimal MSC populations for regenerative medicine purposes. PMID:29765418

In vivo near-infrared fluorescence imaging of CD105 expression during tumor angiogenesis.

PubMed

Yang, Yunan; Zhang, Yin; Hong, Hao; Liu, Glenn; Leigh, Bryan R; Cai, Weibo

2011-11-01

Angiogenesis is an indispensable process during tumor development. The currently accepted standard method for quantifying tumor angiogenesis is to assess microvessel density (MVD) based on CD105 staining, which is an independent prognostic factor for survival in patients with most solid tumor types. The goal of this study is to evaluate tumor angiogenesis in a mouse model by near-infrared fluorescence (NIRF) imaging of CD105 expression. TRC105, a human/murine chimeric anti-CD105 monoclonal antibody, was conjugated to an NIRF dye (IRDye 800CW; Ex: 778 nm; Em: 806 nm). FACS analysis and microscopy studies were performed to compare the CD105 binding affinity of TRC105 and 800CW-TRC105. In vivo/ex vivo NIRF imaging, blocking studies, and ex vivo histology were performed on 4T1 murine breast tumor-bearing mice to evaluate the ability of 800CW-TRC105 to target tumor angiogenesis. Another chimeric antibody, cetuximab, was used as an isotype-matched control. FACS analysis of human umbilical vein endothelial cells (HUVECs) revealed no difference in CD105 binding affinity between TRC105 and 800CW-TRC105, which was further validated by fluorescence microscopy. 800CW conjugation of TRC105 was achieved in excellent yield (> 85%), with an average of 0.4 800CW molecules per TRC105. Serial NIRF imaging after intravenous injection of 800CW-TRC105 revealed that the 4T1 tumor could be clearly visualized as early as 30 min post-injection. Quantitative region of interest (ROI) analysis showed that the tumor uptake peaked at about 16 h post-injection. Based on ex vivo NIRF imaging at 48 h post-injection, tumor uptake of 800CW-TRC105 was higher than most organs, thus providing excellent tumor contrast. Blocking experiments, control studies with 800CW-cetuximab and 800CW, as well as ex vivo histology all confirmed the in vivo target specificity of 800CW-TRC105. This is the first successful NIRF imaging study of CD105 expression in vivo. Fast, prominent, persistent, and CD105-specific uptake of the probe during tumor angiogenesis was observed in a mouse model. 800CW-TRC105 may be used in the clinic for imaging tumor angiogenesis within the lesions close to the skin surface, tissues accessible by endoscopy, or during image-guided surgery.

Regeneration of dental pulp by stem cells.

PubMed

Nakashima, M; Iohara, K

2011-07-01

Angiogenesis/vasculogenesis and neurogenesis are essential for pulp regeneration. Two subfractions of side-population (SP) cells, CD31(-)/CD146(-) SP cells and CD105(+) cells with angiogenic and neurogenic potential, were isolated by flow cytometry from canine dental pulp. In an experimental model of mouse hindlimb ischemia, transplantation of these cell populations resulted in an increase in blood flow, including high-density capillary formation. In a model of rat cerebral ischemia, stem cell transplantations enhanced neuronal regeneration and recovery from motor disability. Autologous transplantation of the CD31(-)/CD146(-) SP cells into an in vivo model of amputated pulp resulted in complete regeneration of pulp tissue with vascular and neuronal processes within 14 days. The transplanted cells expressed pro-angiogenic factors, implying trophic action on endothelial cells. Autologous transplantation of CD31(-)/CD146(-) SP cells or CD105(+) cells with stromal-cell-derived factor-1 (SDF-1) into root canals after whole pulp removal of mature teeth resulted in complete regeneration of pulp replete with nerves and vasculature by day 14, followed by dentin formation along the dentinal wall by day 35. Therefore, the potential utility of fractionated SP cells and CD105(+) cells in angiogenesis and neurogenesis was demonstrated by treatment of limb and cerebral ischemia following pulpotomy and pulpectomy.

Lack of ecto-5'-nucleotidase (CD73) promotes arteriogenesis.

PubMed

Böring, Yang Chul; Flögel, Ulrich; Jacoby, Christoph; Heil, Matthias; Schaper, Wolfgang; Schrader, Jürgen

2013-01-01

Adenosine can stimulate angiogenesis, but its role in the distinct process of arteriogenesis is unknown. We have previously reported that mice lacking ecto-5'-nucleotidase (CD73-/-) show enhanced monocyte adhesion to the endothelium after ischaemia, which is considered to be an important trigger for arteriogenesis. Hindlimb ischaemia was induced in wild-type (WT) and CD73-/- mice to study the role of extracellularly formed adenosine in arteriogenesis. Magnetic resonance angiography (MRA) was performed for serial visualization of newly developed vessels at a spatial resolution of 1 nL, and high-energy phosphates (HEP) were quantified by (31)P MR spectroscopy (MRS). MRA of CD73-/- mice revealed substantially enhanced collateral artery conductance at day 7 [CD73-/-: 0.73 ± 0.11 a.u. (arbitrary units); WT: 0.44 ± 0.13 a.u.; P < 0.01, n = 6], and MRS of the affected hindlimb showed a faster restoration of HEP in correlation with enhanced functional recovery in the mutant. Additionally, histology showed no differences in capillary density between the groups but showed an increased monocyte infiltration in hindlimbs of CD73-/- mice. Serial assessment of dynamic changes of vessel growth and metabolism in the process of arteriogenesis demonstrate that the lack of CD73-derived adenosine importantly promotes arteriogenesis but does not alter angiogenesis in our model of hindlimb ischaemia.

Effect of colorectal cancer on the number of normal stem cells circulating in peripheral blood.

PubMed

Marlicz, Wojciech; Sielatycka, Katarzyna; Serwin, Karol; Kubis, Ewa; Tkacz, Marta; Głuszko, Rafał; Białek, Andrzej; Starzyńska, Teresa; Ratajczak, Mariusz Z

2016-12-01

Bone marrow (BM) residing stem cells are mobilized from their BM niches into peripheral blood (PB) in several pathological situations including tissue organ injury and systemic inflammation. We recently reported that the number of BM-derived stem cells (SCs) increases in patients with pancreatic and stomach cancer. Accordingly, we observed higher numbers of circulating very small embryonic/epiblast‑like stem cells (VSELs) and mesenchymal stem cells (MSCs) that were associated with the activation of pro-mobilizing complement cascade and an elevated level of sphingosine-1 phosphate (S1P) in PB plasma. We wondered if a similar correlation occurs in patients with colorectal cancer (CRC). A total of 46 patients were enrolled in this study: 17 with CRC, 18 with benign colonic adenomas (BCA) and 11 healthy individuals. By employing fluorescence-activated cell sorting (FACS) we evaluated the number of BM-derived SCs circulating in PB: i) CD34+/Lin-/CD45- and CD133-/Lin-/CD45- VSELs; ii) CD45-/CD105+/CD90+/CD29+ MSCs; iii) CD45-/CD34+/CD133+/KDR+ endothelial progenitor cells (EPCs); and iv) CD133+/Lin-/CD45+ or CD34+/Lin-/CD45+ cells enriched for hematopoietic stem/progenitor cells (HSPCs). In parallel, we measured in the PB parameters regulating the egress of SCs from BM into PB. In contrast to pancreatic and gastric cancer patients, CRC subjects presented neither an increase in the number of circulating SCs nor the activation of pro-mobilizing factors such as complement, coagulation and fibrinolytic cascade, circulating stromal derived factor 1 (SDF‑1), vascular endothelial growth factor (VEGF) and intestinal permeability marker (zonulin). In conclusion, mobilization of SCs in cancer patients depends on the type of malignancy and its ability to activate pro-mobilization cascades.

Adenosinergic Immunosuppression by Human Mesenchymal Stromal Cells Requires Co-Operation with T cells.

PubMed

Kerkelä, Erja; Laitinen, Anita; Räbinä, Jarkko; Valkonen, Sami; Takatalo, Maarit; Larjo, Antti; Veijola, Johanna; Lampinen, Milla; Siljander, Pia; Lehenkari, Petri; Alfthan, Kaija; Laitinen, Saara

2016-03-01

Mesenchymal stem/stromal cells (MSCs) have the capacity to counteract excessive inflammatory responses. MSCs possess a range of immunomodulatory mechanisms, which can be deployed in response to signals in a particular environment and in concert with other immune cells. One immunosuppressive mechanism, not so well-known in MSCs, is mediated via adenosinergic pathway by ectonucleotidases CD73 and CD39. In this study, we demonstrate that adenosine is actively produced from adenosine 5'-monophosphate (AMP) by CD73 on MSCs and MSC-derived extracellular vesicles (EVs). Our results indicate that although MSCs express CD39 at low level and it colocalizes with CD73 in bulge areas of membranes, the most efficient adenosine production from adenosine 5'-triphosphate (ATP) requires co-operation of MSCs and activated T cells. Highly CD39 expressing activated T cells produce AMP from ATP and MSCs produce adenosine from AMP via CD73 activity. Furthermore, adenosinergic signaling plays a role in suppression of T cell proliferation in vitro. In conclusion, this study shows that adenosinergic signaling is an important immunoregulatory mechanism of MSCs, especially in situations where ATP is present in the extracellular environment, like in tissue injury. An efficient production of immunosuppressive adenosine is dependent on the concerted action of CD39-positive immune cells with CD73-positive cells such as MSCs or their EVs. © 2016 AlphaMed Press.

CD73 Regulates Stemness and Epithelial-Mesenchymal Transition in Ovarian Cancer-Initiating Cells.

PubMed

Lupia, Michela; Angiolini, Francesca; Bertalot, Giovanni; Freddi, Stefano; Sachsenmeier, Kris F; Chisci, Elisa; Kutryb-Zajac, Barbara; Confalonieri, Stefano; Smolenski, Ryszard T; Giovannoni, Roberto; Colombo, Nicoletta; Bianchi, Fabrizio; Cavallaro, Ugo

2018-04-10

Cancer-initiating cells (CICs) have been implicated in tumor development and aggressiveness. In ovarian carcinoma (OC), CICs drive tumor formation, dissemination, and recurrence, as well as drug resistance, thus accounting for the high death-to-incidence ratio of this neoplasm. However, the molecular mechanisms that underlie such a pathogenic role of ovarian CICs (OCICs) remain elusive. Here, we have capitalized on primary cells either from OC or from its tissues of origin to obtain the transcriptomic profile associated with OCICs. Among the genes differentially expressed in OCICs, we focused on CD73, which encodes the membrane-associated 5'-ectonucleotidase. The genetic inactivation of CD73 in OC cells revealed that this molecule is causally involved in sphere formation and tumor initiation, thus emerging as a driver of OCIC function. Furthermore, functional inhibition of CD73 via either a chemical compound or a neutralizing antibody reduced sphere formation and tumorigenesis, highlighting the druggability of CD73 in the context of OCIC-directed therapies. The biological function of CD73 in OCICs required its enzymatic activity and involved adenosine signaling. Mechanistically, CD73 promotes the expression of stemness and epithelial-mesenchymal transition-associated genes, implying a regulation of OCIC function at the transcriptional level. CD73, therefore, is involved in OCIC biology and may represent a therapeutic target for innovative treatments aimed at OC eradication. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

CD73 expression identifies a subset of IgM+ antigen-experienced cells with memory attributes that is T cell and CD40 signalling dependent.

PubMed

D'Souza, Lucas; Gupta, Sneh Lata; Bal, Vineeta; Rath, Satyajit; George, Anna

2017-12-01

B-cell memory was long characterized as isotype-switched, somatically mutated and germinal centre (GC)-derived. However, it is now clear that the memory pool is a complex mixture that includes unswitched and unmutated cells. Further, expression of CD73, CD80 and CD273 has allowed the categorization of B-cell memory into multiple subsets, with combinatorial expression of the markers increasing with GC progression, isotype-switching and acquisition of somatic mutations. We have extended these findings to determine whether these markers can be used to identify IgM memory phenotypically as arising from T-dependent versus T-independent responses. We report that CD73 expression identifies a subset of antigen-experienced IgM + cells that share attributes of functional B-cell memory. This subset is reduced in the spleens of T-cell-deficient and CD40-deficient mice and in mixed marrow chimeras made with mutant and wild-type marrow, the proportion of CD73 + IgM memory is restored in the T-cell-deficient donor compartment but not in the CD40-deficient donor compartment, indicating that CD40 ligation is involved in its generation. We also report that CD40 signalling supports optimal expression of CD73 on splenic T cells and age-associated B cells (ABCs), but not on other immune cells such as neutrophils, marginal zone B cells, peritoneal cavity B-1 B cells and regulatory T and B cells. Our data indicate that in addition to promoting GC-associated memory generation during B-cell differentiation, CD40-signalling can influence the composition of the unswitched memory B-cell pool. They also raise the possibility that a fraction of ABCs may represent T-cell-dependent IgM memory. © 2017 John Wiley & Sons Ltd.

Inhibition of heat-shock protein 90 sensitizes liver cancer stem-like cells to magnetic hyperthermia and enhances anti-tumor effect on hepatocellular carcinoma-burdened nude mice

PubMed Central

Yang, Rui; Tang, Qiusha; Miao, Fengqin; An, Yanli; Li, Mengfei; Han, Yong; Wang, Xihui; Wang, Juan; Liu, Peidang; Chen, Rong

2015-01-01

Purpose To explore the thermoresistance and expression of heat-shock protein 90 (HSP90) in magnetic hyperthermia-treated human liver cancer stem-like cells (LCSCs) and the effects of a heat-shock protein HSP90 inhibitor 17-allylamino-17-demethoxgeldanamycin (17-AAG) on hepatocellular carcinoma-burdened nude mice. Methods CD90+ LCSCs were isolated by magnetic-activated cell sorting from BEL-7404. Spheroid formation, proliferation, differentiation, drug resistance, and tumor formation assays were performed to identify stem cell characteristics. CD90-targeted thermosensitive magnetoliposomes (TMs)-encapsulated 17-AAG (CD90@17-AAG/TMs) was prepared by reverse-phase evaporation and its characteristics were studied. Heat tolerance in CD90+ LCSCs and the effect of CD90@17-AAG/TMs-mediated heat sensitivity were examined in vitro and in vivo. Results CD90+ LCSCs showed significant stem cell-like properties. The 17-AAG/TMs were successfully prepared and were spherical in shape with an average size of 128.9±7.7 nm. When exposed to magnetic hyperthermia, HSP90 was up-regulated in CD90+ LCSCs. CD90@17-AAG/TMs inhibited the activity of HSP90 and increased the sensitivity of CD90+ LCSCs to magnetic hyperthermia. Conclusion The inhibition of HSP90 could sensitize CD90+ LCSCs to magnetic hyperthermia and enhance its anti-tumor effects in vitro and in vivo. PMID:26677324

Biomarkers for Prostate and Bladder Cancer — EDRN Public Portal

Cancer.gov

CD26+ cancer cells and CD90+ tumor-associated stromal cells were sorted from tumor tissue. Dataset analysis with transcriptomes of CD26+ luminal, CD104+ basal, CD49a+ stromal and CD31+ endothelial was carried out to identify candidates: CD90, AGR2, BCMP11, CEACAM5, CRISP3. Quantitative protomics was applied to measure these proteins in urine samples.

CD90/THY1 is over-expressed in prostate cancer-associated fibroblasts and could serve as a cancer biomarker

PubMed Central

True, Lawrence D; Zhang, Hui; Ye, Mingliang; Huang, Chung-Ying; Nelson, Peter S; von Haller, Priska D; Tjoelker, Larry W; Kim, Jong-Seo; Qian, Wei-Jun; Smith, Richard D; Ellis, William J; Liebeskind, Emily S; Liu, Alvin Y

2010-01-01

A by-product in the processing of prostate tissue for cell sorting by collagenase digestion is the media supernatant that remains after the cells are harvested. These supernatants contain proteins made by the cells within the tissue. Quantitative proteomic analysis of N-glycosylated proteins detected an increased amount of CD90/THY1 in cancer supernatants compared to non-cancer supernatants. Immunohistochemistry showed that in all carcinomas, regardless of Gleason grade, a layer of CD90-positive stromal fibroblastic cells, approximately 5-to-10 cells deep, was localized to tumor glands. In contrast, a no more than 1-cell wide girth of CD90-positive stromal cells was found around benign glands. The increased number of CD90-positive stromal cells in cancer correlated with overexpression of CD90 mRNA detected by gene expression analysis of stromal cells obtained by laser-capture microdissection. There is increasing evidence that cancer-associated stroma plays a role in both tumor progression and carcinogenesis. Most experiments to identify cancer biomarkers have focused on the cancer cells. CD90, being a marker for prostate cancer-associated stroma, might be a potential biomarker for this cancer. A non-invasive test could be provided by a urine test. Proteomic analysis of urine from patients with prostate cancer identified CD90; conversely, CD90 was not detected in the urine of post-prostatectomy patients. Furthermore, this urinary CD90 protein was a variant CD90 protein not known to be expressed by such cells as lymphocytes that express CD90. These CD90 results were obtained from ∼90 cases consisting of proteomic analysis of tissue and urine, immunohistochemistry, Western blot analysis of tissue media, flow cytometry of cells from digested tissue, and reverse transcriptase polymerase chain reaction analysis of isolated stromal cells. PMID:20562849

Intraportal injection of insulin-producing cells generated from human bone marrow mesenchymal stem cells decreases blood glucose level in diabetic rats.

PubMed

Tsai, Pei-Jiun; Wang, Hwai-Shi; Lin, Chi-Hung; Weng, Zen-Chung; Chen, Tien-Hua; Shyu, Jia-Fwu

2014-01-01

We studied the process of trans-differentiation of human bone marrow mesenchymal stem cells (hBM-MSCs) into insulin-producing cells. Streptozotocin (STZ)-induced diabetic rat model was used to study the effect of portal vein transplantation of these insulin-producing cells on blood sugar levels. The BM-MSCs were differentiated into insulin-producing cells under defined conditions. Real-time PCR, immunocytochemistry and glucose challenge were used to evaluate in vitro differentiation. Flow cytometry showed that hBM-MSCs were strongly positive for CD44, CD105 and CD73 and negative for hematopoietic markers CD34, CD38 and CD45. Differentiated cells expressed C-peptide as well as β-cells specific genes and hormones. Glucose stimulation increased C-peptide secretion in these cells. The insulin-producing, differentiated cells were transplanted into the portal vein of STZ-induced diabetic rats using a Port-A catheter. The insulin-producing cells were localized in the liver of the recipient rat and expressed human C-peptide. Blood glucose levels were reduced in diabetic rats transplanted with insulin-producing cells. We concluded that hBM-MSCs could be trans-differentiated into insulin-producing cells in vitro. Portal vein transplantation of insulin-producing cells alleviated hyperglycemia in diabetic rats.

The SHH/Gli axis regulates CD90-mediated liver cancer stem cell function by activating the IL6/JAK2 pathway.

PubMed

Zhang, Ketao; Che, Siyao; Pan, Chuzhi; Su, Zheng; Zheng, Shangyou; Yang, Shanglin; Zhang, Huayao; Li, Wenda; Wang, Weidong; Liu, Jianping

2018-05-02

The cell surface antigen CD90 has recently been established as a promising marker for liver cancer stem cells. This study aimed to investigate potential implications of SHH/Gli signalling in CD90+ liver cancer stem cells. Correlation of the expression of SHH signalling components and CD90 in liver cancer cells and clinical tissues, as well as in enriched CD90+ liver cancer stem cells and the TCGA database, were analysed by quantitative RT-PCR, Western blotting and flow cytometry. Functional analysis was conducted by siRNA-mediated CD90, Gli1 and Gli3 gene knockdown, SHH treatment and application of the JAK2 inhibitor AZD1480 and IL6 neutralizing antibody in CD90+ liver cancer stem cells, followed by cell proliferation, migration, sphere formation and tumorigenicity assays. CD90 expression exhibited a high positive correlation with Gli1 and Gli3 in multiple liver cancer cell lines and human cancerous liver tissues, both of which showed a significant increase in liver cancer. Analysis of TCGA data revealed an association of CD90, Gli1 and Gli3 with a short overall survival and positive correlation between CD90 expression and Gli3 expression level. The stem cell potentials of CD90+ 97L liver cancer cells were greatly impaired by Gli1/3 knockdown with siRNA but enhanced by SHH treatment. Application of the JAK2 inhibitor AZD1480 and IL6 neutralizing antibody showed the CD90 and SHH/Gli-regulated liver cancer stem cell functions were mediated by the IL6/JAK2/STAT3 pathway. The stem cell properties of CD90+ liver cancer cells are regulated by the downstream SHH/Gli and IL6/JAK2/STAT3 signalling pathways. © 2018 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Purification of human adipose-derived stem cells from fat tissues using PLGA/silk screen hybrid membranes.

PubMed

Chen, Da-Chung; Chen, Li-Yu; Ling, Qing-Dong; Wu, Meng-Hsueh; Wang, Ching-Tang; Suresh Kumar, S; Chang, Yung; Munusamy, Murugan A; Alarfajj, Abdullah A; Wang, Han-Chow; Hsu, Shih-Tien; Higuchi, Akon

2014-05-01

The purification of human adipose-derived stem cells (hADSCs) from human adipose tissue cells (stromal vascular fraction) was investigated using membrane filtration through poly(lactide-co-glycolic acid)/silk screen hybrid membranes. Membrane filtration methods are attractive in regenerative medicine because they reduce the time required to purify hADSCs (i.e., less than 30 min) compared with conventional culture methods, which require 5-12 days. hADSCs expressing the mesenchymal stem cell markers CD44, CD73, and CD90 were concentrated in the permeation solution from the hybrid membranes. Expression of the surface markers CD44, CD73, and CD99 on the cells in the permeation solution from the hybrid membranes, which were obtained using 18 mL of feed solution containing 50 × 10⁴ cells, was statistically significantly higher than that of the primary adipose tissue cells, indicating that the hADSCs can be purified in the permeation solution by the membrane filtration method. Cells expressing the stem cell-associated marker CD34 could be successfully isolated in the permeation solution, whereas CD34⁺ cells could not be purified by the conventional culture method. The hADSCs in the permeation solution demonstrated a superior capacity for osteogenic differentiation based on their alkali phosphatase activity, their osterix gene expression, and the results of mineralization analysis by Alizarin Red S and von Kossa staining compared with the cells from the suspension of human adipose tissue. These results suggest that the hADSCs capable of osteogenic differentiation preferentially permeate through the hybrid membranes. Copyright © 2014 Elsevier Ltd. All rights reserved.

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.

Expression of CD73 slows down migration of skin dendritic cells, affecting the sensitization phase of contact hypersensitivity reactions in mice.

PubMed

Neuberger, A; Ring, S; Silva-Vilches, C; Schrader, J; Enk, A; Mahnke, K

2017-09-01

Application of haptens to the skin induces release of immune stimulatory ATP into the extracellular space. This "danger" signal can be converted to immunosuppressive adenosine (ADO) by the action of the ectonucleotidases CD39 and CD73, expressed by skin and immune cells. Thus, the expression and regulation of CD73 by skin derived cells may have crucial influence on the outcome of contact hypersensitivity (CHS) reactions. To investigate the role of CD73 expression during 2,4,6-trinitrochlorobenzene (TNCB) induced CHS reactions. Wild type (wt) and CD73 deficient mice were subjected to TNCB induced CHS. In the different mouse strains the resulting ear swelling reaction was recorded along with a detailed phenotypic analysis of the skin migrating subsets of dendritic cells (DC). In CD73 deficient animals the motility of DC was higher as compared to wt animals and in particular after sensitization we found increased migration of Langerin + DC from skin to draining lymph nodes (LN). In the TNCB model this led to a stronger sensitization as indicated by increased frequency of interferon-γ producing T cells in the LN and an increased ear thickness after challenge. CD73 derived ADO production slows down migration of Langerin + DC from skin to LN. This may be a crucial mechanism to avoid over boarding immune reactions against haptens. Copyright © 2017 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.

Effects of Maternal Exposure to Cadmium Oxide Nanoparticles During Pregnancy on Maternal and Offspring Kidney Injury Markers Using a Murine Model.

PubMed

Blum, Jason L; Edwards, Joshua R; Prozialeck, Walter C; Xiong, Judy Q; Zelikoff, Judith T

2015-01-01

Nanoparticles (NP) are pervasive in many areas of modern life, with little known about their potential toxicities. One commercially important NP is cadmium oxide (CdO), which is used to synthesize other Cd-containing NP, such as quantum dots. Cadmium (Cd) is a well-known nephrotoxicant, but the nephrotoxic potential of CdO NP remains unknown, particularly when exposure occurs during pregnancy. Therefore, pregnant CD-1 mice were used to examine the effects of inhaled CdO NP (230 μg CdO NP/m(3)) on maternal and neonatal renal function by examining urinary creatinine and urinary biomarkers of kidney injury, including kidney injury molecule-1 (Kim-1) and neutrophil gelatinase-associated lipocalin (NGAL). Inhalation of CdO NP by dams produced a fivefold increase in urinary Kim-1 with no marked effect on urinary creatinine levels. Kim-1 mRNA expression peaked by gestational day (GD) 10.5, and NGAL expression increased from GD 10.5 to 17.5. In addition, histological analyses revealed proximal tubular pathology at GD 10.5. Neonatal Kim-1 mRNA expression rose between postnatal days (PND) 7 and 14, with mammary glands/milk being the apparent source of Cd for offspring. These studies demonstrate that, similar to what is seen with other Cd forms, Cd associated with inhaled CdO NP results in renal injury to both directly exposed dam and offspring. As commercial uses for nanotechnology continue to expand throughout the world, risks for unintentional exposure in the workplace increase. Given the large number of women in the industrial workforce, care needs to be taken to protect these already vulnerable populations.

Pluripotent Nontumorigenic Adipose Tissue-Derived Muse Cells have Immunomodulatory Capacity Mediated by Transforming Growth Factor-β1.

PubMed

Gimeno, María L; Fuertes, Florencia; Barcala Tabarrozzi, Andres E; Attorressi, Alejandra I; Cucchiani, Rodolfo; Corrales, Luis; Oliveira, Talita C; Sogayar, Mari C; Labriola, Leticia; Dewey, Ricardo A; Perone, Marcelo J

2017-01-01

Adult mesenchymal stromal cell-based interventions have shown promising results in a broad range of diseases. However, their use has faced limited effectiveness owing to the low survival rates and susceptibility to environmental stress on transplantation. We describe the cellular and molecular characteristics of multilineage-differentiating stress-enduring (Muse) cells derived from adipose tissue (AT), a subpopulation of pluripotent stem cells isolated from human lipoaspirates. Muse-AT cells were efficiently obtained using a simple, fast, and affordable procedure, avoiding cell sorting and genetic manipulation methods. Muse-AT cells isolated under severe cellular stress, expressed pluripotency stem cell markers and spontaneously differentiated into the three germ lineages. Muse-AT cells grown as spheroids have a limited proliferation rate, a diameter of ∼15 µm, and ultrastructural organization similar to that of embryonic stem cells. Muse-AT cells evidenced high stage-specific embryonic antigen-3 (SSEA-3) expression (∼60% of cells) after 7-10 days growing in suspension and did not form teratomas when injected into immunodeficient mice. SSEA-3 + -Muse-AT cells expressed CD105, CD29, CD73, human leukocyte antigen (HLA) class I, CD44, and CD90 and low levels of HLA class II, CD45, and CD34. Using lipopolysaccharide-stimulated macrophages and antigen-challenged T-cell assays, we have shown that Muse-AT cells have anti-inflammatory activities downregulating the secretion of proinflammatory cytokines, such as interferon-γ and tumor necrosis factor-α. Muse-AT cells spontaneously gained transforming growth factor-β1 expression that, in a phosphorylated SMAD2-dependent manner, might prove pivotal in their observed immunoregulatory activity through decreased expression of T-box transcription factor in T cells. Collectively, the present study has demonstrated the feasibility and efficiency of obtaining Muse-AT cells that can potentially be harnessed as immunoregulators to treat immune-related disorders. Stem Cells Translational Medicine 2017;6:161-173. © 2016 The Authors Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

Investigation of the Cell Surface Proteome of Human Periodontal Ligament Stem Cells

PubMed Central

Xiong, Jimin; Menicanin, Danijela; Marino, Victor

2016-01-01

The present study examined the cell surface proteome of human periodontal ligament stem cells (PDLSC) compared to human fibroblasts. Cell surface proteins were prelabelled with CyDye before processing to extract the membrane lysates, which were separated using 2D electrophoresis. Selected differentially expressed protein “spots” were identified using Mass spectrometry. Four proteins were selected for validation: CD73, CD90, Annexin A2, and sphingosine kinase 1 previously associated with mesenchymal stem cells. Flow cytometric analysis found that CD73 and CD90 were highly expressed by human PDLSC and gingival fibroblasts but not by keratinocytes, indicating that these antigens could be used as potential markers for distinguishing between mesenchymal cells and epithelial cell populations. Annexin A2 was also found to be expressed at low copy number on the cell surface of human PDLSC and gingival fibroblasts, while human keratinocytes lacked any cell surface expression of Annexin A2. In contrast, sphingosine kinase 1 expression was detected in all the cell types examined using immunocytochemical analysis. These proteomic studies form the foundation to further define the cell surface protein expression profile of PDLSC in order to better characterise this cell population and help develop novel strategies for the purification of this stem cell population. PMID:27579043

Investigation of the Cell Surface Proteome of Human Periodontal Ligament Stem Cells.

PubMed

Xiong, Jimin; Menicanin, Danijela; Zilm, Peter S; Marino, Victor; Bartold, P Mark; Gronthos, Stan

2016-01-01

The present study examined the cell surface proteome of human periodontal ligament stem cells (PDLSC) compared to human fibroblasts. Cell surface proteins were prelabelled with CyDye before processing to extract the membrane lysates, which were separated using 2D electrophoresis. Selected differentially expressed protein "spots" were identified using Mass spectrometry. Four proteins were selected for validation: CD73, CD90, Annexin A2, and sphingosine kinase 1 previously associated with mesenchymal stem cells. Flow cytometric analysis found that CD73 and CD90 were highly expressed by human PDLSC and gingival fibroblasts but not by keratinocytes, indicating that these antigens could be used as potential markers for distinguishing between mesenchymal cells and epithelial cell populations. Annexin A2 was also found to be expressed at low copy number on the cell surface of human PDLSC and gingival fibroblasts, while human keratinocytes lacked any cell surface expression of Annexin A2. In contrast, sphingosine kinase 1 expression was detected in all the cell types examined using immunocytochemical analysis. These proteomic studies form the foundation to further define the cell surface protein expression profile of PDLSC in order to better characterise this cell population and help develop novel strategies for the purification of this stem cell population.

Endogenous adenosine produced during hypoxia attenuates neutrophil accumulation: coordination by extracellular nucleotide metabolism.

PubMed

Eltzschig, Holger K; Thompson, Linda F; Karhausen, Jorn; Cotta, Richard J; Ibla, Juan C; Robson, Simon C; Colgan, Sean P

2004-12-15

Hypoxia is a well-documented inflammatory stimulus and results in tissue polymorphonuclear leukocyte (PMN) accumulation. Likewise, increased tissue adenosine levels are commonly associated with hypoxia, and given the anti-inflammatory properties of adenosine, we hypothesized that adenosine production via adenine nucleotide metabolism at the vascular surface triggers an endogenous anti-inflammatory response during hypoxia. Initial in vitro studies indicated that endogenously generated adenosine, through activation of PMN adenosine A(2A) and A(2B) receptors, functions as an antiadhesive signal for PMN binding to microvascular endothelia. Intravascular nucleotides released by inflammatory cells undergo phosphohydrolysis via hypoxia-induced CD39 ectoapyrase (CD39 converts adenosine triphosphate/adenosine diphosphate [ATP/ADP] to adenosine monophosphate [AMP]) and CD73 ecto-5'-nucleotidase (CD73 converts AMP to adenosine). Extensions of our in vitro findings using cd39- and cd73-null animals revealed that extracellular adenosine produced through adenine nucleotide metabolism during hypoxia is a potent anti-inflammatory signal for PMNs in vivo. These findings identify CD39 and CD73 as critical control points for endogenous adenosine generation and implicate this pathway as an innate mechanism to attenuate excessive tissue PMN accumulation.

Successful vitrification of human amnion-derived mesenchymal stem cells.

PubMed

Moon, Jeong Hee; Lee, Jung Ryeol; Jee, Byung Chul; Suh, Chang Suk; Kim, Seok Hyun; Lim, Hyun Jung; Kim, Hae Kwon

2008-08-01

A cryopreservation protocol for human amnion-derived mesenchymal stem cells (HAMs) is required because these cells cannot survive for long periods in culture. The aim of this study was to determine whether vitrification is a useful freezing method for storage of HAMs. HAMs were cryopreserved using vitrification method. The morphology and viability of thawed HAMs was evaluated by Trypan Blue staining. The expression of several embryonic stem cell (ESC) markers was evaluated using flow cytometry, RT-PCR and immunocytochemistry. Von Kossa, Oil Red O and Alcian Blue staining were used to asses the differentiation potential of thawed HAMs. The post-thawing viability of HAMs was 84.3 +/- 3.2% (Mean +/- SD, n = 10). The thawed HAMs showed morphological characteristics indistinguishable from the non-vitrified fresh HAMs. The expression of surface antigens (strong positive for CD44, CD49d, CD59, CD90, CD105 and HLA-ABC; weak positive for HLA-G; negative for CD31, CD34, CD45, CD106, CD117 and HLA-DR) and the expression of ESC markers [CK18, fibroblast growth factor-5, GATA-4, neural cell adhesion molecule, Nestin, Oct-4, stem cell factor, HLA-ABC, Vimentin, bone morphogenetic protein (BMP) 4, hepatocyte nuclear factor 4 alpha (HNF-4 alpha), Pax-6, alpha-fetoprotein, Brachyury, BMP-2, TRA-1-60, stage-specific embryonic antigen (SSEA-3, SSEA-4)] were maintained in the vitrified-thawed HAMs. The thawed HAMs retained ability to differentiate into osteoblasts, adipocytes and chondrocytes under appropriate culture conditions. Our results suggest that vitrification is a reliable and effective method for cryopreservation of HAMs.

[Pooled Umbilical Cord Blood Plasma for Culturing UCMSC and Ex Vivo Expanding Umbilical Cord Blood CD34⁺ Cells].

PubMed

Wu, Jie-Ying; Lu, Yan; Chen, Jin-Song; Wu, Shao-Qing; Tang, Xue-Wei; Li, Yan

2015-08-01

To investigate the feasibility of umbilical cord blood plasma (UCP) as a replacement for fetal bovine serum (FBS) for culturing mesenchymal stem cells (MSC) derived from umbilical cord, and to observe the supporting effects of these cells (served as a feeder layer) on ex vivo expanding of human umbilical cord blood CD34(+) cells. Umbilical cord blood (UCB) units were suitable if the Guangzhou cord blood bank donor selection criteria strictly were fulfilled. UCP were ready to use after the collection from the plasma depletion/reduction during the processing and pooling of suitable UCB units (at least 30 units were screened for pathogens and microorganisms, and qualified). Umbilical cord mesenchymal stem cells (UCMSC) were harvested from the umbilical cord tissue of health full-term newborns after delivery by enzyme digestion and divided into 3 groups: group 1 and 2 were cultured in the presence of DMEM/F12 containing either FBS or UCP; and group 3 was cultured in serum-free medium (StemPro® MSC SFM CTS™). Morphology, proliferation and surface marker expression were examined by flow cytometry, and the differentiation toward adipogenic and osteogenic lineages was used for investigating the effect of media on UCMSC after 3-5 passages. Next, the cells cultured in the three different media were cryopreserved and thawed, then prepared as feeder layers with the name of UCMSC(FBS), UCMSC(UCP), and UCMSC(SFM), respectively. The CD34⁺ cells were separated from UCB by magnetic activated cell sorting (MACS) and divided into 4 groups cultured in StemPro(-34) SFM medium added with hematopoietic cytokine combination (StemSpan® CC100). The control group included only CD34⁺ cells as group A (blank control) and experimental groups included UCMSC(FBS) + CD34⁺ cells as group B, UCMSC(UCP) + CD34⁺ cells as group C, UCMSC(SFM) + CD34⁺ cells as group D, and cells in all groups were cultured ex vivo for 7 days. The nucleated cell (NC) number was counted by cell counter, CD34⁺ cells were measured by flow cytometry, and clonogenic assay was conducted at day 0 and 7 of culture. The expansion efficiency was assessed. The morphology (spindle-shaped and plastic-adherent), the immunophenotype (high positive percentage of CD73, CD90, CD105 and CD166) and the differentiation potential (osteogenic and adipogenic) were almost indistinguishable among the cells cultured in any of these three media except for the expression of CD105 in group 3 (serum-free medium) was lower than that in other 2 groups (P < 0.05). UCMSC grown in UCP medium demonstrated significantly higher proliferation rates than that in media containing FBS or commercial serum-free supplement (P < 0.05). After co-culture for 7 days, the CD34⁺ cell percentage decreased in all the groups, while NC were amplified effectively and the CD34⁺ cell number increased with the same order as group C or D group B or A (control group) (P < 0.05). As compared with the colony-forming unit (CFU) number at day 0, there was no significant difference in the expansion multiple between group C and D, while the expansion of CFU in group C were higher than that in group B and A. The UCP can be used as a better animal-free serum supplement for growth, maintenance and differentiation of UCMSC, thus would be a safe choice for clinical-scale production of human MSC.

Subchondral mesenchymal stem cells from osteoarthritic knees display high osteogenic differentiation capacity through microRNA-29a regulation of HDAC4.

PubMed

Lian, Wei-Shiung; Wu, Ren-Wen; Lee, Mel S; Chen, Yu-Shan; Sun, Yi-Chih; Wu, Shing-Long; Ke, Huei-Jing; Ko, Jih-Yang; Wang, Feng-Sheng

2017-12-01

Subchondral bone deterioration and osteophyte formation attributable to excessive mineralization are prominent features of end-stage knee osteoarthritis (OA). The cellular events underlying subchondral integrity diminishment remained elusive. This study was undertaken to characterize subchondral mesenchymal stem cells (SMSCs) isolated from patients with end-stage knee OA who required total knee arthroplasty. The SMSCs expressed surface antigens CD29, CD44, CD73, CD90, CD105, and CD166 and lacked CD31, CD45, and MHCII expression. The cell cultures exhibited higher proliferation and greater osteogenesis and chondrogenesis potencies, whereas their population-doubling time and adipogenic lineage commitment were lower than those of bone marrow MSCs (BMMSCs). They also displayed higher expressions of embryonic stem cell marker OCT3/4 and osteogenic factors Wnt3a, β-catenin, and microRNA-29a (miR-29a), concomitant with lower expressions of joint-deleterious factors HDAC4, TGF-β1, IL-1β, TNF-α, and MMP3, in comparison with those of BMMSCs. Knockdown of miR-29a lowered Wnt3a expression and osteogenic differentiation of the SMSCs through elevating HDAC4 translation, which directly regulated the 3'-untranslated region of HDAC4. Likewise, transgenic mice that overexpressed miR-29a in osteoblasts exhibited a high bone mass in the subchondral region. SMSCs in the transgenic mice showed a higher osteogenic differentiation and lower HDAC4 signaling than those in wild-type mice. Taken together, high osteogenesis potency existed in the SMSCs in the osteoarthritic knee. The miR-29a modulation of HDAC4 and Wnt3a signaling was attributable to the increase in osteogenesis. This study shed an emerging light on the characteristics of SMSCs and highlighted the contribution of SMSCs in the exacerbation of subchondral integrity in end-stage knee OA. Subchondral MSCs (SMSCs) from OA knee expressed embryonic stem cell marker Oct3/4. The SMSCs showed high proliferation and osteogenic and chondrogenic potencies. miR-29a regulated osteogenesis of the SMSCs through modulation of HDAC4 and Wnt3a. A high osteogenic potency of the SMSCs existed in mice overexpressing miR-29a in bone. Aberrant osteogenesis in SMSCs provides a new insight to subchondral damage in OA.

5-FU resistant EMT-like pancreatic cancer cells are hypersensitive to photochemical internalization of the novel endoglin-targeting immunotoxin CD105-saporin.

PubMed

Lund, Kaja; Olsen, Cathrine Elisabeth; Wong, Judith Jing Wen; Olsen, Petter Angell; Solberg, Nina Therese; Høgset, Anders; Krauss, Stefan; Selbo, Pål Kristian

2017-12-19

Development of resistance to 5-fluorouracil (5-FU) is a major problem in treatment of various cancers including pancreatic cancer. In this study, we reveal important resistance mechanisms and photochemical strategies to overcome 5-FU resistance in pancreatic adenocarcinoma. 5-FU resistant (5-FUR), epithelial-to-mesenchymal-like sub-clones of the wild type pancreatic cancer cell line Panc03.27 were previously generated in our lab. We investigated the cytotoxic effect of the endosomal/lysosomal-localizing photosensitizer TPCS 2a (fimaporfin) combined with light (photochemical treatment, PCT) using MTS viability assay, and used fluorescence microscopy to show localization of TPCS 2a and to investigate the effect of photodamage of lysosomes. Flow cytometric analysis was performed to investigate uptake of photosensitizer and to assess intracellular ROS levels. Expression and localization of LAMP1 was assessed using RT-qPCR, western blotting, and structured illumination microscopy. MTS viability assay was used to assess the effect of combinations of 5-FU, chloroquine (CQ), and photochemical treatment. Expression of CD105 was investigated using RT-qPCR, western blotting, flow cytometry, and fluorescence microscopy, and co-localization of TPCS 2a and anti-CD105-saporin was assessed using microscopy. Lastly, the MTS assay was used to investigate cytotoxic effects of photochemical internalization (PCI) of the anti-CD105-immunotoxin. The 5-FUR cell lines display hypersensitivity to PCT, which was linked to increased uptake of TPCS 2a , altered lysosomal distribution, lysosomal photodamage and increased expression of the lysosomal marker LAMP-1 in the 5-FUR cells. We show that inhibition of autophagy induced by either chloroquine or lysosomal photodamage increases the sensitivity to 5-FU in the resistant cells. The three 5-FUR sub-clones overexpress Endoglin (CD105). Treatment with the immunotoxin anti-CD105-saporin alone significantly reduced the viability of the CD105-expressing 5-FUR cells, whereas little effect was seen in the CD105-negative non-resistant parental cancer cell lines. Strikingly, using the intracellular drug delivery method photochemical internalization (PCI) by combining light-controlled activation of the TPCS 2a with nanomolar levels of CD105-saporin resulted in strong cytotoxic effects in the 5-FUR cell population. Our findings suggested that autophagy is an important resistance mechanism against the chemotherapeutic drug 5-FU in pancreatic cancer cells, and that inhibition of the autophagy process, either by CQ or lysosomal photodamage, can contribute to increased sensitivity to 5-FU. For the first time, we demonstrate the promise of PCI-based targeting of CD105 in site-specific elimination of 5-FU resistant pancreatic cancer cells in vitro. In conclusion, PCI-based targeting of CD105 may represent a potent anticancer strategy and should be further evaluated in pre-clinical models.

CD105+-mesenchymal stem cells migrate into osteoarthritis joint: An animal model.

PubMed

Fernandez-Pernas, Pablo; Rodríguez-Lesende, Iván; de la Fuente, Alexandre; Mateos, Jesús; Fuentes, Isaac; De Toro, Javier; Blanco, Fco J; Arufe, M C

2017-01-01

Mesenchymal stem cells are being the focus of connective tissue technology and regenerative medicine, presenting a good choice cell source for improving old and well recognized techniques of cartilage defect repair. For instance, the autologous chondrocyte transplantation using new concepts of regenerative medicine. The present study investigated the risk of xenogenicity of human synovial membrane-derived MSCs, injected into the monkeys using intravenous and intra-articular administration. The animal models used were adult monkeys Rhesus which had been injured into the left knee to create an Osteoarthritis (OA) animal model. CD105+-MSCs were injected twice into the OA monkeys with an interval of one week between them. The animals were euthanized one month after treatment. Immunohistochemistry analysis of different organs: spleen, heart, fat, liver, gut, pancreas, lung, skeletal muscle and kidney from the animals revealed that CD105+-MSCs migrated towards the injured knee joint. MSCs naive were found statistically significant increased in the injured knee in front of healthy one. CD105+-MSCs were negatives for CD68 and the area where CD105+-MSCs were found presented SDF-1 increased levels in front of healthy knee. We concluded that a characterized MSCs subset could be a safe alternative for cell therapy in clearly localized pathologies.

CD105+-mesenchymal stem cells migrate into osteoarthritis joint: An animal model

PubMed Central

Fernandez-Pernas, Pablo; Rodríguez-Lesende, Iván; de la Fuente, Alexandre; Mateos, Jesús; Fuentes, Isaac; De Toro, Javier; Blanco, Fco J.

2017-01-01

Mesenchymal stem cells are being the focus of connective tissue technology and regenerative medicine, presenting a good choice cell source for improving old and well recognized techniques of cartilage defect repair. For instance, the autologous chondrocyte transplantation using new concepts of regenerative medicine. The present study investigated the risk of xenogenicity of human synovial membrane-derived MSCs, injected into the monkeys using intravenous and intra-articular administration. The animal models used were adult monkeys Rhesus which had been injured into the left knee to create an Osteoarthritis (OA) animal model. CD105+-MSCs were injected twice into the OA monkeys with an interval of one week between them. The animals were euthanized one month after treatment. Immunohistochemistry analysis of different organs: spleen, heart, fat, liver, gut, pancreas, lung, skeletal muscle and kidney from the animals revealed that CD105+-MSCs migrated towards the injured knee joint. MSCs naive were found statistically significant increased in the injured knee in front of healthy one. CD105+-MSCs were negatives for CD68 and the area where CD105+-MSCs were found presented SDF-1 increased levels in front of healthy knee. We concluded that a characterized MSCs subset could be a safe alternative for cell therapy in clearly localized pathologies. PMID:29190645

Cell Homogeneity Indispensable for Regenerative Medicine by Cultured Human Corneal Endothelial Cells.

PubMed

Hamuro, Junji; Toda, Munetoyo; Asada, Kazuko; Hiraga, Asako; Schlötzer-Schrehardt, Ursula; Montoya, Monty; Sotozono, Chie; Ueno, Morio; Kinoshita, Shigeru

2016-09-01

To identify the subpopulation (SP) among heterogeneous cultured human corneal endothelial cells (cHCECs) devoid of cell-state transition applicable for cell-based therapy. Subpopulation presence in cHCECs was confirmed via surface CD-marker expression level by flow cytometry. CD markers effective for distinguishing distinct SPs were selected by analyzing those on established cHCECs with a small cell area and high cell density. Contrasting features among three typical cHCEC SPs was confirmed by PCR array for extracellular matrix (ECM). Combined analysis of CD markers was performed to identify the SP (effector cells) applicable for therapy. ZO-1 and Na+/K+ ATPase, CD200, and HLA expression were compared among heterogeneous SPs. Flow cytometry analysis identified the effector cell expressing CD166+CD105-CD44-∼+/-CD26-CD24-, but CD200-, and the presence of other SPs with CD166+ CD105-CD44+++ (CD26 and CD24, either + or -) was confirmed. PCR array revealed three distinct ECM expression profiles. Some SPs expressed ZO-1 and Na+/K+ ATPase at comparable levels with effector cells, while only one SP expressed CD200, but not on effector cells. Human leukocyte antigen expression was most reduced in the effector SP. The proportion of effector cells (E-ratio) inversely paralleled donor age and decreased during prolonged culture passages. The presence of Rho-associated protein kinase (ROCK) inhibitor increased the E-ratio in cHCECs. The average area of effector cells was approximately 200∼220 μm2, and the density of cHCECs exceeded 2500 cells/mm2. A specified cultured effector cell population sharing the surface phenotypes with mature HCECs in corneal tissues may serve as an alternative to donor corneas for the treatment of corneal endothelial dysfunction.

Equine mesenchymal stem cells from bone marrow, adipose tissue and umbilical cord: immunophenotypic characterization and differentiation potential

PubMed Central

2014-01-01

Introduction Studies with mesenchymal stem cells (MSCs) are increasing due to their immunomodulatory, anti-inflammatory and tissue regenerative properties. However, there is still no agreement about the best source of equine MSCs for a bank for allogeneic therapy. The aim of this study was to evaluate the cell culture and immunophenotypic characteristics and differentiation potential of equine MSCs from bone marrow (BM-MSCs), adipose tissue (AT-MSCs) and umbilical cord (UC-MSCs) under identical in vitro conditions, to compare these sources for research or an allogeneic therapy cell bank. Methods The BM-MSCs, AT-MSCs and UC-MSCs were cultured and evaluated in vitro for their osteogenic, adipogenic and chondrogenic differentiation potential. Additionally, MSCs were assessed for CD105, CD44, CD34, CD90 and MHC-II markers by flow cytometry, and MHC-II was also assessed by immunocytochemistry. To interpret the flow cytometry results, statistical analysis was performed using ANOVA. Results The harvesting and culturing procedures of BM-MSCs, AT-MSCs and UC-MSCs were feasible, with an average cell growth until the third passage of 25 days for BM-MSCs, 15 days for AT-MSCs and 26 days for UC-MSCs. MSCs from all sources were able to differentiate into osteogenic (after 10 days for BM-MSCs and AT-MSCs and 15 days for UC-MSCs), adipogenic (after 8 days for BM-MSCs and AT-MSCs and 15 days for UC-MSCs) and chondrogenic (after 21 days for BM-MSCs, AT-MSCs and UC-MSCs) lineages. MSCs showed high expression of CD105, CD44 and CD90 and low or negative expression of CD34 and MHC-II. The MHC-II was not detected by immunocytochemistry techniques in any of the MSCs studied. Conclusions The BM, AT and UC are feasible sources for harvesting equine MSCs, and their immunophenotypic and multipotency characteristics attained minimal criteria for defining MSCs. Due to the low expression of MHC-II by MSCs, all of the sources could be used in clinical trials involving allogeneic therapy in horses. However, the BM-MSCs and AT-MSCs showed fastest ‘‘in vitro’’ differentiation and AT-MSCs showed highest cell growth until third passage. These findings suggest that BM and AT may be preferable for cell banking purposes. PMID:24559797

Proteolysis-a characteristic of tumor-initiating cells in murine metastatic breast cancer

PubMed Central

Hillebrand, Larissa E.; Bengsch, Fee; Hochrein, Jochen; Hülsdünker, Jan; Bender, Julia; Follo, Marie; Busch, Hauke; Boerries, Melanie; Reinheckel, Thomas

2016-01-01

Tumor initiating cells (TICs) have been identified and functionally characterized in hematological malignancies as well as in solid tumors such as breast cancer. In addition to their high tumor-initiating potential, TICs are founder cells for metastasis formation and are involved in chemotherapy resistance. In this study we explored molecular pathways which enable this tumor initiating potential for a cancer cell subset of the transgenic MMTV-PyMT mouse model for metastasizing breast cancer. The cell population, characterized by the marker profile CD24+CD90+CD45−, showed a high tumorigenicity compared to non-CD24+CD90+CD45− cancer cells in colony formation assays, as well as upon orthotopic transplantation into the mammary fat pad of mice. In addition, these orthotopically grown CD24+CD90+CD45− TICs metastasized to the lungs. The transcriptome of TICs freshly isolated from primary tumors by cell sorting was compared with that of sorted non-CD24+CD90+CD45− cancer cells by RNA-seq. In addition to more established TIC signatures, such as epithelial-to-mesenchymal transition or mitogen signaling, an upregulated gene set comprising several classes of proteolytic enzymes was uncovered in the TICs. Accordingly, TICs showed high intra- and extracellular proteolytic activity. Application of a broad range of protease inhibitors to TICs in a colony formation assay reduced anchorage independent growth and had an impact on colony morphology in 3D cell culture assays. We conclude that CD24+CD90+CD45− cells of the MMTV- PyMT mouse model possess an upregulated proteolytic signature which could very well represent a functional hallmark of metastatic TICs from mammary carcinomas. PMID:27542270

Endoglin (CD105) expression in sinonasal polyposis.

PubMed

Ottaviano, Giancarlo; Cappellesso, Rocco; Mylonakis, Ioannis; Lionello, Marco; Favaretto, Niccolò; Giacomelli, Luciano; Spoladore, Cristiano; Marchese-Ragona, Rosario; Marino, Filippo; Staffieri, Alberto; Martini, Alessandro; Marioni, Gino

2015-11-01

Despite appropriate surgical therapy, 5-10 % of patients with chronic rhinosinusitis (CRS) and nasal polyps (NP) experience disease recurrences. It has been suggested that angiogenesis may relate to the pathogenesis and prognosis of CRS with NP. Endoglin (CD105) is a component of the receptor complex of transforming growth factor-beta, a pleiotropic cytokine that modulates angiogenesis. A series of patients treated surgically for CRS with NP was analyzed to assess the relationship between CD105 expression, main clinicopathological features, and recurrence rate. The immunohistochemical expression of CD105 was assessed in 70 patients consecutively operated for CRS with NP. In the univariate setting, the presence of CD105 (1/0) showed a trend towards a significant association with increasing NP dimensions (p = 0.054). Intensity of CD105 reaction was also significantly associated with NP size (0.04) and with an eosinophilic histology (p = 0.048). In our multivariate setting, only asthma (p = 0.016), hypereosinophilia (p = 0.022), and preoperative polyposis score (p = 0.046) retained their independent prognostic significance in relation to NP recurrence. Further efforts are needed to elucidate the biological, angiogenic and proliferative mechanisms behind recurrent NP. Our preliminary results support the clinical utility of extra postoperative care, in terms of closer follow-ups and medication with oral anti-histamines, topical and/or oral steroids, and antileukotrienes in patients with asthma, advanced nasal polyposis at presentation, and serum hypereosinophilia.

Normal T-cell activation in elite controllers with preserved CD4+ T-cell counts.

PubMed

Bansal, Anju; Sterrett, Sarah; Erdmann, Nathan; Westfall, Andrew O; Dionne-Odom, Jodie; Overton, Edgar T; Goepfert, Paul A

2015-11-01

HIV elite controllers suppress HIV viremia without antiretroviral therapy (ART), yet previous studies demonstrated that elite controllers maintain an activated T-cell phenotype. Chronic immune activation has detrimental consequences and thus ART has been advocated for all elite controllers. However, elite controllers are not a clinically homogenous group. Since CD4% is among the best predictors of AIDS-related events, in the current study, we assessed whether this marker can be used to stratify elite controllers needing ART. Sixteen elite controllers were divided into two groups based on CD4% (EC > 40% and EC ≤40%), and T-cell subsets were analyzed for markers of memory/differentiation (CD45RA, CCR7, CD28), activation (CD38/HLA-DR), immunosenescence (CD57), costimulation (CD73, CD28) and exhaustion (PD-1, CD160, Tim-3). Monocyte subsets (CD14, CD16) were also analyzed and sCD14 levels were quantified using ELISA. In the EC group, expression of activation, exhaustion, and immunosensescence markers on T cells were significantly reduced compared with the EC group and similar to the seronegative controls. The EC group expressed higher levels of costimulatory molecules CD28 and CD73 and had lower levels of monocyte activation (HLA-DR expression) with a reduced frequency of inflammatory monocyte (CD14 CD16) subset. Furthermore, the EC group maintained a stable CD4% during a median follow-up of 6 years. Elite controllers with preserved CD4T cells (EC) have normal T-cell and monocyte phenotypes and therefore may have limited benefit from ART. CD4% can be an important marker for evaluating future studies aimed at determining the need for ART in this group of individuals.

PET Imaging of Abdominal Aortic Aneurysm with 64Cu-Labeled Anti-CD105 Antibody Fab Fragment.

PubMed

Shi, Sixiang; Orbay, Hakan; Yang, Yunan; Graves, Stephen A; Nayak, Tapas R; Hong, Hao; Hernandez, Reinier; Luo, Haiming; Goel, Shreya; Theuer, Charles P; Nickles, Robert J; Cai, Weibo

2015-06-01

The critical challenge in abdominal aortic aneurysm (AAA) research is the accurate diagnosis and assessment of AAA progression. Angiogenesis is a pathologic hallmark of AAA, and CD105 is highly expressed on newly formed vessels. Our goal was to use (64)Cu-labeled anti-CD105 antibody Fab fragment for noninvasive assessment of angiogenesis in the aortic wall in a murine model of AAA. Fab fragment of TRC105, a mAb that specifically binds to CD105, was generated by enzymatic papain digestion and conjugated to NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid) for (64)Cu labeling. The binding affinity/specificity of NOTA-TRC105-Fab was evaluated by flow cytometry and various ex vivo studies. BALB/c mice were anesthetized and treated with calcium phosphate to induce AAA and underwent weekly PET scans using (64)Cu-NOTA-TRC105-Fab. Biodistribution and autoradiography studies were also performed to confirm the accuracy of PET results. NOTA-TRC105-Fab exhibited high purity and specifically bound to CD105 in vitro. Uptake of (64)Cu-NOTA-TRC105-Fab increased from a control level of 3.4 ± 0.1 to 9.5 ± 0.4 percentage injected dose per gram (%ID/g) at 6 h after injection on day 5 and decreased to 7.2 ± 1.4 %ID/g on day 12, which correlated well with biodistribution and autoradiography studies (i.e., much higher tracer uptake in AAA than normal aorta). Of note, enhanced AAA contrast was achieved, due to the minimal background in the abdominal area of mice. Degradation of elastic fibers and highly expressed CD105 were observed in ex vivo studies. (64)Cu-NOTA-TRC105-Fab cleared rapidly through the kidneys, which enabled noninvasive PET imaging of the aorta with enhanced contrast and showed increased angiogenesis (CD105 expression) during AAA. (64)Cu-NOTA-TRC105-Fab PET may potentially be used for future diagnosis and prognosis of AAA. © 2015 by the Society of Nuclear Medicine and Molecular Imaging, Inc.

Adenosine signalling mediates the anti-inflammatory effects of the COX-2 inhibitor nimesulide.

PubMed

Caiazzo, Elisabetta; Maione, Francesco; Morello, Silvana; Lapucci, Andrea; Paccosi, Sara; Steckel, Bodo; Lavecchia, Antonio; Parenti, Astrid; Iuvone, Teresa; Schrader, Jürgen; Ialenti, Armando; Cicala, Carla

2016-07-15

Extracellular adenosine formation from ATP is controlled by ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase/CD39) and ecto-5'-nucleotidase (e-5NT/CD73); the latter converts AMP to adenosine and inorganic phosphate, representing the rate limiting step controlling the ratio between extracellular ATP and adenosine. Evidence that cellular expression and activity of CD39 and CD73 may be subject to changes under pathophysiological conditions has identified this pathway as an endogenous modulator in several diseases and was shown to be involved in the molecular mechanism of drugs, such as methotrexate, salicylates , interferon-β. We evaluated whether CD73/adenosine/A2A signalling pathway is involved in nimesulide anti-inflammatory effect, in vivo and in vitro. We found that the adenosine A2A agonist, 4-[2-[[6-amino-9-(N-ethyl-β-d-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl]benzenepropanoic acid hydrochloride (CGS21680, 2mg/kg ip.), inhibited carrageenan-induced rat paw oedema and the effect was reversed by co-administration of the A2A antagonist -(2-[7-amino-2-[2-furyl][1,2,4]triazolo[2,3-a][1,3,5]triazin-5-yl-amino]ethyl)phenol (ZM241385; 3mg/kg i.p.). Nimesulide (5mg/kg i.p.) anti-inflammatory effect was inhibited by pre-treatment with ZM241385 (3mg/kg i.p.) and by local administration of the CD73 inhibitor, adenosine 5'-(α,β-methylene)diphosphate (APCP; 400μg/paw). Furthermore, we found increased activity of 5'-nucleotidase/CD73 in paws and plasma of nimesulide treated rats, 4h following oedema induction. In vitro, the inhibitory effect of nimesulide on nitrite and prostaglandin E2 production by lipopolysaccharide-activated J774 cell line was reversed by ZM241385 and APCP. Furthermore, nimesulide increased CD73 activity in J774 macrophages while it did not inhibit nitrite accumulation by lipopolysaccharide-activated SiRNA CD73 silenced J774 macrophages. Our data demonstrate that the anti-inflammatory effect of nimesulide in part is mediated by CD73-derived adenosine acting on A2A receptors. Copyright © 2016 Elsevier Inc. All rights reserved.

Comparison Between the Effects of Intravenous Morphine, Tramadol, and Ketorolac on Stress and Immune Responses in Patients Undergoing Modified Radical Mastectomy.

PubMed

Bakr, Mohamed A-E-M; Amr, Samy A-E R; Mohamed, Sahar A; Hamed, Hosny B; Abd El-Rahman, Ahmad M; Mostafa, Mohamed A M; El Sherif, Fatma A

2016-10-01

Analgesics had been suspected of impairing various immune functions either directly or indirectly. Our primary objective was to compare the effects of intravenous (IV) morphine, tramadol, and ketorolac on stress and immune responses in patients who underwent modified radical mastectomy. Sixty patients randomly assigned to receive IV morphine 5 mg (group M, n=20), tramadol 100 mg (group T, n=20), or ketorolac 60 mg (group K, n=20) at the end of surgery. Serum cortisol, prolactin were measured immediately, 40 minutes, and 24 hours postoperatively. Expressions of peripheral T lymphocytes (CD3, CD3CD4, CD3CD8) and natural killer cells (CD3, CD56) were measured as percentages of total lymphocytes by flow cytometry immediately, 90 minutes, and 24 hours postoperatively. After 40 minutes, cortisol level increased but prolactin decreased significantly (P=0.001), then both decreased after 24 hours (P=0.001) compared with baseline within the 3 groups. CD3, CD4, CD8, and CD56 significantly decreased at 90 minutes and 24 hours (P≤0.033) compared with baseline in the 3 groups. CD4, CD8, and CD56 significantly decreased in group M, compared with group T and K (P≤0.016) and CD3, CD8, and CD56 in group T compared with group K at 90 minutes (P≤0.024) postoperatively. After 24 hours, CD4, and CD8 decreased in group M compared with group T (P≤0.048) and CD4 and CD56 in groups M and T compared with group K (P≤0.049). IV morphine, tramadol, and ketorolac suppressed stress and immune responses. Ketorolac was the least immunosuppressive among the 3 drugs.

Combined influence of basal media and fibroblast growth factor on the expansion and differentiation capabilities of adipose-derived stem cells.

PubMed

Ahearne, Mark; Lysaght, Joanne; Lynch, Amy P

2014-01-01

Interest in adipose-derived stem cells (ASCs) has increased in recent years due to their multi-linage differentiation capabilities. While much work has been done to optimize the differentiation media, few studies have focused on examining the influence of different expansion media on cell behavior. In this study, three basal media (low glucose Dulbecco's modified Eagle's medium (DMEM), high glucose DMEM and DMEM-F12) supplemented with or without fibroblast growth factor 2 (FGF) were examined to assess their suitability for expanding ASCs. Flow cytometry, colony-forming unit assays (CFU-Fs) and differentiation assays were utilized to study cell behavior. High glucose media CFU-Fs produced fewest colonies while the addition of FGF increased colony size. By passage 2, the majority of cells were positive for CD44, 45, 73, 90 and 105 and negative for CD14, 31 and 45, indicating a mesenchymal phenotype. A sub-population of CD34 positive cells was present among passage 2 cells; however, by passage 4 the cells were negative for CD34. FGF has a negative effective on passage 4 ASC adipogenesis and high glucose media plus FGF-enhanced osteogenic capacity of passage 4 ASCs. FGF supplemented basal media were most suitable for chondrogenesis. High glucose media plus FGF appeared to be the most beneficial for priming ASCs to induce a keratocyte phenotype. These findings demonstrate the reciprocal effect FGF and basal media have on ASCs. This research has implications for those interested regenerating bone, cartilage, cornea or adipose tissues.

Performance and Safety Characteristics of Sanyo NiCd Cells

NASA Technical Reports Server (NTRS)

Deng, Yi; Jeevarajan, Judith; Bragg, Bobby; Zhang, Wenlin

2002-01-01

NiCd batteries are widely used for high drain applications like power tools and also in other portable equipment like cameras, PCs, etc. NASA and Dreamtime Holdings, Inc. worked together to have the capability of a High Definition TV (HDTV) on the ISS and Space Shuttle. The Sanyo HD camcorder was used on the STS 105 fight in July, 2001 . The camcorder used two versions of a NiCd battery. One was a cOlnmercial off-the-shelf Sony BP90 battery pack that had Sanyo NiCd D cells. The other was a modified battery (FBP-90) made by Frezzi Energy, which also had the same Sanyo NiCd D cells. The battery has 10 NiCd D cells in series to form a 12 V pack with 5.0 Ah capacity. Our current study involved the perforn1ance and abuse tests on the Sanyo NiCd 5.0 Ah D cells. The best combination of charge/discharge current rate is 0.3C for charge and 1/2e for discharge within 200 cycles. No significant changes in capacity were observed in 200 cycles. The cell also showed capability of 5C (25.0A) high rate discharge. In overcharge and overdischarge tests, all tested cells passed the tests without venting. In imbalance tests, the battery pack could be charged and discharged only at relatively low current. At charge current of 1.0A or less, the imbalanced cells in the battery pack displayed relatively high temperatures during charge or discharge. The cells functioned normally during internal short and no mishap occurred during external short. Cells passed exposure tests at 80 C and no leakage till 150 C during heat-tovent tests.

Extracellular adenosine production by ecto-5′-nucleotidase (CD73) enhances radiation-induced lung fibrosis

PubMed Central

Wirsdörfer, Florian; de Leve, Simone; Cappuccini, Federica; Eldh, Therese; Meyer, Alina V.; Gau, Eva; Thompson, Linda F.; Chen, Ning-Yuan; Karmouty-Quintana, Harry; Fischer, Ute; Kasper, Michael; Klein, Diana; Ritchey, Jerry W.; Blackburn, Michael R.; Westendorf, Astrid M.; Stuschke, Martin; Jendrossek, Verena

2016-01-01

Radiation-induced pulmonary fibrosis is a severe side effect of thoracic irradiation, but its pathogenesis remains poorly understood and no effective treatment is available. In this study, we investigated the role of the extracellular adenosine as generated by the ecto-5'-nucleotidase CD73 in fibrosis development after thoracic irradiation. Exposure of wild-type C57BL/6 mice to a single dose (15 Gray) of whole thorax irradiation triggered a progressive increase in CD73 activity in the lung between 3 and 30 weeks post-irradiation. In parallel, adenosine levels in bronchoalveolar lavage fluid (BALF) were increased by approximately three-fold. Histological evidence of lung fibrosis was observed by 25 weeks after irradiation. Conversely, CD73-deficient mice failed to accumulate adenosine in BALF and exhibited significantly less radiation-induced lung fibrosis (P<0.010). Furthermore, treatment of wild-type mice with pegylated adenosine deaminase (PEG-ADA) or CD73 antibodies also significantly reduced radiation-induced lung fibrosis. Taken together, our findings demonstrate that CD73 potentiates radiation-induced lung fibrosis, suggesting that existing pharmacological strategies for modulating adenosine may be effective in limiting lung toxicities associated with the treatment of thoracic malignancies. PMID:26921334

Structural Basis of Immune Evasion at the Site of CD4 Attachment on HIV-1 gp120

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

Chen, Lei; Kwon, Young Do; Zhou, Tongqing

2010-01-13

The site on HIV-1 gp120 that binds to the CD4 receptor is vulnerable to antibodies. However, most antibodies that interact with this site cannot neutralize HIV-1. To understand the basis of this resistance, we determined co-crystal structures for two poorly neutralizing, CD4-binding site (CD4BS) antibodies, F105 and b13, in complexes with gp120. Both antibodies exhibited approach angles to gp120 similar to those of CD4 and a rare, broadly neutralizing CD4BS antibody, b12. Slight differences in recognition, however, resulted in substantial differences in F105- and b13-bound conformations relative to b12-bound gp120. Modeling and binding experiments revealed these conformations to be poorlymore » compatible with the viral spike. This incompatibility, the consequence of slight differences in CD4BS recognition, renders HIV-1 resistant to all but the most accurately targeted antibodies.« less

Extracellular matrix metalloproteinase inducer (EMMPRIN) expression correlates positively with active angiogenesis and negatively with basic fibroblast growth factor expression in epithelial ovarian cancer.

PubMed

Szubert, Sebastian; Szpurek, Dariusz; Moszynski, Rafal; Nowicki, Michal; Frankowski, Andrzej; Sajdak, Stefan; Michalak, Slawomir

2014-03-01

The primary aim of this paper was to evaluate the expression of extracellular matrix metalloproteinase inducer (EMMPRIN) and its relationship with proangiogenic factors and microvessel density (MVD) in ovarian cancer. The study group included 58 epithelial ovarian cancers (EOCs), 35 benign ovarian tumors, and 21 normal ovaries. The expression of EMMPRIN, vascular endothelial growth factor (VEGF), and basic fibroblast growth factor (bFGF) was assessed by ELISA of tissue homogenates. Antibodies against CD105, CD31, and CD34 were used to immunohistochemically assess MVD. We have found significantly higher EMMPRIN expression in EOC than in benign ovarian tumors and normal ovaries. Similarly, the VEGF expression was higher in EOC than in benign ovarian tumors and normal ovaries. By contrast, bFGF expression was lower in EOC than in benign ovarian tumors and ovary samples. EMMPRIN expression in EOC was directly correlated with VEGF expression and CD105-MVD, but inversely correlated with bFGF expression. Grade 2/3 ovarian cancers had increased expression of EMMPRIN and VEGF, increased CD105-MVD, and lowered expression of bFGF compared to grade 1 ovarian cancers. Moreover, EMMPRIN expression was higher in advanced (FIGO III and IV) ovarian cancer. The upregulation of EMMPRIN and VEGF expression is correlated with increased CD105-MVD and silenced bFGF, which suggests early and/or reactivated angiogenesis in ovarian cancer. Aggressive EOC is characterized by the following: high expression of EMMPRIN and VEGF, high CD105-MVD, and low expression of bFGF.

Tumoral immune-infiltrate (IF), PD-L1 expression and role of CD8/TIA-1 lymphocytes in localized osteosarcoma patients treated within protocol ISG-OS1.

PubMed

Palmerini, Emanuela; Agostinelli, Claudio; Picci, Piero; Pileri, Stefano; Marafioti, Teresa; Lollini, Pier-Luigi; Scotlandi, Katia; Longhi, Alessandra; Benassi, Maria Serena; Ferrari, Stefano

2017-12-19

We hypothesized that immune-infiltrates were associated with superior survival, and examined a primary osteosarcoma tissue microarrays (TMAs) to test this hypothesis. 129 patients (pts) with localized osteosarcoma treated within protocol ISG-OS1 were included in the study. Clinical characteristics, expression of CD8, CD3, FOXP3, CD20, CD68/CD163 (tumor associated macrophage, TAM), Tia-1 (cytotoxic T cell), CD303 (plasmacytoid dendritic cells: pDC), Arginase-1 (myeloid derived suppressor cells: MDSC), PD-1 on immune-cells (IC), and PD-L1 on tumoral cells (TC) and IC were analysed and correlated with outcome. Most of the cases presented tumor infiltrating lymphocytes (TILs) (CD3+ 90%; CD8+ 86%). Tia-1 was detected in 73% of the samples. PD-L1 expression was found in 14% patients in IC and 0% in TC; 22% showed PD-1 expression in IC.With a median follow-up of 8 years (range 1-13), the 5-year overall survival (5-year OS) was 74% (95% CI 64-85). Univariate analysis showed better 5-year OS for: a) pts with a good histologic response to neoadjuvant chemotherapy (p = 0.0001); b) pts with CD8/Tia1 tumoral infiltrates (p = 0.002); c) pts with normal alkaline phosphatas (sALP) (p = 0.04). After multivariate analysis, histologic response (p = 0.007) and CD8/Tia1 infiltration (p = 0.01) were independently correlated with survival. In the subset of pts with CD8+ infiltrate, worse (p 0.02) OS was observed for PD-L1(IC)+ cases. Our findings support the hypothesis that CD8/Tia1 infiltrate in tumor microenvironment at diagnosis confers superior survival for pts with localized osteosarcoma, while PD-L1 expression is associated with worse survival.

Effects of Different Cell-Detaching Methods on the Viability and Cell Surface Antigen Expression of Synovial Mesenchymal Stem Cells.

PubMed

Tsuji, Kunikazu; Ojima, Miyoko; Otabe, Koji; Horie, Masafumi; Koga, Hideyuki; Sekiya, Ichiro; Muneta, Takeshi

2017-06-09

Flow cytometric analysis of cell surface antigens is a powerful tool for the isolation and characterization of stem cells residing in adult tissues. In contrast to the collection of hematopoietic stem cells, the process of enzymatic digestion is usually necessary to prepare mesenchymal stem cells (MSCs) suspensions, which can influence the expression of cell surface markers. In this study, we examined the effects of various cell-detaching reagents and digestion times on the expression of stem cell-related surface antigens and MSC functions. Human MSCs were detached from dishes using four different reagents: trypsin, TrypLE, collagenase, and a nonenzymatic cell dissociation reagent (C5789; Sigma-Aldrich). Following dissociation reagent incubations ranging from 5 to 120 min, cell surface markers were analyzed by flow cytometry. Trypsin and TrypLE quickly dissociated the cells within 5 min, while collagenase and C5789 required 60 min to obtain maximum cell yields. C5789 significantly decreased cell viability at 120 min. Trypsin treatment significantly reduced CD44+, CD55+, CD73+, CD105+, CD140a+, CD140b+, and CD201+ cell numbers within 30 min. Collagenase treatment reduced CD140a expression by 30 min. In contrast, TrypLE treatment did not affect the expression of any cell surface antigens tested by 30 min. Despite the significant loss of surface antigen expression after 60 min of treatment with trypsin, adverse effects of enzymatic digestion on multipotency of MSCs were limited. Overall, our data indicated that TrypLE is advantageous over other cell dissociation reagents tested for the rapid preparation of viable MSC suspensions.

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.

The associations between serum VEGF, bFGF and endoglin levels with microvessel density and expression of proangiogenic factors in malignant and benign ovarian tumors.

PubMed

Szubert, Sebastian; Moszynski, Rafal; Michalak, Slawomir; Nowicki, Michal; Sajdak, Stefan; Szpurek, Dariusz

2016-09-01

To investigate whether serum levels of VEGF, bFGF and endoglin correlate with tumor VEGF and bFGF expression or microvessel density (MVD) in ovarian cancer. Forty five patients with epithelial ovarian cancers (EOCs) and 38 patients with benign ovarian tumors (BOTs) were included into the study. Serum levels of VEGF, bFGF and endoglin were assessed using ELISA. The expression of VEGF and bFGF in tumor samples were evaluated using ELISA of supernatants obtained from tumor homogenization. MVD was analyzed using immunohistochemistry with antibodies against CD31, CD34 and CD105. Serum VEGF levels were significantly higher in EOCs than in BOTs (436.6pg/ml [19.67-2860] vs 295.5pg/ml [123-539], P=0.025). Serum endoglin levels were lowered in the group EOCs when compared to BOTs (33,720g/ml [12,220-73,940] vs 42,390pg/ml [19,380-56,910], P=0.015). There were no differences in bFGF levels between studied groups. EOCs have significantly higher CD105 MVD (25 vessels/mm2 [0-57] vs 6 vessels/mm2 [0-70], P<0.001) and tumor VEGF (405.9pg/mg protein [0-3000] vs 2.225 [0-634.7], P<0.001) expression than BOTs, while, bFGF expression was higher in BOTs than in EOCs (2076pg/mg protein [668.1-8718] vs 847.3pg/mg protein [188.9-8333], P=0.003). In patients with EOCs we have observed negative correlation between serum VEGF concentration and its tissue expression (r Spearman=-0.571, P=0.0261), and serum VEGF concentration correlated positively with CD34-MVD (r Spearman=0.545, P=0.0289). In a multiple regression analysis we have observed only the negative correlation between serum VEGF and CD105-MVD (r=-0.5288, P=0.0427). Serum VEGF is a useful marker for prediction of ovarian cancer MVD and tumor VEGF expression. Copyright © 2016 Elsevier Inc. All rights reserved.

Mesenchymal Stem Cells Induce Expression of CD73 in Human Monocytes In Vitro and in a Swine Model of Myocardial Infarction In Vivo.

PubMed

Monguió-Tortajada, Marta; Roura, Santiago; Gálvez-Montón, Carolina; Franquesa, Marcella; Bayes-Genis, Antoni; Borràs, Francesc E

2017-01-01

The ectoenzymes CD39 and CD73 regulate the purinergic signaling through the hydrolysis of adenosine triphosphate (ATP)/ADP to AMP and to adenosine (Ado), respectively. This shifts the pro-inflammatory milieu induced by extracellular ATP to the anti-inflammatory regulation by Ado. Mesenchymal stem cells (MSCs) have potent immunomodulatory capabilities, including monocyte modulation toward an anti-inflammatory phenotype aiding tissue repair. In vitro , we observed that human cardiac adipose tissue-derived MSCs (cATMSCs) and umbilical cord MSCs similarly polarize monocytes toward a regulatory M2 phenotype, which maintained the expression of CD39 and induced expression of CD73 in a cell contact dependent fashion, correlating with increased functional activity. In addition, the local treatment with porcine cATMSCs using an engineered bioactive graft promoted the in vivo CD73 expression on host monocytes in a swine model of myocardial infarction. Our results suggest the upregulation of ectonucleotidases on MSC-conditioned monocytes as an effective mechanism to amplify the long-lasting immunomodulatory and healing effects of MSCs delivery.

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.

Deficits in Endogenous Adenosine Formation by Ecto-5′-Nucleotidase/CD73 Impair Neuromuscular Transmission and Immune Competence in Experimental Autoimmune Myasthenia Gravis

PubMed Central

Cristina Costa, Ana; Guerra-Gomes, Sónia; Ferreirinha, Fátima; Magalhães-Cardoso, Maria Teresa; Correia-de-Sá, Paulo

2015-01-01

AMP dephosphorylation via ecto-5′-nucleotidase/CD73 is the rate limiting step to generate extracellular adenosine (ADO) from released adenine nucleotides. ADO, via A2A receptors (A2ARs), is a potent modulator of neuromuscular and immunological responses. The pivotal role of ecto-5′-nucleotidase/CD73, in controlling extracellular ADO formation, prompted us to investigate its role in a rat model of experimental autoimmune myasthenia gravis (EAMG). Results show that CD4+CD25+FoxP3+ regulatory T cells express lower amounts of ecto-5′-nucleotidase/CD73 as compared to controls. Reduction of endogenous ADO formation might explain why proliferation of CD4+ T cells failed upon blocking A2A receptors activation with ZM241385 or adenosine deaminase in EAMG animals. Deficits in ADO also contribute to neuromuscular transmission failure in EAMG rats. Rehabilitation of A2AR-mediated immune suppression and facilitation of transmitter release were observed by incubating the cells with the nucleoside precursor, AMP. These findings, together with the characteristic increase in serum adenosine deaminase activity of MG patients, strengthen our hypothesis that the adenosinergic pathway may be dysfunctional in EAMG. Given that endogenous ADO formation is balanced by ecto-5′-nucleotidase/CD73 activity and that A2ARs exert a dual role to restore use-dependent neurocompetence and immune suppression in myasthenics, we hypothesize that stimulation of the two mechanisms may have therapeutic potential in MG. PMID:25691808

Maintenance of “stem cell” features of cartilage cell sub-populations during in vitro propagation

PubMed Central

2013-01-01

Background The discovery of mesenchymal stem cells (MSCs) or MSC-like cells in cartilage tissue does not tie in well with the established view that MSCs derive from a perivascular niche. The presence of MSCs may raise concerns about specificity and application safety, particularly in terms of the regulatory site. The aim of the present study was to investigate the benefits or possible risks of the MSC-like properties of cells isolated from cartilage in the context of autologous chondrocyte implantation. Methods Chondrocytic cells were isolated from cartilage or intervertebral disc tissue. Flow cytometry was used to analyze the expression of cell surface antigens. MSC-like cells were either enriched or depleted by means of magnetic cell sorting (MACS) involving the monoclonal antibodies W5C5/SUSD2 and W8B2/MSCA-1. We addressed the issues of prolonged expansion of such cells as well as the influence of culture medium as a trigger for selecting a single cell type. Established protocols were used to study in vitro differentiation. In addition to histological and biochemical assessment, the acquired phenotypes were also evaluated on the mRNA transcript level. Results In the studied cells, we found strongly analogous expression of antigens typically expressed on MSCs, including CD49e, CD73, CD90, CD105, CD140b and CD166. The expression of W5C5 and W8B2 antigens in cartilage cell sub-populations did not correlate with multi-potency. We demonstrated that a chondroid precursor, but not a bona fide multipotent mesenchymal, cell type can be obtained under established in vitro culture conditions. The culture media used for expansion influenced the cell phenotype. Conclusions The risk of adverse adipose or osseous differentiation is not posed by expanded chondrocyte cultures, even after enrichment of putative MSC-like cell populations by MACS. It is possible that this limited “stemness” in chondrocytes, expanded for use in ACI, may instead be beneficial as it allows re-differentiation under appropriate conditions despite prolonged times in culture. PMID:23363653

Induction of dopaminergic neurons from human Wharton's jelly mesenchymal stem cell by forskolin.

PubMed

Paldino, Emanuela; Cenciarelli, Carlo; Giampaolo, Adele; Milazzo, Luisa; Pescatori, Mario; Hassan, Hamisa Jane; Casalbore, Patrizia

2014-02-01

The purpose of this study was to investigate the Wharton's jelly mesenchymal stem cells differentiation ability toward neuronal fate. Human Wharton's jelly mesenchymal stem cells (hWJMSC) have been isolated from human umbilical cord of full-term births and characterized by flow cytometry analysis for their stem mesenchymal properties through specific surface markers expression (CD73, CD90, and CD105). hWJMSC mesodermal lineage differentiation ability and karyotype analysis were assessed. The trans-differentiation of hWJMSC into neural lineage was investigated in presence of forskolin, an agent known to increase the intracellular levels of cAMP. A molecular profile of differentiated hWJMSC was performed by microarray technology which revealed 1,532 statistically significant modulated genes respect to control cells. Most of these genes are mainly involved in functional neuronal signaling pathways and part of them are specifically required for the neuronal dopaminergic induction. The acquisition of the dopaminergic phenotype was evaluated via immunocytochemistry and Western blot analysis revealed the significant induction of Nurr1, NeuroD1, and TH proteins expression in forskolin-induced hWJMSC. Moreover, the treatment with forskolin promoted, in hWJMSC, a strong upregulation of the neurotrophin Trk receptors related to the high release of brain-derived neurotrophic factor. Taken together these findings show that hWJMSC may be represent an optimal therapeutic strategy for neurological diseases. © 2013 Wiley Periodicals, Inc.

HIERARCHICAL ORGANIZATION OF OSTEOBLASTS REVEALS THE SIGNIFICANT ROLE OF CD166 IN HEMATOPOIETIC STEM CELL MAINTANANCE AND FUNCTION

PubMed Central

Chitteti, Brahmananda R.; Cheng, Ying-Hua; Kacena, Melissa A.; Srour, Edward F.

2013-01-01

The role of osteoblasts (OB) in maintaining hematopoietic stem cells (HSC) in their niche is well elucidated, but the exact definition, both phenotypically and hierarchically of OB responsible for these functions is not clearly known. We previously demonstrated that OB maturational status influences HSC function whereby immature OB with high Runx2 expression promote hematopoietic expansion. Here, we show that Activated Leukocyte Cell Adhesion Molecule (ALCAM) or CD166 expression on OB is directly correlated with Runx2 expression and high hematopoiesis enhancing activity (HEA). Fractionation of OB with lineage markers: Sca1, osteopontin (OPN), CD166, CD44, and CD90 revealed that Lin-Sca1-OPN+CD166+ cells (CD166+) and their subpopulations fractionated with CD44 and CD90 expressed high levels of Runx2 and low levels of osteocalcin (OC) demonstrating the relatively immature status of these cells. Conversely, the majority of the Lin-Sca1-OPN+CD166− cells (CD166−) expressed high OC levels suggesting that CD166− OB are more mature. In vitro hematopoietic potential of LSK cells co-cultured for 7 days with fresh OB or OB pre-cultured for 1, 2, or 3 weeks declined precipitously with increasing culture duration concomitant with loss of CD166 expression. Importantly, LSK cells co-cultured with CD166+CD44+CD90+ OB maintained their in vivo repopulating potential through primary and secondary transplantation, suggesting that robust HEA activity is best mediated by immature CD166+ OB with high Runx2 and low OC expression. These studies begin to define the hierarchical organization of osteoblastic cells and provide a more refined definition of OB that can mediate HEA. PMID:23369988

Steroid resistance in COPD is associated with impaired molecular chaperone Hsp90 expression by pro-inflammatory lymphocytes.

PubMed

Hodge, Greg; Roscioli, Eugene; Jersmann, Hubertus; Tran, Hai B; Holmes, Mark; Reynolds, Paul N; Hodge, Sandra

2016-10-21

Corticosteroid resistance is a major barrier to effective treatment of COPD. We have shown that the resistance is associated with decreased expression of glucocorticoid receptor (GCR) by senescent CD28nullCD8+ pro-inflammatory lymphocytes in peripheral blood of COPD patients. GCR must be bound to molecular chaperones heat shock proteins (Hsp) 70 and Hsp90 to acquire a high-affinity steroid binding conformation, and traffic to the nucleus. We hypothesized a loss of Hsp70/90 from these lymphocytes may further contribute to steroid resistance in COPD. Blood was collected from COPD (n = 10) and aged-matched controls (n = 10). To assess response to steroids, cytotoxic mediators, intracellular pro-inflammatory cytokines, CD28, GCR, Hsp70 and Hsp90 were determined in T and NKT-like cells in the presence of ± 10 μM prednisolone and 2.5 ng/mL cyclosporine A (binds to GCR-Hsp70/90 complex) using flow cytometry, western blot and fluorescence microscopy. A loss of expression of Hsp90 and GCR from CD28null CD8+ T and NKT-like cells in COPD was noted (Hsp70 unchanged). Loss of Hsp90 expression correlated with the percentage of CD28null CD8+ T and NKT-like cells producing IFNγ or TNFα in all subjects (eg, COPD: R = -0.763, p = 0.007 for T-cell IFNγ). Up-regulation of Hsp90 and associated decrease in pro-inflammatory cytokine production was found in CD28nullCD8+ T and NKT-like cells in the presence of 10 μM prednisolone and 2.5 ng/mL cyclosporine A. Loss of Hsp90 from cytotoxic/pro-inflammatory CD28nullCD8+ T and NKT-like cells could contribute to steroid resistance in COPD. Combination prednisolone and low-dose cyclosporine A therapy inhibits these pro-inflammatory cells and may reduce systemic inflammation in COPD.

Biodistribution of Yttrium-90-Labeled Anti-CD45 Antibody in a Nonhuman Primate Model

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

Nemecek, Eneida; Hamlin, Donald K.; Fisher, Darrell R.

2005-01-15

Radioimmunotherapy may improve the outcome of hematopoietic cell transplantation for hematologic malignancies by delivering targeted radiation to hematopoietic organs while relatively sparing nontarget organs. We evaluated the organ localization of yttrium-90-labeled anti-CD45 (90Y-anti-CD45) antibody in macaques, a model that had previously predicted iodine-131-labeled anti-CD-45 (131I-anti-CD45) antibody biodistribution in humans. Experimental Design: Twelve Macaca nemestrina primates received anti-CD45 antibody labeled with 1 to 2 mCi of 90Y followed by serial blood sampling and marrow and lymph node biopsies, and necropsy. The content of 90Y per gram of tissue was determined by liquid scintillation spectrometry. Time-activity curves were constructed using average isotopemore » concentrations in each tissue at measured time points to yield the fractional residence time and estimate radiation absorbed doses for each organ per unit of administered activity. The biodistribution of 90Y-anti-CD45 antibody was then compared with that previously obtained with 131I-anti-CD45 antibody in macaques. Results: The spleen received 2,120, marrow 1,060, and lymph nodes 315 cGy/mCi of 90Y injected. The liver and lungs were the nontarget organs receiving the highest radiation absorbed doses (440 and 285 cGy/mCi, respectively). Ytrrium-90-labeled anti-CD45 antibody delivered 2.5- and 3.7-fold more radiation to marrow than to liver and lungs, respectively. The ratios previously observed with 131I-antiCD45 antibody were 2.5-and 2.2-fold more radiation to marrow than to liver and lungs, respectively. Conclusions: This study shows that 90Y-anti-CD45 antibody can deliver relatively selective radiation to hematopoietic tissues, with similar ratios of radiation delivered to target versus nontarget organs, as compared with the 131I immunoconjugate in the same animal model.« less

Quantifying CD4 receptor protein in two human CD4+ lymphocyte preparations for quantitative flow cytometry.

PubMed

Wang, Meiyao; Misakian, Martin; He, Hua-Jun; Bajcsy, Peter; Abbasi, Fatima; Davis, Jeffrey M; Cole, Kenneth D; Turko, Illarion V; Wang, Lili

2014-01-01

In our previous study that characterized different human CD4+ lymphocyte preparations, it was found that both commercially available cryopreserved peripheral blood mononuclear cells (PBMC) and a commercially available lyophilized PBMC (Cyto-Trol™) preparation fulfilled a set of criteria for serving as biological calibrators for quantitative flow cytometry. However, the biomarker CD4 protein expression level measured for T helper cells from Cyto-Trol was about 16% lower than those for cryopreserved PBMC and fresh whole blood using flow cytometry and mass cytometry. A primary reason was hypothesized to be due to steric interference in anti- CD4 antibody binding to the smaller sized lyophilized control cells. Targeted multiple reaction monitoring (MRM) mass spectrometry (MS) is used to quantify the copy number of CD4 receptor protein per CD4+ lymphocyte. Scanning electron microscopy (SEM) is utilized to assist searching the underlying reasons for the observed difference in CD4 receptor copy number per cell determined by MRM MS and CD4 expression measured previously by flow cytometry. The copy number of CD4 receptor proteins on the surface of the CD4+ lymphocyte in cryopreserved PBMCs and in lyophilized control cells is determined to be (1.45 ± 0.09) × 10(5) and (0.85 ± 0.11) × 10(5), respectively, averaged over four signature peptides using MRM MS. In comparison with cryopreserved PBMCs, there are more variations in the CD4 copy number in lyophilized control cells determined based on each signature peptide. SEM images of CD4+ lymphocytes from lyophilized control cells are very different when compared to the CD4+ T cells from whole blood and cryopreserved PBMC. Because of the lyophilization process applied to Cyto-Trol control cells, a lower CD4 density value, defined as the copy number of CD4 receptors per CD4+ lymphocyte, averaged over three different production lots is most likely explained by the loss of the CD4 receptors on damaged and/or broken microvilli where CD4 receptors reside. Steric hindrance of antibody binding and the association of CD4 receptors with other biomolecules likely contribute significantly to the nearly 50% lower CD4 receptor density value for cryopreserved PBMC determined from flow cytometry compared to the value obtained from MRM MS.

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

Analyses of cell surface molecules on hepatic stem/progenitor cells in mouse fetal liver.

PubMed

Kakinuma, Sei; Ohta, Haruhiko; Kamiya, Akihide; Yamazaki, Yuji; Oikawa, Tsunekazu; Okada, Ken; Nakauchi, Hiromitsu

2009-07-01

Hepatic stem/progenitor cells possess active proliferative ability and the capacity for differentiation into hepatic and cholangiocytic lineages. Our group and others have shown that a prospectively defined population in mid-gestational fetal liver contains hepatic stem/progenitor cells. However, the phenotypes of such cells are incompletely elucidated. We analyzed the profile of cell-surface molecules on primary hepatic stem/progenitor cells. Expression of cell surface molecules on primary hepatic stem/progenitor cells in mouse mid-gestational fetal liver was analyzed using flow cytometric multicolor analyses and colony-formation assays. The potential of the cells for liver repopulation was examined by transplantation assay. We found that CD13 (aminopeptidase N) was detected on the cells of the previously reported (Dlk/Pref-1(+)) hepatic stem/progenitor fraction. Colony-formation assays revealed that the CD13(+) fraction, compared with the Dlk(+) fraction, of non-hematopoietic cells in fetal liver was enriched in hepatic stem/progenitor cells. Transplantation assay showed the former fraction exhibited repopulating potential in regenerating liver. Moreover, flow cytometric analysis for over 90 antigens demonstrated enrichment of hepatic stem/progenitor cells using several positive selection markers, including (hitherto unknown) CD13, CD73, CD106, and CD133. Our data indicated that CD13 is a positive selection marker for hepatic stem/progenitor cells in mid-gestational fetal liver.

Augmenting Trastuzumab Therapy Against Breast Cancer Through Selective Activation of NK Cells

DTIC Science & Technology

2013-10-01

selection and assessed for purity (>90% purity as defined by CD3-CD56+ flow cytometry ) and activation (>50% expression of CD137). Breast cancer cell lines...at a ratio of 1:1. After 24 hours, NK cells were isolated by negative selection and assessed for purity (>90% purity as defined by CD3-CD56+ flow ... cytometry ) and activation (>50% expression of CD137). Chromium-labeled breast cancer cell lines including MCF7 (A), BT474M1 (B), HER18 (C), and SKBR3

Different culture media affect growth characteristics, surface marker distribution and chondrogenic differentiation of human bone marrow-derived mesenchymal stromal cells.

PubMed

Hagmann, Sebastien; Moradi, Babak; Frank, Sebastian; Dreher, Thomas; Kämmerer, Peer Wolfgang; Richter, Wiltrud; Gotterbarm, Tobias

2013-07-30

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) play an important role in modern tissue engineering, while distinct variations of culture media compositions and supplements have been reported. Because MSCs are heterogeneous regarding their regenerative potential and their surface markers, these parameters were compared in four widely used culture media compositions. MSCs were isolated from bone marrow and expanded in four established cell culture media. MSC yield/1000 MNCs, passage time and growth index were observed. In P4, typical MSC surface markers were analysed by fluorescence cytometry. Additionally, chondrogenic, adipogenic and osteogenic differentiation potential were evaluated. Growth index and P0 cell yield varied importantly between the media. The different expansion media had a significant influence on the expression of CD10, CD90, CD105, CD140b CD146 and STRO-1. While no significant differences were observed regarding osteogenic and adipogenic differentiation, chondrogenic differentiation was superior in medium A as reflected by GAG/DNA content. The choice of expansion medium can have a significant influence on growth, differentiation potential and surface marker expression of mesenchymal stromal cells, which is of fundamental importance for tissue engineering procedures.

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.

Yolk Sac Mesenchymal Progenitor Cells from New World Mice (Necromys lasiurus) with Multipotent Differential Potential

PubMed Central

Favaron, Phelipe Oliveira; Mess, Andrea; Will, Sônia Elisabete; Maiorka, Paulo César; de Oliveira, Moacir Franco; Miglino, Maria Angelica

2014-01-01

Fetal membranes are abundant, ethically acceptable and readily accessible sources of stem cells. In particular, the yolk sac is a source of cell lineages that do not express MHCs and are mainly free from immunological incompatibles when transferred to a recipient. Although data are available especially for hematopoietic stem cells in mice and human, whereas other cell types and species are dramatically underrepresented. Here we studied the nature and differentiation potential of yolk sac derived mesenchymal stem cells from a New World mouse, Necromys lasiurus. Explants from mid-gestation were cultured in DMEM-High glucose medium with 10% defined fetal bovine serum. The cells were characterized by standard methods including immunophenotyping by fluorescence and flow cytometry, growth and differentiation potential and tumorigenicity assays. The first adherent cells were observed after 7 days of cell culture and included small, elongated fibroblast-like cells (92.13%) and large, round epithelial-like cells with centrally located nuclei (6.5%). Only the fibroblast-like cells survived the first passages. They were positive to markers for mesenchymal stem cells (Stro-1, CD90, CD105, CD73) and pluripotency (Oct3/4, Nanog) as well as precursors of hematopoietic stem cells (CD117). In differentiation assays, they were classified as a multipotent lineage, because they differentiated into osteogenic, adipogenic, and chondrogenic lineages and, finally, they did not develop tumors. In conclusion, mesenchymal progenitor cells with multipotent differentiation potential and sufficient growth and proliferation abilities were able to be obtained from Necromys yolk sacs, therefore, we inferred that these cells may be promising for a wide range of applications in regenerative medicine. PMID:24918429

Mesenchymal stem cells with osteogenic potential in human maxillary sinus membrane: an in vitro study.

PubMed

Berbéri, Antoine; Al-Nemer, Fatima; Hamade, Eva; Noujeim, Ziad; Badran, Bassam; Zibara, Kazem

2017-06-01

The aim of our study is to prove and validate the existence of an osteogenic progenitor cell population within the human maxillary Schneiderian sinus membrane (hMSSM) and to demonstrate their potential for bone formation. Ten hMSSM samples of approximately 2 × 2 cm were obtained during a surgical nasal approach for treatment of chronic rhinosinusitis and were retained for this study. The derived cells were isolated, cultured, and assayed at passage 3 for their osteogenic potential using the expression of Alkaline phosphatase, alizarin red and Von Kossa staining, flow cytometry, and quantitative real-time polymerase chain reaction. hMSSM-derived cells were isolated, showed homogenous spindle-shaped fibroblast-like morphology, characteristic of mesenchymal progenitor cells (MPCs), and demonstrated very high expression of MPC markers such as STRO-1, CD44, CD90, CD105, and CD73 in all tested passages. In addition, von Kossa and Alizarin red staining showed significant mineralization, a typical feature of osteoblasts. Moreover, alkaline phosphatase (ALP) activity was significantly increased at days 7, 14, 21, and 28 of culture in hMSSM-derived cells grown in osteogenic medium, in comparison to controls. Furthermore, osteogenic differentiation significantly upregulated the transcriptional expression of osteogenic markers such as ALP, Runt-related transcription factor 2 (Runx-2), bone morphogenetic protein (BMP)-2, osteocalcin (OCN), osteonectin (ON), and osteopontin (OPN), confirming that hMSSM-derived cells are of osteoprogenitor origin. Finally, hMSSM-derived cells were also capable of producing OPN proteins upon culturing in an osteogenic medium. Our data showed that hMSSM holds mesenchymal osteoprogenitor cells capable of differentiating to the osteogenic lineage. hMSSM contains potentially multipotent postnatal stem cells providing a promising clinical application in preimplant and implant therapy.

Single- and two-color infrared focal plane arrays made by MBE in HgCdTe

NASA Astrophysics Data System (ADS)

Zanatta, Jean-Paul; Ferret, P.; Loyer, R.; Petroz, G.; Cremer, S.; Chamonal, Jean-Paul; Bouchut, Philippe; Million, Alain; Destefanis, Gerard L.

2000-12-01

We present here recent developments obtained at LETI infrared laboratory in the field of infrared detectors made in HgCdTe material and using the molecular beam epitaxial growth technique (MBE). We discuss the metallurgical points (growth temperature and flux control) that lead to achieve excellent quality epitaxial layers grown by MBE. We show a run-to-run reproducibility measured on growth run of more than 15 layers. The crystalline quality, surface morphology, and composition uniformity are excellent. The etch pits density (EPD) are in the low 105.cm-2 when HgCdTe grows on a CdZnTe substrate. Transport properties reveal a low n-type carrier concentration in the 1014 to 1015.cm-3 range with a carrier mobility in excess of 105 cm2/V/sec at 77K for epilayers grown with 10 micrometers cutoff wavelength. We describe the performances of several kinds of our HgCdTe- MBE devices: single color MWIR and LWIR detectors on HgCdTe/CdZnTe operating at 77K in respectively (3-5 micrometers ) and (8-12 micrometers ) wavelength range; single color MWIR detectors on HgCdTe grown on germanium heterosubstrate operating at 77K in the (3-5 micrometers ) wavelength range; two color HgCdTe detectors operating within the MWIR (3-5 micrometers ) band.

The prevalence of abnormal celiac antibodies and celiac disease in patients with suspected irritable bowel syndrome: a prospective multi-center US study

PubMed Central

Cash, Brooks D.; Rubenstein, Joel H.; Young, Patrick E.; Gentry, Andrew; Nojkov, Borko; Lee, Dong; Andrews, A. Hirsohi; Dobhan, Richard; Chey, William D.

2011-01-01

Background & Aims Guidelines recommend that patients with symptoms of non-constipated inflammatory bowel syndrome (NC-IBS) undergo testing for celiac disease (CD). We evaluated the prevalence of CD antibodies and biopsy confirmed CD among patients with NC-IBS in a large US population. Methods In a study conducted at 4 sites, from 2003 to 2008, we compared data from 492 patients with symptoms of NC-IBS to 458 asymptomatic individuals who underwent colonoscopy examinations for cancer screening or polyp surveillance (controls). All participants provided blood samples for specific and non-specific CD-associated antibodies. Additionally, patients with IBS were analyzed for complete blood cell counts, metabolic factors, erythrocyte sedimentation rates, and levels of C-reactive protein and thyroid-stimulating hormone. Any subjects found to have CD-associated antibodies were offered esophagogastroduodenoscopy and duodenal biopsy analysis. Results Of patients with NC-IBS, 7.3% had abnormal results in tests for CD-associated antibodies, compared to 4.8% of controls (adjusted odds ratio=1.49; 95% confidence interval, 0.76–2.90. P=.25). Within the NC-IBS group, 6.51% had antibodies against gliadin, 1.22% against tissue transglutaminase, and 0.61% against endomysium (P>.05 vs controls for all antibodies tested). CD was confirmed in 0.41% of patients in the NC-IBS group and 0.44% of controls (P>0.99). Conclusions Although CD-associated antibodies are relatively common, the prevalence of CD among patients with NC-IBS is similar to that among controls in a large US population. These findings challenge recommendations to routinely screen patients with NC-IBS for CD. More than 7% of patients with NC-IBS had CD-associated antibodies, indicating that gluten sensitivity might mediate IBS symptoms; further studies are needed. PMID:21762658

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.

Positron emission tomography imaging of angiogenesis in a murine hindlimb ischemia model with 64Cu-labeled TRC105.

PubMed

Orbay, Hakan; Zhang, Yin; Hong, Hao; Hacker, Timothy A; Valdovinos, Hector F; Zagzebski, James A; Theuer, Charles P; Barnhart, Todd E; Cai, Weibo

2013-07-01

The goal of this study was to assess ischemia-induced angiogenesis with (64)Cu-NOTA-TRC105 positron emission tomography (PET) in a murine hindlimb ischemia model of peripheral artery disease (PAD). CD105 binding affinity/specificity of NOTA-conjugated TRC105 (an anti-CD105 antibody) was evaluated by flow cytometry, which exhibited no difference from unconjugated TRC105. BALB/c mice were anesthetized, and the right femoral artery was ligated to induce hindlimb ischemia, with the left hindlimb serving as an internal control. Laser Doppler imaging showed that perfusion in the ischemic hindlimb plummeted to ∼ 20% of the normal level after surgery and gradually recovered to near normal level on day 24. Ischemia-induced angiogenesis was noninvasively monitored and quantified with (64)Cu-NOTA-TRC105 PET on postoperative days 1, 3, 10, 17, and 24. (64)Cu-NOTA-TRC105 uptake in the ischemic hindlimb increased significantly from the control level of 1.6 ± 0.2 %ID/g to 14.1 ± 1.9 %ID/g at day 3 (n = 3) and gradually decreased with time (3.4 ± 1.9 %ID/g at day 24), which correlated well with biodistribution studies performed on days 3 and 24. Blocking studies confirmed the CD105 specificity of tracer uptake in the ischemic hindlimb. Increased CD105 expression on days 3 and 10 following ischemia was confirmed by histology and reverse transcription polymerase chain reaction (RT-PCR). This is the first report of PET imaging of CD105 expression during ischemia-induced angiogenesis. (64)Cu-NOTA-TRC105 PET may play multiple roles in future PAD-related research and improve PAD patient management by identifying the optimal timing of treatment and monitoring the efficacy of therapy.

Amount of cadmium associated with Cd-binding protein in roots of young plants. [Agrostis gigantea

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

Rauser, W.E.

1986-04-01

The partitioning of Cd between roots and shoots was determined for young plants exposed to Cd in nutrient solution. The intentionally high concentration of 3 ..mu..m Cd was used to assess the role of root Cd-binding protein (Cd-BP) in Cd detoxification. The roots of tomatoes exposed to Cd retained 60-84% of the plant Cd from day 2 through day 9 without toxicity symptoms evident. Cd-BP did not contribute to Cd retention over the initial 4 days, only 1-4% of the root Cd was in this protein fraction. Maize roots retained 59-66% of the plant Cd from day 1 through daymore » 7. The Cd-BP fraction bound 8-19% of the root Cd on day 1 and 31-55% by day 7. Cd toxicity symptoms occurred in leaves by 4 days. In the grass Agrostis gigantea the roots retained 73-85% of the seedling Cd after 1 day and for another 6 days. A high proportion of the root Cd(34-68%) was in the Cd-BP fraction after one day and continued to be so to day 7 (46-64%). No Cd toxicity symptoms were evident. Only the specific pattern of rapid, early and sustained production of Cd-BP observed in Agrostis was consistent with the putative detoxification role for Cd-BP.« less

Safety of nevirapine in HIV-infected pregnant women initiating antiretroviral therapy at higher CD4 counts: a systematic review and meta-analysis.

PubMed

Bera, Ebrahim; Mia, Rafiq

2012-10-08

The package insert for nevirapine (NVP) cautions use in HIV-infected women (including pregnant women) with CD4 counts ≥250 cells/µl. However, recent studies showed that the CD4 count of pregnant women receiving antiretroviral therapy (ART) was not predictive of NVP toxicity. To determine whether ART-naive pregnant women initiating NVP-based ART at higher CD4 counts experience greater toxicity compared with pregnant women at lower CD4 counts. We reviewed studies comparing serious adverse NVP-related events among ART-naive pregnant women who commenced therapy at higher v. lower CD4 counts. Relevant studies were extracted from PubMed, SCOPUS and EMBASE, major journals and conference proceedings prior to December 2011. Authors were contacted for additional data. Data were independently extracted and entered into Review Manager. Fourteen studies (2 663 participants) were included for analysis. The odds ratio (OR) for overall NVP toxicity among pregnant women with CD4 <250 cells/µl was 0.61 (95% confidence interval (CI) 0.43 - 0.85). When analysis was restricted to prospective studies only (7 studies, 1 318 participants), the results were consistent for overall NVP toxicity (OR 0.43; 95% CI 0.25 - 0.73) and severe hepatotoxicity (OR 0.45; 95% CI 0.22 - 0.90), but not for severe cutaneous reaction (OR 0.53; 95% CI 0.26 - 1.10). Initiating NVP-based ART during pregnancy at CD4 ≥250 cells/µl increases toxicity risk and should be avoided, necessitating urgent revision of current guidelines supporting this practice.

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

Tan Guosheng; Xiang Xianhong; Guo Wenbo

PurposeTo investigate the influence of uterine artery embolization (UAE) on endometrial microvessel density (MVD) and angiogenesis.MethodsSixty female guinea pigs were divided into two groups, the control group (n = 15) and the UAE treatment group (n = 45). In the UAE group, tris-acryl gelatin microspheres were used to generate embolization. Animals were further divided into three subgroups, A1, A2, and A3 (n = 15 for each subgroup), with uterine specimens collected at 7-15, 16-30, and 31-45 days after UAE, respectively. Immunostaining for factor VIII and CD105 was performed to identify total endometrial MVD (MVD{sub FVIII}) and CD105-positive angiogenesis (MVD{sub CD105})more » at the indicated time points after UAE.ResultsQuantitative analysis revealed that MVD{sub FVIII} significantly decreased in the A1 (11.40 {+-} 2.76, p < 0.05) and A2 (15.37 {+-} 3.06, p < 0.05) groups compared to the control group (19.40 {+-} 2.50), and was restored to normal in the A3 group (18.77 {+-} 2.69). UAE caused a temporal up-regulation of MVD{sub CD105}-positive angiogenesis in the A1 group (9.33 {+-} 2.37, p < 0.05) and the A2 group (11.63 {+-} 1.56, p < 0.05) compared to the control group (7.12 {+-} 1.67), and the MVD{sub CD105} value returned to normal in the A3 group (8.07 {+-} 1.97).ConclusionUAE caused a temporal decrease in endometrial MVD that reversed over time as a result of the increase of CD105-positive angiogenesis. Although the UAE-induced reduction of endometrial MVD was reversible, its long-term effect on endometrial receptivity still needs further study.« less

Hyperglycemic Stress Impairs the Stemness Capacity of Kidney Stem Cells in Rats.

PubMed

Yang, Guang; Jia, Yali; Li, Chunlin; Cheng, Qingli; Yue, Wen; Pei, Xuetao

2015-01-01

The incidence of acute kidney injury in patients with diabetes is significantly higher than that of patients without diabetes, and may be associated with the poor stemness capacity of kidney stem cells (KSCs) and limited recovery of injured renal tubules. To investigate the effects of hyperglycemic stress on KSC stemness, KSCs were isolated from the rat renal papilla and analyzed for their self-renewal and differentiation abilities. Our results showed that isolated KSCs expressed the mesenchymal stem cell markers N-cadherin, Nestin, CD133, CD29, CD90, and CD73. Moreover, KSCs co-cultured with hypoxia-injured renal tubular epithelial cell (RTECs) induced the expression of the mature epithelial cell marker CK18, suggesting that the KSCs could differentiate into RTECs in vitro. However, KSC proliferation, differentiation ability and tolerance to hypoxia were decreased in high-glucose cultures. Taken together, these results suggest the high-glucose microenvironment can damage the reparative ability of KSCs. It may result in a decreased of recovery capability of renal tubules from injury.

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.

Reduced numbers of mucosal DR(int) macrophages and increased numbers of CD103(+) dendritic cells during anti-TNF-α treatment in patients with Crohn's disease.

PubMed

Dige, Anders; Magnusson, Maria K; Öhman, Lena; Hvas, Christian Lodberg; Kelsen, Jens; Wick, Mary Jo; Agnholt, Jørgen

2016-01-01

Anti-TNF-α treatment constitutes a mainstay in the treatment of Crohn's disease (CD), but its mechanisms of action are not fully understood. We aimed to investigate the effects of adalimumab, a human monoclonal TNF-α antibody, on macrophage (MQ) and dendritic cell (DC) subsets in mucosal biopsies and peripheral blood. Intestinal biopsies and blood samples were obtained from 12 different CD patients both before and 4 weeks after the initiation of the induction of adalimumab treatment. Endoscopic disease activity was estimated by the Simple Endoscopic Score for Crohn's Disease. Biopsies were obtained from inflamed and non-inflamed areas. The numbers of lamina propria CD14 (+) DR(int) and CD14 (+) DR(hi) MQs, CD141(+), CD141(-) and CD103(+) DCs subsets, and circulating monocytes and DCs were analyzed using flow cytometry. At baseline, we observed higher numbers of DR(int) MQs and lower numbers of CD103(+) DCs in inflamed versus non-inflamed mucosa [843 vs. 391/10(5) lamina propria mononuclear cells (LPMCs) (p < 0.05) and 9 vs. 19 × 10(5) LPMCs (p = 0.01), respectively]. After four weeks of adalimumab treatment, the numbers of DR(int) MQs decreased [843 to 379/10(5) LPMCs (p = 0.03)], whereas the numbers of CD103(+) DCs increased [9-20 × 10(5) LPMCs (p = 0.003)] compared with baseline. In peripheral blood, no alterations were observed in monocyte or DC numbers between baseline and week 4. In CD, mucosal inflammation is associated with high numbers of DR(int) MQs and low numbers of CD103(+) DCs. This composition of intestinal myeloid subsets is reversed by anti-TNF-α treatment. These results suggest that DR(int) MQs play a pivotal role in CD inflammation.

Separation of human CD4+CD39+ T cells by magnetic beads reveals two phenotypically and functionally different subsets

PubMed Central

Schuler, Patrick J.; Harasymczuk, Malgorzata; Schilling, Bastian; Lang, Stephan; Whiteside, Theresa L.

2011-01-01

Objective The ectonucleotidase CD39 is an enzyme involved in adenosine production. Its surface expression on human regulatory T cells (Treg) allows for their flow-cytometry-based isolation from peripheral blood. To further develop and improve this method on a scale supporting translational studies, we introduced capture of CD39+ Treg on magnetic immunobeads. Methods Peripheral blood mononuclear cells (PBMC) obtained from healthy donors were used for negative selection of CD4+ T cells on AutoMACS using antibodies (Abs) specific for all lineage+ cells. CD4+CD39+ Treg were captured by biotin-conjugated anti-CD39 Abs and anti-biotin Ab-coated magnetic beads. Isolated CD4+CD39+ T cells were phenotyped by flow cytometry for Treg-associated markers: CD39, CD73, FOXP3, CD25, CTLA-4, CCR4, CD45RO and CD121a or for the absence of CD127 and CD49d. CFSE-based proliferation assays and ATP hydrolysis were used to measure Treg functions. Results The purity, recovery and viability of the separated CD4+CD39+ T cells were satisfactory. The isolated CD4+CD39+ T cell population consisted of FOXP3+CD25+ T cells which hydrolyzed exogenous ATP and suppressed autologous CD4+ T cell proliferation and of FOXP3negCD25neg T cells without suppressor function. The same two subsets were detectable by flow cytometry in normal PBMC, gating on CD4+CD39+, CD4+CD127neg, CD4+CD49dneg or CD4+CD25high Treg. Conclusion CD4+CD39+ Treg capture on immunobeads led to a discovery of two CD39+ subsets. Similar to CD39+ Treg in the peripheral blood, half of these cells are CD25+FOXP3+ active suppressor cells, while the other half are CD25negFOXP3neg and do not mediate suppression. PMID:21513715

Early endothelial damage detected by circulating particles in baboons fed a diet high in simple carbohydrates in conjunction with saturated or unsaturated fat.

PubMed

Shi, Qiang; Hodara, Vida; Meng, Qinghe; Voruganti, V Saroja; Rice, Karen; Michalek, Joel E; Comuzzie, Anthony G; VandeBerg, John L

2014-01-01

Studies have shown that high-fat diets cause blood vessel damage, however, assessing pathological effects accurately and efficiently is difficult. In this study, we measured particle levels of static endothelium (CD31+ and CD105+) and activated endothelium (CD62E+, CD54+ and CD106+) in plasma. We determined individual responses to two dietary regimens in two groups of baboons. One group (n = 10), was fed a diet high in simple carbohydrates and saturated fats (the HSF diet) and the other (n = 8) received a diet high in simple carbohydrates and unsaturated fats (the HUF diet). Plasma samples were collected at 0, 3, and 7 weeks. The percentages of CD31+ and CD62E+ particles were elevated at 3 weeks in animals fed either diet, but these elevations were statistically significant only in animals fed the HUF diet. Surprisingly, both percentages and counts of CD31+ particles were significantly lower at week 7 compared to week 0 and 3 in the HSF group. The median absolute counts of CD105+ particles were progressively elevated over time in the HSF group with a significant increase from week 0 to 7; the pattern was somewhat different for the HUF group with significant increase from week 3 to 7. The counts of CD54+ particles exhibited wide variation in both groups during the dietary challenge, while the median counts of CD106+ particles were significantly lower at week 3 than at week 0 and week 7. Endothelial particles exhibited time-dependent changes, suggesting they were behaving as quantifiable surrogates for the early detection of vascular damage caused by dietary factors.

Clinical Utility of Urinary CD90 as a Biomarker for Prostate Cancer Detection — EDRN Public Portal

Cancer.gov

Tumor-associated stromal cells differ from normal gland-associated stromal cells in gene expression. Genes up-regulated in these stromal cells are potential cancer biomarkers, especially those encoding secreted or extracellular proteins. These proteins might be detected in urine. CD90/THY1 is one such candidate. A clinical test based on urinary CD90 would be useful in reducing the number of unnecessary biopsies done because of abnormal serum PSA and/or DRE finding. Elevated CD90 protein is found in tumor tissue and urine.

Screening of a new cadmium hyperaccumulator, Galinsoga parviflora, from winter farmland weeds using the artificially high soil cadmium concentration method.

PubMed

Lin, Lijin; Jin, Qian; Liu, Yingjie; Ning, Bo; Liao, Ming'an; Luo, Li

2014-11-01

A new method, the artificially high soil cadmium (Cd) concentration method, was used to screen for Cd hyperaccumulators among winter farmland weeds. Galinsoga parviflora was the most promising remedial plant among 5 Cd accumulators or hyperaccumulators. In Cd concentration gradient experiments, as soil Cd concentration increased, root and shoot biomass decreased, and their Cd contents increased. In additional concentration gradient experiments, superoxide dismutase and peroxidase activities increased with soil Cd concentrations up to 75 mg kg(-1) , while expression of their isoenzymes strengthened. Catalase (CAT) activity declined and CAT isoenzyme expression weakened at soil Cd concentrations less than 50 mg kg(-1) . The maxima of Cd contents in shoots and roots were 137.63 mg kg(-1) and 105.70 mg kg(-1) , respectively, at 100 mg kg(-1) Cd in soil. The root and shoot bioconcentration factors exceeded 1.0, as did the translocation factor. In a field experiment, total extraction of Cd by shoots was 1.35 mg m(-2) to 1.43 mg m(-2) at soil Cd levels of 2.04 mg kg(-1) to 2.89 mg kg(-1) . Therefore, the artificially high soil Cd concentration method was effective for screening Cd hyperaccumulators. Galinsoga parviflora is a Cd hyperaccumulator that could be used to efficiently remediate Cd-contaminated farmland soil. © 2014 SETAC.

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.

T cell costimulation blockade promotes transplantation tolerance in combination with sirolimus and post-transplantation cyclophosphamide for haploidentical transplantation in children with severe aplastic anemia.

PubMed

Jaiswal, Sarita Rani; Bhakuni, Prakash; Zaman, Shamsuz; Bansal, Satish; Bharadwaj, Priyanka; Bhargava, Sneh; Chakrabarti, Suparno

2017-08-01

We conducted a pilot study employing extended T cell costimulation blockade (COSBL) with Abatacept along with sirolimus and post-transplantation cyclophosphamide (PTCy) in 10 patients (median age 12) with severe aplastic anemia (SAA). Nine patients engrafted in the COSBL group, compared to all 10 patients (median 14 vs 13days) treated on PTCy protocols without abatacept (CONTROL group). The incidence of acute graft-versus-host disease (GVHD) was 10.5% in the COSBL group compared to 50% in the CONTROL group (p=0.04). Chronic GVHD (12.5% vs 56%, p=0.02) and CMV reactivation (30% vs 80%, p=0.03) were also reduced in the COSBL group. T and NK cell subset analysis revealed higher CD56 bright CD16 - NK cells in the CONTROL group (p=0.004), but similar CD56 dim CD16 + NK cells in both groups at day+30. Tregs (CD4 + CD25 + CD127 dim/- FoxP3+) were markedly higher in the COSBL group at day+30 (8.4% vs 1.1%) and the trend was maintained through day+90 (p<0.01). The GVHD and Disease-free survival at one year in the COSBL group was 80% vs. 30% in the CONTROL group (p=0.05). Our preliminary findings suggest that COSBL in combination with PTCy and sirolimus might augment transplantation tolerance in children with SAA, probably due to synergistic effect on early recovery of Tregs. Copyright © 2017 Elsevier B.V. All rights reserved.

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.

Extracellular Nucleotide Hydrolysis in Dermal and Limbal Mesenchymal Stem Cells: A Source of Adenosine Production.

PubMed

Naasani, Liliana I Sous; Rodrigues, Cristiano; de Campos, Rafael Paschoal; Beckenkamp, Liziane Raquel; Iser, Isabele C; Bertoni, Ana Paula Santin; Wink, Márcia R

2017-08-01

Human Limbal (L-MSCs) and Dermal Mesenchymal Stem Cell (D-MSCs) possess many properties that increase their therapeutic potential in ophthalmology and dermatology. It is known that purinergic signaling plays a role in many aspects of mesenchymal stem cells physiology. They release and respond to purinergic ligands, altering proliferation, migration, differentiation, and apoptosis. Therefore, more information on these processes would be crucial for establishing future clinical applications using their differentiation potential, but without undesirable side effects. This study evaluated and compared the expression of ecto-nucleotidases, the enzymatic activity of degradation of extracellular nucleotides and the metabolism of extracellular ATP in D-MSCs and L-MSCs, isolated from discard tissues of human skin and sclerocorneal rims. The D-MSCs and L-MSCs showed a differentiation potential into osteogenic, adipogenic, and chondrogenic lineages and the expression of markers CD105 + , CD44 + , CD14 - , CD34 - , CD45 - , as expected. Both cells hydrolyzed low levels of extracellular ATP and high levels of AMP, leading to adenosine accumulation that can regulate inflammation and tissue repair. These cells expressed mRNA for ENTPD1, 2, 3, 5 and 6, and CD73 that corresponded to the observed enzymatic activities. Thus, considering the degradation of ATP and adenosine production, limbal MSCs are very similar to dermal MSCs, indicating that from the aspect of extracellular nucleotide metabolism L-MSCs are very similar to the characterized D-MSCs. J. Cell. Biochem. 118: 2430-2442, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Does pretransplant soluble CD30 serum concentration affect deceased-donor kidney graft function 3 years after transplantation?

PubMed

Kovac, J; Arnol, M; Vidan-Jeras, B; Bren, A F; Kandus, A

2008-06-01

Elevated serum concentrations of soluble CD30 molecule (sCD30) have been related to acute cellular rejection and poor graft outcomes in kidney transplantation. This historical cohort study investigated the association of pretransplant sCD30 serum concentrations with kidney graft function expressed as estimated glomerular filtration rate (GFR) at 3 years after transplantation. Pretransplant sera from 176 adult deceased-donor kidney graft recipients were tested for sCD30 content using a commercially available automated enzyme-linked immunosorbent assay. The immunosuppression consisted of induction therapy with monoclonal anti-CD25 antibodies and a maintenance regimen of cyclosporine (CsA)-based therapy. GFR was estimated (eGFR) by the four-variable Modification of Diet in Renal Disease (MDRD) Study equation. According to the distribution of pretransplant sCD30 levels (median 66.7 U/mL; interquartile range, 46.6 to 98.6 U/mL), a concentration of 66 U/mL or higher was defined as high (n = 89) and below 66 U/mL as low (n = 87). Three years after transplantation, eGFR was not significantly different among recipients in high versus low sCD30 groups (69 +/- 23 mL/min/1.73m2 vs 66 +/- 21 mL/min/1.73m2; P = .327) and there was no correlation between eGFR and pretransplant sCD30 levels (r2 = 0.001; P = .73). Upon multivariate regression analysis, donor age, recipient body mass index at transplantation, and acute rejection episodes were independent variables affecting eGFR at 3 years after transplantation. This study showed that pretransplant sCD30 serum concentrations were not associated with deceased-donor kidney graft function at 3 years after transplantation. The immunosuppression with anti-CD25 antibodies and a triple CsA-based maintenance regimen could possibly be decisive for our findings.

[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.

Microvessel density in head and neck squamous cell carcinoma.

PubMed

Szafarowski, Tomasz; Sierdzinski, Janusz; Szczepanski, Miroslaw J; Whiteside, Theresa L; Ludwig, Nils; Krzeski, Antoni

2018-05-10

Microvessel density (MVD) corresponds to the intensity of neo-angiogenesis. MVD assessments are based on the expression levels of the vascular endothelium markers such as, e.g., CD34 or CD105. The goal of this study was to assess MVD among patients with head and neck squamous cell carcinoma (HNSCC), and to evaluate the predictive value of MVD in head and neck cancers. The study included 49 patients treated for HNSCC and 11 patients with dysplasia of the upper respiratory tract epithelium. Control tissues consisted of 12 normal mucous membranes of the throat. Expression levels of MVD markers were assessed by immunohistochemistry (IHC) using tissue microarrays (TMA). Clinicopathological factors and patients' survival over the 5-year follow-up period were analyzed. The MVD/CD34 values were found to be significantly elevated in the HNSCCs compared to the non-malignant control tissues (p = 0.001) and to dysplastic tissues. (p = 0.02). Significantly higher MVD/CD105 values were also seen in the tumor compared to the control tissues (p = 0.001) or the dysplastic tissues (p = 0.001). Unexpectedly, significantly lower MVD/CD34 values were seen in the tumor tissues of patients with the T3-T4 tumors compared to those with T1-T2 tumors (p = 0.01). HNSCCs have statistically higher MVD values compared to dysplasia of the upper respiratory tract epithelium. However, the MVD/CD34 values did not correlate with local invasiveness (the T feature) of HNSCCs. This counterintuitive observation suggests that assessments of MVD as performed on TMA by IHC using anti-CD34 or anti-CD105 antibodies considered to be specific for endothelial cell markers might underestimate the extent of the tumor vascularity in HNSCC.

Regulation of PGE2 signaling pathways and TNF-alpha signaling pathways on the function of bone marrow-derived dendritic cells and the effects of CP-25.

PubMed

Li, Ying; Sheng, Kangliang; Chen, Jingyu; Wu, Yujing; Zhang, Feng; Chang, Yan; Wu, Huaxun; Fu, Jingjing; Zhang, Lingling; Wei, Wei

2015-12-15

This study was to investigate PGE2 and TNF-alpha signaling pathway involving in the maturation and activation of bone marrow dendritic cells (DCs) and the effect of CP-25. Bone marrow DCs were isolated and stimulated by PGE2 and TNF-alpha respectively. The markers of maturation and activation expressed on DCs, such as CD40, CD80, CD83, CD86, MHC-II, and the ability of antigen uptake of DCs were analyzed by flow cytometry. The proliferation of T cells co-cultured with DCs, the signaling pathways of PGE2-EP4-cAMP and TNF-alpha-TRADD-TRAF2-NF-κB in DCs were analyzed. The results showed that both PGE2 and TNF-alpha up-regulated the expressions of CD40, CD80, CD83, CD86, and MHC-II, decreased the antigen uptake of DCs, and DCs stimulated by PGE2 or TNF-alpha could increase T cell proliferation. CP-25 (10(-5), 10(-6), and 10(-7)mol/l) decreased significantly the expressions of CD40, CD80, CD83, CD86 and MHC-II, increased the antigen uptake of DCs, and suppressed T cell proliferation induced by DCs. PGE2 increased the expressions of EP4, NF-κB and down-regulated cAMP level of DCs. TNF-alpha could also up-regulate TNFR1, TRADD, TRAF2, and NF-κB expression of DCs. CP-25 (10(-5), 10(-6), and 10(-7)mol/l) decreased the expressions of EP4 and NF-κB, increased cAMP level in DCs stimulated by PGE2. CP-25 (10(-5), 10(-6), and 10(-7)mol/l) also could down-regulate significantly TNFR1, TRADD, TRAF2, and NF-κB expression in DCs stimulated by TNF-alpha. These results demonstrate that PGE2 and TNF-alpha could enhance DCs functions by mediating PGE2-EP4-cAMP pathway, TNF-alpha-TNFR1-TRADD-TRAF2-NF-κB pathway respectively. CP-25 might inhibit the function of DCs through regulating PGE2-EP4-cAMP and TNF-alpha-TNFR1-TRADD-TRAF2-NF-κB pathways. Copyright © 2015 Elsevier B.V. All rights reserved.

Assessment of CD-105 as an Angiogenic Modulator in Odontogenic Myxomas and Dental Follicles.

PubMed

Del Carmen González-Galván, María; Aguirre-Urizar, José Manuel; Bologna-Molina, Ronell; Farfán-Morales, J Eduardo; Gainza-Cirauqui, Maria Luisa; Marichalar-Mendia, Xabier; Mosqueda-Taylor, Adalberto

2016-06-01

Aim Odontogenic myxoma is a benign intraosseous neoplasm of the jaws, with a locally aggressive behavior and a high recurrence rate. CD-105 is a homodimeric cell membrane glycoprotein and is a component of the TGF-β1 growth factor receptor complex that modulates angiogenesis by regulating the proliferation, differentiation and cellular migration. The aim of this study is to quantify the microvascular density of the odontogenic myxoma based on the expression of CD-105. Materials and Methods The analysis included 18 odontogenic myxoma and 18 dental follicles as controls. A standard immunohistochemical procedure was performed with the CD-105 antibody. Five representative fields (40×) of the odontogenic myxoma and the dental follicles were selected to determine the microvascular density, which was then followed by a descriptive and comparative statistical analysis. Results Dental follicles presented a significantly higher microvascular density compared with odontogenic myxoma (P = .001). The odontogenic myxoma smaller than 3 cm showed a greater microvascular density than those larger than 3 cm in size (P > .05), and the microvascular density was lower in large odontogenic myxomas as compared with the dental follicles (P = .003). Conclusion A weaker expression of CD-105 in odontogenic myxoma might indicate a lower angiogenic activity, suggesting that vascular proliferation has a limited role in the growth mechanisms and in the aggressive behavior of this neoplasm. © The Author(s) 2016.

Induction of hsp70, hsp90, and catalase activity in planarian Dugesia japonica exposed to cadmium.

PubMed

Zhang, Xiufang; Mo, Yehua; Zhou, Luming; Wang, Yinan; Wang, Zhongchen; Zhao, Bosheng

2016-08-01

The hsp70 and hsp90 expression patterns and catalase (CAT) activity in the freshwater planaria Dugesia japonica exposed to cadmium (Cd) under laboratory conditions were investigated. Planaria were exposed to a range of Cd concentrations (0-150 μg Cd/L) for 24 h. The expression levels of hsp70 and hsp90 were determined by relative quantitative real-time polymerase chain reaction. Within the overall dose range in the experiment, the expression level of hsp70 and the activity of CAT in D. japonica were altered significantly. Hsp70 was induced in D. japonica upon Cd exposure concentrations as low as 9.375 μg Cd/L. No significant effect on the expression level of hsp90 was observed. Our findings demonstrated that stress gene hsp70, but not hsp90, was responsive to Cd contamination in D. japonica CAT activity was significantly induced at concentrations of 18.75, 37.5, and 75 μg Cd/L after 24-h exposure. We recommend that the use of hsp70 as a biomarker should be complemented by evidence of changes in other parameters, such as CAT activity, in D. japonica. © The Author(s) 2014.

The prognostic and clinicopathologic characteristics of CD147 and esophagus cancer: A meta-analysis.

PubMed

Li, Hui; Jiang, Chunxiang; Wu, Dongwen; Shi, Shupeng; Liao, Mengting; Wang, Jing; Li, Yanwen; Xu, Zihao

2017-01-01

The prognostic significance of CD147 expression in esophageal cancer patients remains controversial. Using a meta-analysis, we investigated the prognostic and clinicopathologic characteristics of CD147 in esophageal cancer. A comprehensive literature search of the 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) was performed to identify studies of all esophageal cancer subtypes. Correlations between CD147 expression and survival outcomes and clinicopathological features were analyzed using meta-analysis methods. Seventeen studies were included. High CD147 expression reduced the 3-year survival rate (OR = 3.26, 95% CI = (1.53, 6.93), p = 0.02) and 5-year survival rate(OR = 4.35, 95% CI = (2.13, 8.90), p < 0.0001). High CD147 expression reduced overall survival in esophageal cancer (HR = 1.60, 95% CI = (1.19, 2.15), p = 0.02). Additionally, higher CD147 expression was detected in esophageal cancer tissues than noncancerous tissues (OR = 9.45, 95% CI = (5.39, 16.59), p < 0.00001), normal tissues (OR = 12.73, 95% CI = (3.49, 46.46), p = 0.0001), para-carcinoma tissues (OR = 12.80, 95% CI = (6.57, 24.92), p < 0.00001), and hyperplastic tissues (OR = 3.27, 95% CI = (1.47, 7.29), p = 0.004). CD147 expression was associated with TNM stage (OR = 3.66, 95% CI = (2.20, 6.09), p < 0.00001), tumor depth (OR = 7.97, 95% CI = (4.13, 15.38), p < 0.00001), and lymph node status (OR = 5.14, 95% CI = (2.03,13.01), p = 0.0005), but not with tumor differentiation, age, or sex. Our meta-analysis suggests that CD147 is an efficient prognostic factor in esophageal cancer. High CD147 expression in patients with esophageal cancer was associated with worse survival outcomes and common clinicopathological indicators of poor prognosis.

CD15s/CD62E Interaction Mediates the Adhesion of Non-Small Cell Lung Cancer Cells on Brain Endothelial Cells: Implications for Cerebral Metastasis

PubMed Central

Jassam, Samah A.; Maherally, Zaynah; Ashkan, Keyoumars; Roncaroli, Federico; Fillmore, Helen L.; Pilkington, Geoffrey J.

2017-01-01

Expression of the cell adhesion molecule (CAM), Sialyl Lewis X (CD15s) correlates with cancer metastasis, while expression of E-selectin (CD62E) is stimulated by TNF-α. CD15s/CD62E interaction plays a key role in the homing process of circulating leukocytes. We investigated the heterophilic interaction of CD15s and CD62E in brain metastasis-related cancer cell adhesion. CD15s and CD62E were characterised in human brain endothelium (hCMEC/D3), primary non-small cell lung cancer (NSCLC) (COR-L105 and A549) and metastatic NSCLC (SEBTA-001 and NCI-H1299) using immunocytochemistry, Western blotting, flow cytometry and immunohistochemistry in human brain tissue sections. TNF-α (25 pg/mL) stimulated extracellular expression of CD62E while adhesion assays, under both static and physiological flow live-cell conditions, explored the effect of CD15s-mAb immunoblocking on adhesion of cancer cell–brain endothelium. CD15s was faintly expressed on hCMEC/D3, while high levels were observed on primary NSCLC cells with expression highest on metastatic NSCLC cells (p < 0.001). CD62E was highly expressed on hCMEC/D3 cells activated with TNF-α, with lower levels on primary and metastatic NSCLC cells. CD15s and CD62E were expressed on lung metastatic brain biopsies. CD15s/CD62E interaction was localised at adhesion sites of cancer cell–brain endothelium. CD15s immunoblocking significantly decreased cancer cell adhesion to brain endothelium under static and shear stress conditions (p < 0.001), highlighting the role of CD15s–CD62E interaction in brain metastasis. PMID:28698503

CD15s/CD62E Interaction Mediates the Adhesion of Non-Small Cell Lung Cancer Cells on Brain Endothelial Cells: Implications for Cerebral Metastasis.

PubMed

Jassam, Samah A; Maherally, Zaynah; Smith, James R; Ashkan, Keyoumars; Roncaroli, Federico; Fillmore, Helen L; Pilkington, Geoffrey J

2017-07-10

Expression of the cell adhesion molecule (CAM), Sialyl Lewis X (CD15s) correlates with cancer metastasis, while expression of E-selectin (CD62E) is stimulated by TNF-α. CD15s/CD62E interaction plays a key role in the homing process of circulating leukocytes. We investigated the heterophilic interaction of CD15s and CD62E in brain metastasis-related cancer cell adhesion. CD15s and CD62E were characterised in human brain endothelium (hCMEC/D3), primary non-small cell lung cancer (NSCLC) (COR-L105 and A549) and metastatic NSCLC (SEBTA-001 and NCI-H1299) using immunocytochemistry, Western blotting, flow cytometry and immunohistochemistry in human brain tissue sections. TNF-α (25 pg/mL) stimulated extracellular expression of CD62E while adhesion assays, under both static and physiological flow live-cell conditions, explored the effect of CD15s-mAb immunoblocking on adhesion of cancer cell-brain endothelium. CD15s was faintly expressed on hCMEC/D3, while high levels were observed on primary NSCLC cells with expression highest on metastatic NSCLC cells ( p < 0.001). CD62E was highly expressed on hCMEC/D3 cells activated with TNF-α, with lower levels on primary and metastatic NSCLC cells. CD15s and CD62E were expressed on lung metastatic brain biopsies. CD15s/CD62E interaction was localised at adhesion sites of cancer cell-brain endothelium. CD15s immunoblocking significantly decreased cancer cell adhesion to brain endothelium under static and shear stress conditions ( p < 0.001), highlighting the role of CD15s-CD62E interaction in brain metastasis.

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.

Validation of the minimal citrate tube fill volume for routine coagulation tests on ACL TOP 500 CTS®.

PubMed

Ver Elst, K; Vermeiren, S; Schouwers, S; Callebaut, V; Thomson, W; Weekx, S

2013-12-01

CLSI recommends a minimal citrate tube fill volume of 90%. A validation protocol with clinical and analytical components was set up to determine the tube fill threshold for international normalized ratio of prothrombin time (PT-INR), activated partial thromboplastin time (aPTT) and fibrinogen. Citrated coagulation samples from 16 healthy donors and eight patients receiving vitamin K antagonists (VKA) were evaluated. Eighty-nine tubes were filled to varying volumes of >50%. Coagulation tests were performed on ACL TOP 500 CTS(®) . Receiver Operating Characteristic (ROC) plot, with Total error (TE) and critical difference (CD) as possible acceptance criteria, was used to determine the fill threshold. Receiving Operating Characteristic was the most accurate with CD for PT-INR and TE for aPTT resulting in thresholds of 63% for PT and 80% for aPTT. By adapted ROC, based on threshold setting at a point of 100% sensitivity at a maximum specificity, CD was best for PT and TE for aPTT resulting in thresholds of 73% for PT and 90% for aPTT. For fibrinogen, the method was only valid with the TE criterion at a 63% fill volume. In our study, we validated the minimal citrate tube fill volumes of 73%, 90% and 63% for PT-INR, aPTT and fibrinogen, respectively. © 2013 John Wiley & Sons Ltd.

Derivation of Mesenchymal Stromal Cells from Canine Induced Pluripotent Stem Cells by Inhibition of the TGFβ/Activin Signaling Pathway

PubMed Central

Frith, Jessica E.; Frith, Thomas J.R.; Ovchinnikov, Dmitry A.; Cooper-White, Justin J.; Wolvetang, Ernst J.

2014-01-01

In this study we have generated canine mesenchymal stromal cells (MSCs), also known as mesenchymal stem cells, from canine induced pluripotent stem cells (ciPSCs) by small-molecule inhibition of the transforming growth factor beta (TGFβ)/activin signaling pathway. These ciPSC-derived MSCs (ciPSC-MSCs) express the MSC markers CD73, CD90, CD105, STRO1, cPDGFRβ and cKDR, in addition to the pluripotency factors OCT4, NANOG and REX1. ciPSC-MSCs lack immunostaining for H3K27me3, suggesting that they possess two active X chromosomes. ciPSC-MSCs are highly proliferative and undergo robust differentiation along the osteo-, chondro- and adipogenic pathways, but do not form teratoma-like tissues in vitro. Of further significance for the translational potential of ciPSC-MSCs, we show that these cells can be encapsulated and maintained within injectable hydrogel matrices that, when functionalized with bound pentosan polysulfate, dramatically enhance chondrogenesis and inhibit osteogenesis. The ability to efficiently derive large numbers of highly proliferative canine MSCs from ciPSCs that can be incorporated into injectable, functionalized hydrogels that enhance their differentiation along a desired lineage constitutes an important milestone towards developing an effective MSC-based therapy for osteoarthritis in dogs, but equally provides a model system for assessing the efficacy and safety of analogous approaches for treating human degenerative joint diseases. PMID:25055193

CRISPR/Cas9-based genetic correction for recessive dystrophic epidermolysis bullosa

PubMed Central

Webber, Beau R; Osborn, Mark J; McElroy, Amber N; Twaroski, Kirk; Lonetree, Cara-lin; DeFeo, Anthony P; Xia, Lily; Eide, Cindy; Lees, Christopher J; McElmurry, Ron T; Riddle, Megan J; Kim, Chong Jai; Patel, Dharmeshkumar D; Blazar, Bruce R; Tolar, Jakub

2016-01-01

Recessive dystrophic epidermolysis bullosa (RDEB) is a severe disorder caused by mutations to the COL7A1 gene that deactivate production of a structural protein essential for skin integrity. Haematopoietic cell transplantation can ameliorate some of the symptoms; however, significant side effects from the allogeneic transplant procedure can occur and unresponsive areas of blistering persist. Therefore, we employed genome editing in patient-derived cells to create an autologous platform for multilineage engineering of therapeutic cell types. The clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 system facilitated correction of an RDEB-causing COL7A1 mutation in primary fibroblasts that were then used to derive induced pluripotent stem cells (iPSCs). The resulting iPSCs were subsequently re-differentiated into keratinocytes, mesenchymal stem cells (MSCs) and haematopoietic progenitor cells using defined differentiation strategies. Gene-corrected keratinocytes exhibited characteristic epithelial morphology and expressed keratinocyte-specific genes and transcription factors. iPSC-derived MSCs exhibited a spindle morphology and expression of CD73, CD90 and CD105 with the ability to undergo adipogenic, chondrogenic and osteogenic differentiation in vitro in a manner indistinguishable from bone marrow-derived MSCs. Finally, we used a vascular induction strategy to generate potent definitive haematopoietic progenitors capable of multilineage differentiation in methylcellulose-based assays. In totality, we have shown that CRISPR/Cas9 is an adaptable gene-editing strategy that can be coupled with iPSC technology to produce multiple gene-corrected autologous cell types with therapeutic potential for RDEB. PMID:28250968

The Role of Extracellular Adenosine Generation in the Development of Autoimmune Diseases

PubMed Central

Rizzo, R.; Malavasi, F.

2018-01-01

Adenosine (ADO) is an immunosuppressive molecule, which suppresses the immune responses by interacting with specific receptors expressed by immune effector cells. ADO is produced from ATP through the enzymatic activities of CD39 and CD73. Alternatively, ADO can be generated starting from NAD+, which is metabolized by the concerted action of CD38, CD203a/PC-1, and CD73. The role of ADO in immunity has been characterized in the last years in physiology and in pathological settings. This review examines a panel of reports focused on the functions of ADO in the context of human autoimmune/inflammatory diseases and the selected animal models. The final aim is to consider the role of adenosinergic ectoenzymes and ADO receptors as novel therapeutic targets for selected diseases. PMID:29769837

Mobilizing peripheral blood stem cells with high-dose G-CSF alone is as effective as with Dexa-BEAM plus G-CSF in lymphoma patients.

PubMed

Kröger, N; Zeller, W; Fehse, N; Hassan, H T; Krüger, W; Gutensohn, K; Lölliger, C; Zander, A R

1998-09-01

We compared retrospectively the efficacy of granulocyte colony stimulating factor (G-CSF) alone with chemotherapy plus G-CSF in mobilizing CD34-positive cells in patients with malignant lymphoma. 35 patients underwent peripheral blood stem cell (PBSC) collection following mobilization either with 24 microg/kg G-CSF for 4 consecutive days (n = 18) or Dexa-BEAM chemotherapy plus 5 microg/kg G-CSF (n = 17). High-dose G-CSF was well tolerated with only slight bone pain and/or myalgia. The Dexa-BEAM therapy required hospitalization with a median duration of 21 d. The median number of apheresis procedures in both groups was two (range two to four), resulting in a median of 5.3 and 5.1 x 10(6) CD34+ cells/kg. No patients in the G-CSF group, but one in the Dexa-BEAM group, failed to reach the target of collecting >2.0 x 10(6) CD34+ cells/kg. The number of CFU-GM (10.4 v 6.0 x 10(5)/kg) and of BFU-E (10.6 v 4.5 x 10(5)/kg; P = 0.04) was higher in the G-CSF group than in the Dexa-BEAM group. A subset analysis of CD34+ cells was performed in 16 patients showing a higher mean of Thy-1 (CD90w) coexpression in the G-CSF than in the Dexa-BEAM group (4.8 v 1.8%, P = 0.12). Additionally the percentage of CD34+/CD38- cells was higher in the G-CSF group (10.66% v 8.8%). However, these differences were not statistically significant. The median time to leucocyte and platelet engraftment after high-dose chemotherapy was slightly shorter in the G-CSF than in the Dexa-BEAM group (9 v 10 and 12 v 13.5 d, respectively). These results demonstrate that high-dose G-CSF is as effective as Dexa-BEAM plus G-CSF in mobilizing peripheral blood stem cells and produces prompt engraftment. The major advantages of G-CSF mobilization were the safe outpatient self-application and the fixed-day apheresis.

CD28 T-cell costimulatory molecule expression in pemphigus vulgaris.

PubMed

Alecu, M; Ursaciuc, C; Surcel, M; Coman, G; Ciotaru, D; Dobre, M

2009-03-01

CD28 superfamily of immune costimulatory molecules could play an important role in autotolerance control. CD28 costimulation seems to be necessary for regulatory T cell (Treg) activation and successive suppressive activities involved in autoimmunity protection. This study investigates CD28 expression, especially inducible costimulator fraction, on T lymphocytes in pemphigus vulgaris (PV) patients. CD28 expression on T lymphocytes was assessed in 16 PV patients during acute attack. All patients and 10 healthy control subjects were tested for lymphocyte populations, T-cell subpopulations (T-CD4+, T-CD8+), Treg and CD28 expression on T-cell subpopulations. T, B and natural killer cells average values in PV patients were close to the control group values. Compared with control group, PV values showed lower Treg (2.2% compared with 4.7%), slightly decreased CD4+ CD28+ T cells (91% compared with 95%), higher CD4+ CD28- T cells (9% compared with 5%), decreased CD8+ CD28+ T cells (57% and 73%, respectively) and significantly enhanced CD8+ CD28- T cells (43% compared with 27%). These data suggest that Treg-mediated suppressor T-cell effects could be diminished in PV, together with an abnormal or ineffective subsequent helper T-cell suppression. CD28 high expression on helper T cells and low expression on suppressor T cells are arguments for a potential CD28 role in PV autoimmune response mechanism.

CD22-targeted CAR T cells induce remission in B-ALL that is naive or resistant to CD19-targeted CAR immunotherapy.

PubMed

Fry, Terry J; Shah, Nirali N; Orentas, Rimas J; Stetler-Stevenson, Maryalice; Yuan, Constance M; Ramakrishna, Sneha; Wolters, Pamela; Martin, Staci; Delbrook, Cindy; Yates, Bonnie; Shalabi, Haneen; Fountaine, Thomas J; Shern, Jack F; Majzner, Robbie G; Stroncek, David F; Sabatino, Marianna; Feng, Yang; Dimitrov, Dimiter S; Zhang, Ling; Nguyen, Sang; Qin, Haiying; Dropulic, Boro; Lee, Daniel W; Mackall, Crystal L

2018-01-01

Chimeric antigen receptor (CAR) T cells targeting CD19 mediate potent effects in relapsed and/or refractory pre-B cell acute lymphoblastic leukemia (B-ALL), but antigen loss is a frequent cause of resistance to CD19-targeted immunotherapy. CD22 is also expressed in most cases of B-ALL and is usually retained following CD19 loss. We report results from a phase 1 trial testing a new CD22-targeted CAR (CD22-CAR) in 21 children and adults, including 17 who were previously treated with CD19-directed immunotherapy. Dose-dependent antileukemic activity was observed, with complete remission obtained in 73% (11/15) of patients receiving ≥1 × 10 6 CD22-CAR T cells per kg body weight, including 5 of 5 patients with CD19 dim or CD19 - B-ALL. Median remission duration was 6 months. Relapses were associated with diminished CD22 site density that likely permitted CD22 + cell escape from killing by CD22-CAR T cells. These results are the first to establish the clinical activity of a CD22-CAR in B-ALL, including leukemia resistant to anti-CD19 immunotherapy, demonstrating potency against B-ALL comparable to that of CD19-CAR at biologically active doses. Our results also highlight the critical role played by antigen density in regulating CAR function.

Investigation of the structural, optical and electrical transport properties of n-doped CdSe thin films

NASA Astrophysics Data System (ADS)

Ali, H. M.; Abd El-Ghanny, H. A.

2008-04-01

Thin films of (CdSe)90(In2O3)10, (CdSe)90(SnO2)10 and (CdSe)90(ZnO)10 have been grown on glass substrates by the electron beam evaporation technique. It has been found that undoped and Sn or In doped CdSe films have two direct transitions corresponding to the energy gaps Eg and Eg+Δ due to spin-orbit splitting of the valence band. The electrical resistivity for n-doped CdSe thin films as a function of light exposure time has been studied. The influence of doping on the structural, optical and electrical characteristics of In doped CdSe films has been investigated in detail. The lattice parameters, grain size and dislocation were determined from x-ray diffraction patterns. The optical transmittance and band gap of these films were determined using a double beam spectrophotometer. The DC conductivity of the films was measured in vacuum using a two-probe technique.

In Vitro Expression of Cytokeratin 19 in Adipose-Derived Stem Cells Is Induced by Epidermal Growth Factor.

PubMed

Chen, Shangliang; Wang, Mingzhu; Chen, Xinglu; Chen, Shaolian; Liu, Li; Zhu, Jianbin; Wang, Jinhui; Yang, Xiaorong; Cai, Xiangsheng

2018-06-21

BACKGROUND Cytokeratin 19 (CK19) is a typical epithelial marker. In this study, we determined whether epidermal growth factor (EGF) or basic fibroblast growth factor (bFGF) could enhance CK19 expression in adipose-derived stem cells (ADSCs), thereby inducing the differentiation of ADSCs into epithelial-like cells. MATERIAL AND METHODS ADSCs were isolated from perinephric fat, and the expression of CD29, CD90, and CD105 was confirmed. Following isolation, ADSCs were cultured in static medium or medium containing EGF or bFGF. RESULTS Flow cytometry revealed that EGF and bFGF could alter mesenchymal stem cell markers as well as the cell cycle of ADSCs. Western blotting and immunofluorescence revealed that after 14 days, EGF treatment enhanced the expression of CK19 in ADSCs. CONCLUSIONS Our findings offer important insight for the clinical use of ADSCs in the generation of epithelial-like cells in the future.

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.

[CD34(+)/CD123(+) cell sorting from the patients with leukemia by Midi MACS method].

PubMed

Wang, Guang-Ping; Cao, Xin-Yu; Xin, Hong-Ya; Li, Qun; Qi, Zhen-Hua; Chen, Fang-Ping

2006-10-01

The aim of this study was to sort the CD34(+)/CD123(+) cells from the bone marrow cells of patients with acute myeloid leukemia (AML) by Midi MACS method. Firstly, the bone marrow mononuclear cells (BMMNC) were isolated from the patients with AML with Ficoll Paque, CD34(+) cells were then isolated by Midi MACS method followed by the isolation of CD34(+)/CD123(+) cells from the fraction of CD34(+) cells. The enrichment and recovery of CD34(+) and CD34(+)/CD123(+) cells were assayed by FACS technique. The results showed that the enrichment of CD34(+) cells was up to 98.73%, its average enrichment was 95.6%, and the recovery of CD34(+) was 84.6%, its average recovery was 51% after the first round sorting, by the second round sorting, the enrichment of CD34(+)/CD123(+) cells was up to 99.23%, its average enrichment was 83%. With regard to BMMNCs before sorting, the recovery of CD34(+)/CD123(+) was 34%. But, on the CD34(+) cells obtained by the first round sorting, its recovery was 56%. In conclusion, these results confirmed that the method of Midi MACS sorting can be applied to sort CD34(+)/CD123(+) cells from the bone marrow cells of AML patients, which give rise to the similar enrichment and recovery of the sorted cells with that of literature reported by the method of FACS.

Advances in all-sputtered CdTe solar cells on flexible substrates

NASA Astrophysics Data System (ADS)

Wieland, Kristopher; Mahabaduge, Hasitha; Vasko, Anthony; Compaan, Alvin

2010-03-01

The University of Toledo II-VI semiconductor group has developed magnetron sputtering (MS) for the deposition of thin films of CdS, CdTe, and related materials for photovoltaic applications. On glass superstrates, we have reached air mass 1.5 efficiencies of 14%.[1] Recently we have studied the use of MS for the fabrication of thin-film CdS/CdTe cells on flexible polyimide superstrates. This takes advantage of the high film quality that can be achieved at substrate temperatures below 300 C when RF MS is used. Our recent CdS/CdTe solar cells have reached 10.5% on flexible polyimide substrates. [2] This all-sputtered cell (except for back contact) has a structure of polyimide/ZnO:Al/ZnO/CdS/CdTe/Cu/Au. The physics of this device will be discussed through the use of spectral quantum efficiency and current-voltage measurements as a function of CdTe layer thickness. Pathways toward further increases in device efficiencies will also be discussed. [1] Appl. Phys. Lett. 85, 684 (2004) [2] Phys. Stat. Sol. (B) 241, No. 3, 779--782 (2004)

Urinary Cadmium Threshold to Prevent Kidney Disease Development.

PubMed

Satarug, Soisungwan; Ruangyuttikarn, Werawan; Nishijo, Muneko; Ruiz, Patricia

2018-05-01

The frequently observed association between kidney toxicity and long-term cadmium (Cd) exposure has long been dismissed and deemed not to be of clinical relevance. However, Cd exposure has now been associated with increased risk of developing chronic kidney disease (CKD). We investigated the link that may exist between kidney Cd toxicity markers and clinical kidney function measure such as estimated glomerular filtration rates (eGFR). We analyzed data from 193 men to 202 women, aged 16−87 years [mean age 48.8 years], who lived in a low- and high-Cd exposure areas in Thailand. The mean (range) urinary Cd level was 5.93 (0.05⁻57) μg/g creatinine. The mean (range) for estimated GFR was 86.9 (19.6−137.8) mL/min/1.73 m². Kidney pathology reflected by urinary β2-microglobulin (β2-MG) levels ≥ 300 μg/g creatinine showed an association with 5.32-fold increase in prevalence odds of CKD ( p = 0.001), while urinary Cd levels showed an association with a 2.98-fold greater odds of CKD prevalence ( p = 0.037). In non-smoking women, Cd in the highest urinary Cd quartile was associated with 18.3 mL/min/1.73 m² lower eGFR value, compared to the lowest quartile ( p < 0.001). Evidence for Cd-induced kidney pathology could thus be linked to GFR reduction, and CKD development in Cd-exposed people. These findings may help prioritize efforts to reassess Cd exposure and its impact on population health, given the rising prevalence of CKD globally.

Intramyocardial autologous CD34+ cell therapy for refractory angina: A meta-analysis of randomized controlled trials.

PubMed

Velagapudi, Poonam; Turagam, Mohit; Kolte, Dhaval; Khera, Sahil; Hyder, Omar; Gordon, Paul; Aronow, Herbert D; Leopold, Jane; Abbott, J Dawn

2018-06-05

Previous studies have demonstrated that intramyocardial human CD34+ cells may relieve symptoms and improve clinical outcomes in chronic refractory angina unresponsive to optimal medical therapy or not amenable to revascularization. We performed a meta-analysis of randomized controlled trials (RCTs) to evaluate the impact of human CD34+ cells compared with placebo in chronic refractory angina. Primary efficacy outcomes in our analysis were angina frequency and exercise time. Primary safety outcomes included major adverse cardiovascular events such as myocardial infarction (MI), stroke and death. Three eligible randomized trials including 269 patients (placebo = 90, CD34+ = 179) were included. Dose of auto-CD34+ cells ranged from 5 × 10 4 to 5 × 10 5  cells/kg. Follow-up ranged from 6 to 24 months. In a pooled analysis, administration of CD34+ cells decreased the risk of all-cause mortality [OR 0.24, 95% CI (0.08-0.73), p = 0.01], reduced angina frequency [mean difference -2.91, 95% CI (-4.57 to -1.25), p = 0.0006] and improved exercise time [mean difference 58.62 s, 95% CI (21.19 to 96.06), p = 0.02] compared with control group. However, there was no significant difference in the risk of myocardial infarction (MI) and stroke between groups. In a meta-analysis, intra-myocardial CD34+ cell therapy was superior to placebo in improving risk of all - cause mortality, angina frequency with an increase in exercise time, without a significant increase in adverse events. This analysis supports further trials of CD34+ cell therapy for ischemic heart disease. Copyright © 2018 Elsevier Inc. All rights reserved.

Stemness and angiogenic gene expression changes of serial-passage human amnion mesenchymal cells.

PubMed

Fatimah, Simat Siti; Tan, Geok Chin; Chua, Kienhui; Fariha, Mohd Manzor Nur; Tan, Ay Eeng; Hayati, Abdul Rahman

2013-03-01

Particular attention has been directed towards human amnion mesenchymal stem cells (HAMCs) due to their accessibility, availability and immunomodulatory properties. Therefore, the aim of the present study was to determine the temporal changes of stemness and angiogenic gene expressions of serial-passage HAMCs. HAMCs were isolated from human term placenta and cultured in serial passages in culture medium supplemented with 10% fetal bovine serum. Morphological analysis, growth kinetic and CFU-F assay of HAMCs were assessed. In vitro differentiation and the immunophenotype of HAMCs at P5 were also analyzed. Quantitative PCR was used to determine the stemness, angiogenic and endothelial gene expression of cultured HAMCs after serial passage. Cultured HAMCs displayed intermediate epitheloid-fibroblastoid morphology at an initial culture and the fibroblastoid features became more pronounced in later passages. They showed high clonogenic activity and faster proliferation at later passages with colony forming efficiency of 0.88%. HAMCs were successfully differentiated into adipocytes, osteocytes and neuron-like cells. Most HAMCs expressed CD9, CD44, CD73, CD90 and HLA-A,B,C but negligibly expressed CD31, CD34, CD45, CD117 and HLA-DR,DP,DQ. After serial passage, stemness genes Oct-3/4, Sox-2, Nanog3, Rex-1, FGF-4 and FZD-9 expressions significantly decreased. Of the angiogenic genes PECAM-1, bFGF, eNOS, VEGFR-2, VEGF, and vWF expressions also decreased significantly except angiopoietin-1 which significantly increased. No significant differences were observed in ABCG-2, BST-1, nestin, PGF and HGF expressions after serial passage. These results suggested that cultured HAMCs could be an alternative source of stem cells and may have the potential for angiogenesis and hence its use in stem-cell based therapy. Copyright © 2012 Elsevier Inc. All rights reserved.

CD73 Inhibition Shifts Cardiac Macrophage Polarization toward a Microbicidal Phenotype and Ameliorates the Outcome of Experimental Chagas Cardiomyopathy.

PubMed

Ponce, Nicolás Eric; Sanmarco, Liliana Maria; Eberhardt, Natalia; García, Mónica Cristina; Rivarola, Héctor Walter; Cano, Roxana Carolina; Aoki, Maria Pilar

2016-08-01

Increasing evidence demonstrates that generation of extracellular adenosine from ATP, which is hydrolyzed by the CD39/CD73 enzyme pair, attenuates the inflammatory response and deactivates macrophage antimicrobial mechanisms. Although CD73 is emerging as a critical pathway and therapeutic target in cardiovascular disorders, the involvement of this ectonucleotidase during myocardial infection has not been explored. Using a murine model of infection with Trypanosoma cruzi, the causal agent of Chagas cardiomyopathy, we observed a sudden switch from the classical M1 macrophage (microbicidal) phenotype toward an alternative M2 (repairing/anti-inflammatory) phenotype that occurred within the myocardium very shortly after BALB/c mice infection. The observed shift in M1/M2 rate correlated with the cardiac cytokine milieu. Considering that parasite persistence within myocardium is a necessary and sufficient condition for the development of the chronic myocarditis, we hypothesized that CD73 activity may counteract cardiac macrophage microbicidal polarization, rendering the local immune response less effective. In fact, a transient treatment with a specific CD73 inhibitor (adenosine 5'-α,β-methylene-diphosphate) enhanced the microbicidal M1 subset predominance, diminished IL-4- and IL-10-producing CD4(+) T cells, promoted a proinflammatory cytokine milieu, and reduced parasite load within the myocardium during the acute phase. As a direct consequence of these events, there was a reduction in serum levels of creatine kinase muscle-brain isoenzyme, a myocardial-specific injury marker, and an improvement in the electrocardiographic characteristics during the chronic phase. Our results demonstrate that this purinergic system drives the myocardial immune response postinfection and harbors a promising potential as a therapeutic target. Copyright © 2016 by The American Association of Immunologists, Inc.

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.

Superior Potential of CD34-Positive Cells Compared to Total Mononuclear Cells for Healing of Nonunion Following Bone Fracture.

PubMed

Fukui, Tomoaki; Mifune, Yutaka; Matsumoto, Tomoyuki; Shoji, Taro; Kawakami, Yohei; Kawamoto, Atsuhiko; Ii, Masaaki; Akimaru, Hiroshi; Kuroda, Tomoya; Horii, Miki; Yokoyama, Ayumi; Alev, Cantas; Kuroda, Ryosuke; Kurosaka, Masahiro; Asahara, Takayuki

2015-01-01

We recently demonstrated that the local transplantation of human peripheral blood (PB) CD34(+) cells, an endothelial/hematopoietic progenitor cell-rich population, contributes to fracture repair via vasculogenesis/angiogenesis and osteogenesis. Human PB mononuclear cells (MNCs) are also considered a potential cell fraction for neovascularization. We have previously shown the feasibility of human PB MNCs to enhance fracture healing. However, there is no report directly comparing the efficacy for fracture repair between CD34(+) cells and MNCs. In addition, an unhealing fracture model, which does not accurately resemble a clinical setting, was used in our previous studies. To overcome these issues, we compared the capacity of human granulocyte colony-stimulating factor-mobilized PB (GM-PB) CD34(+) cells and human GM-PB MNCs in a nonunion model, which more closely resembles a clinical setting. First, the effect of local transplantation of 1 × 10(5) GM-PB CD34(+) cells (CD34(+) group), 1 × 10(7) GM-PB MNCs (containing approximately 1 × 10(5) GM-PB CD34(+) cells) (MNC group), and phosphate-buffered saline (PBS) (PBS group) on nonunion healing was compared. Similar augmentation of blood flow recovery at perinonunion sites was observed in the CD34(+) and MNC groups. Meanwhile, a superior effect on nonunion repair was revealed by radiological, histological, and functional assessment in the CD34(+) group compared with the other groups. Moreover, through in vivo and in vitro experiments, excessive inflammation induced by GM-PB MNCs was confirmed and believed to be one of the mechanisms underlying this potency difference. These results strongly suggest that local transplantation of GM-PB CD34(+) cells is a practical and effective strategy for treatment of nonunion after fracture.

Variation in primary and culture-expanded cells derived from connective tissue progenitors in human bone marrow space, bone trabecular surface and adipose tissue.

PubMed

Qadan, Maha A; Piuzzi, Nicolas S; Boehm, Cynthia; Bova, Wesley; Moos, Malcolm; Midura, Ronald J; Hascall, Vincent C; Malcuit, Christopher; Muschler, George F

2018-03-01

Connective tissue progenitors (CTPs) embody the heterogeneous stem and progenitor cell populations present in native tissue. CTPs are essential to the formation and remodeling of connective tissue and represent key targets for tissue-engineering and cell-based therapies. To better understand and characterize CTPs, we aimed to compare the (i) concentration and prevalence, (ii) early in vitro biological behavior and (iii) expression of surface-markers and transcription factors among cells derived from marrow space (MS), trabecular surface (TS), and adipose tissues (AT). Cancellous-bone and subcutaneous-adipose tissues were collected from 8 patients. Cells were isolated and cultured. Colony formation was assayed using Colonyze software based on ASTM standards. Cell concentration ([Cell]), CTP concentration ([CTP]) and CTP prevalence (P CTP ) were determined. Attributes of culture-expanded cells were compared based on (i) effective proliferation rate and (ii) expression of surface-markers CD73, CD90, CD105, SSEA-4, SSEA-3, SSEA-1/CD15, Cripto-1, E-Cadherin/CD324, Ep-CAM/CD326, CD146, hyaluronan and transcription factors Oct3/4, Sox-2 and Nanog using flow cytometry. Mean [Cell], [CTP] and P CTP were significantly different between MS and TS samples (P = 0.03, P = 0.008 and P= 0.0003), respectively. AT-derived cells generated the highest mean total cell yield at day 6 of culture-4-fold greater than TS and more than 40-fold greater than MS per million cells plated. TS colonies grew with higher mean density than MS colonies (290 ± 11 versus 150 ± 11 cell per mm 2 ; P = 0.0002). Expression of classical-mesenchymal stromal cell (MSC) markers was consistently recorded (>95%) from all tissue sources, whereas all the other markers were highly variable. The prevalence and biological potential of CTPs are different between patients and tissue sources and lack variation in classical MSC markers. Other markers are more likely to discriminate differences between cell populations in biological performance. Understanding the underlying reasons for variation in the concentration, prevalence, marker expression and biological potential of CTPs between patients and source tissues and determining the means of managing this variation will contribute to the rational development of cell-based clinical diagnostics and targeted cell-based therapies. Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Grating coupled SPR microarray analysis of proteins and cells in blood from mice with breast cancer.

PubMed

Mendoza, A; Torrisi, D M; Sell, S; Cady, N C; Lawrence, D A

2016-01-21

Biomarker discovery for early disease diagnosis is highly important. Of late, much effort has been made to analyze complex biological fluids in an effort to develop new markers specific for different cancer types. Recent advancements in label-free technologies such as surface plasmon resonance (SPR)-based biosensors have shown promise as a diagnostic tool since there is no need for labeling or separation of cells. Furthermore, SPR can provide rapid, real-time detection of antigens from biological samples since SPR is highly sensitive to changes in surface-associated molecular and cellular interactions. Herein, we report a lab-on-a-chip microarray biosensor that utilizes grating-coupled surface plasmon resonance (GCSPR) and grating-coupled surface plasmon coupled fluorescence (GCSPCF) imaging to detect circulating tumor cells (CTCs) from a mouse model (FVB-MMTV-PyVT). GCSPR and GCSPCF analysis was accomplished by spotting antibodies to surface cell markers, cytokines and stress proteins on a nanofabricated GCSPR microchip and screening blood samples from FVB control mice or FVB-MMTV-PyVT mice with developing mammary carcinomas. A transgenic MMTV-PyVT mouse derived cancer cell line was also analyzed. The analyses indicated that CD24, CD44, CD326, CD133 and CD49b were expressed in both cell lines and in blood from MMTV-PyVT mice. Furthermore, cytokines such as IL-6, IL-10 and TNF-α, along with heat shock proteins HSP60, HSP27, HSc70(HSP73), HSP90 total, HSP70/HSc70, HSP90, HSP70, HSP90 alpha, phosphotyrosine and HSF-1 were overexpressed in MMTV-PyVT mice.

Positron emission tomography imaging of CD105 expression in a rat myocardial infarction model with (64)Cu-NOTA-TRC105.

PubMed

Orbay, Hakan; Zhang, Yin; Valdovinos, Hector F; Song, Guoqing; Hernandez, Reinier; Theuer, Charles P; Hacker, Timothy A; Nickles, Robert J; Cai, Weibo

2013-01-01

Biological changes following myocardial infarction (MI) lead to increased secretion of angiogenic factors that subsequently stimulate the formation of new blood vessels as a compensatory mechanism to reverse ischemia. The goal of this study was to assess the role of CD105 expression during MI-induced angiogenesis by positron emission tomography (PET) imaging using (64)Cu-labeled TRC105, an anti-CD105 monoclonal antibody. MI was induced by ligation of the left anterior descending (LAD) artery in female rats. Echocardiography and (18)F-fluoro-2-deoxy-D-glucose ((18)F-FDG) PET scans were performed on post-operative day 3 to confirm the presence of MI in the infarct group and intact heart in the sham group, respectively. Ischemia-induced angiogenesis was non-invasively monitored with (64)Cu-NOTA-TRC105 (an extensively validated PET tracer in our previous studies) PET on post-operative days 3, 10, and 17. Tracer uptake in the infarct zone was highest on day 3 following MI, which was significantly higher than that in the sham group (1.41 ± 0.45 %ID/g vs 0.57 ± 0.07 %ID/g; n=3, p<0.05). Subsequently, tracer uptake in the infarct zone decreased over time to the background level on day 17, whereas tracer uptake in the heart of sham rats remained low at all time points examined. Histopathology documented increased CD105 expression following MI, which corroborated in vivo findings. This study indicated that PET imaging of CD105 can be a useful tool for MI-related research, which can potentially improve MI patient management in the future upon clinical translation of the optimized PET tracers.

High frequency of CD34+CD38-/low immature leukemia cells is correlated with unfavorable prognosis in acute myeloid leukemia.

PubMed

Plesa, Adriana; Dumontet, Charles; Mattei, Eve; Tagoug, Ines; Hayette, Sandrine; Sujobert, Pierre; Tigaud, Isabelle; Pages, Marie Pierre; Chelghoum, Youcef; Baracco, Fiorenza; Labussierre, Helene; Ducastelle, Sophie; Paubelle, Etienne; Nicolini, Franck Emmanuel; Elhamri, Mohamed; Campos, Lydia; Plesa, Claudiu; Morisset, Stéphane; Salles, Gilles; Bertrand, Yves; Michallet, Mauricette; Thomas, Xavier

2017-12-26

To evaluate the importance of the CD34+CD38- cell population when compared to the CD34+CD38+/low and CD34+CD38+/high leukemic cell sub-populations and to determine its correlations with leukemia characteristics and known prognostic factors, as well as with response to therapy and survival. Two hundred bone marrow samples were obtained at diagnosis from 200 consecutive patients with newly diagnosed acute myeloid leukemia (AML) were studied between September 2008 and December 2010 at our Institution (Hematology Department, Lyon, France). The CD34/CD38 cell profile was analyzed by multiparameter flowcytometry approach using 8C panels and FACS CANTO and Diva software (BD Bioscience). We analyzed CD34 and CD38 expression in bone marrow samples of 200 AML patients at diagnosis, and investigated the prognostic value of the most immature CD34+CD38- population. Using a cut-off value of 1% of CD34+CD38- from total "bulk leukemic cells" we found that a high (> 1%) level of CD34+CD38- blasts at diagnosis was correlated with advanced age, adverse cytogenetics as well as with a lower rate of complete response after induction and shorter disease-free survival. In a multivariate analysis considering age, leukocytosis, the % of CD34+ blasts cells and the standardized cytogenetic and molecular risk subgroups, a percentage of CD34+CD38- leukemic cells > 1% was an independent predictor of DFS [HR = 2.8 (1.02-7.73), P = 0.04] and OS [HR = 2.65 (1.09-6.43), P = 0.03]. Taken together, these results show that a CD34/CD38 "backbone" for leukemic cell analysis by multicolour flowcytometry at diagnosis provides useful prognostic information.

Activity of a long-acting echinocandin, CD101, determined using CLSI and EUCAST reference methods, against Candida and Aspergillus spp., including echinocandin- and azole-resistant isolates.

PubMed

Pfaller, Michael A; Messer, Shawn A; Rhomberg, Paul R; Jones, Ronald N; Castanheira, Mariana

2016-10-01

The objective of this study was to evaluate the in vitro activity of CD101, a novel echinocandin with a long serum elimination half-life, and comparator (anidulafungin and caspofungin) antifungal agents against a collection of Candida and Aspergillus spp. isolates. CD101 and comparator agents were tested against 106 Candida spp. and 67 Aspergillus spp. isolates, including 27 isolates of Candida harbouring fks hotspot mutations and 12 itraconazole non-WT Aspergillus, using CLSI and EUCAST reference susceptibility broth microdilution (BMD) methods. Against WT and fks mutant Candida albicans, Candida glabrata and Candida tropicalis, the activity of CD101 [MIC90 = 0.06, 0.12 and 0.03 mg/L, respectively (CLSI method values)] was comparable to that of anidulafungin (MIC90 = 0.03, 0.12 and 0.03 mg/L, respectively) and caspofungin (MIC90 = 0.12, 0.25 and 0.12 mg/L, respectively). WT Candida krusei isolates were very susceptible to CD101 (MIC = 0.06 mg/L). CD101 activity (MIC50/90 = 1/2 mg/L) was comparable to that of anidulafungin (MIC50/90 = 2/2 mg/L) against Candida parapsilosis. CD101 (MIC mode = 0.06 mg/L for C. glabrata) was 2- to 4-fold more active against fks hotspot mutants than caspofungin (MIC mode = 0.5 mg/L). CD101 was active against Aspergillus fumigatus, Aspergillus terreus, Aspergillus niger and Aspergillus flavus (MEC90 range = ≤0.008-0.03 mg/L). The essential agreement between CLSI and EUCAST methods for CD101 was 92.0%-100.0% among Candida spp. and 95.0%-100.0% among Aspergillus spp. The activity of CD101 is comparable to that of other members of the echinocandin class for the prevention and treatment of serious fungal infections. Similar results for CD101 activity versus Candida and Aspergillus spp. may be obtained with either CLSI or EUCAST BMD methods. © The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy.

Dendritic-cell-based immunotherapy evokes potent anti-tumor immune responses in CD105+ human renal cancer stem cells.

PubMed

Zhang, Xiao-Fei; Weng, De-Sheng; Pan, Ke; Zhou, Zi-Qi; Pan, Qiu-Zhong; Zhao, Jing-Jing; Tang, Yan; Jiang, Shan-Shan; Chen, Chang-Long; Li, Yong-Qiang; Zhang, Hong-Xia; Chang, Alfred E; Wicha, Max S; Zeng, Yi-Xin; Li, Qiao; Xia, Jian-Chuan

2017-11-01

Cancer stem cells (CSCs) are responsible for tumor initiation, progression, and resistance to therapeutic agents; they are usually less sensitive to conventional cancer therapies, and could cause tumor relapse. An ideal therapeutic strategy would therefore be to selectively target and destroy CSCs, thereby preventing tumor relapse. The aim of the present study was to evaluate the effectiveness of dendritic cells (DCs) pulsed with antigen derived from CD105+ human renal cell carcinoma (RCC) CSCs against renal cancer cells in vitro and in vivo. We identified "stem-like" characteristics of CD105+ cells in two human RCC cell lines: A498 and SK-RC-39. Loading with cell lysates did not change the characteristics of the DCs. However, DCs loaded with lysates derived from CD105+ CSCs induced more functionally specific active T cells and specific antibodies against CSCs, and clearly depressed the tumor growth in mice. Our results could form the basis for a novel strategy to improve the efficacy of DC-based immunotherapy for human RCC. © 2017 Wiley Periodicals, Inc.

Nonlinear refraction of Nd3+-Li+ co-doped CdS-PVP nanostructure

NASA Astrophysics Data System (ADS)

Talwatkar, S. S.; Sunatkari, A. L.; Tamgadge, Y. S.; Muley, G. G.

2018-05-01

Third-order optical nonlinearity of the co-doped CdS nanoparticles embedded in polymer thin films is studied with the Z-scan technique with 632.8 nm excitation using a cw laser. The results show that the co-doped CdS nanocomposite film exhibits enhanced negative nonlinear refractive index in the order of n2 ≈ 10-5 cm2/W, as compared to the undoped CdS nanoparticles. Nonlinear refractive index of co-doped CdS-PVP thin film are found maximum for the sample doped with 5 wt% of Nd3+-Li+ concentration. The underlying mechanism is discussed.

Differential susceptibility of SD and CD rats to a novel rat theilovirus.

PubMed

Drake, Michael T; Riley, Lela K; Livingston, Robert S

2008-10-01

Antibodies to rat theilovirus (RTV) have been detected in rats for many years because of their serologic crossreactivity with strains of Theiler murine encephalomyelitis virus (TMEV) of mice. Little information exists regarding this pathogen, yet it is among the most common viruses detected in serologic surveys of rats used in research. In the study reported here, a novel isolate of RTV, designated RTV1, was cultured from the feces of infected rats. The RTV1 genome contained 8094 nucleotides and had approximately 95% identity with another rat theilovirus, NSG910, and 73% identity with TMEV strains. In addition, the genome size of RTV1 was similar to those of TMEV strains but larger than that reported for NSG910. Oral inoculation of Sprague-Dawley (SD) and CD male rats (n = 10 each group) with RTV1 revealed that SD rats were more susceptible than CD rats to RTV1 infection. At 14 d postinoculation, 100% of SD rats shed virus in the feces, and 70% were positive for RTV serum antibodies. By 56 d postinoculation 30% of SD rats continued to have detectable virus in the feces, and 90% had seroconverted. In contrast, in inoculated CD rats RTV was detected only in the feces at 14 d postinoculation, at which time 40% of CD rats were fecal positive. By 56 d postinoculation only 20% of CD rats had detectable RTV serum antibodies. Our data provide additional sequence information regarding a rat-specific Cardiovirus and indicate that SD rats are more susceptible than CD rats to RTV1 infection.

Cervical cancer cells suppress effector functions of cytotoxic T cells through the adenosinergic pathway.

PubMed

Mora-García, M L; Ávila-Ibarra, L R; García-Rocha, R; Weiss-Steider, B; Hernández-Montes, J; Don-López, C A; Gutiérrez-Serrano, V; Titla-Vilchis, I J; Fuentes-Castañeda, M C; Monroy-Mora, A; Jave-Suárez, L F; Chacón-Salinas, R; Vallejo-Castillo, L; Pérez-Tapia, S M; Monroy-García, A

2017-10-01

The expression of CD73 in tumor cells plays a significant role in the production of adenosine (Ado) that suppresses antitumor effector cells. In this study we analyzed the capability of HPV-positive (HPV+) cervical cancer (CeCa) cell lines CaSki, SiHa, HeLa, and RoVa; and HPV-negative (HPV-) cell lines C33A and ViBo to produce Ado and inhibit effector functions of CD8+ T cells. HPV+ CeCa cells expressed significantly higher levels of CD73 in the membrane (p<0.01) than HPV- CeCa cells and this expression was associated with the production of larger amounts of Ado (>400μM) compared to HPV-CeCa cells (<200μM) in the presence of AMP, as well asa stronger inhibition of (>50%) proliferation, activation, and cytotoxic activity of CD8+ T cells via interaction with A2A adenosine receptor. We also provide evidence that silenced E6/E7 expression in CeCa cells, strongly reduced its CD73 expression level and its capability to generate Ado. This results suggest that HPV infection, which is associated with more than 99% of CeCa cases, may present an increased constitutive expression of CD73 in cervical neoplasia to contribute to the suppression of the immune response mediated by the production of large amounts of Ado. Copyright © 2017 Elsevier Inc. All rights reserved.

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.

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.

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.

Design and validation of a consistent and reproducible manufacture process for the production of clinical-grade bone marrow-derived multipotent mesenchymal stromal cells.

PubMed

Codinach, Margarita; Blanco, Margarita; Ortega, Isabel; Lloret, Mireia; Reales, Laura; Coca, Maria Isabel; Torrents, Sílvia; Doral, Manel; Oliver-Vila, Irene; Requena-Montero, Miriam; Vives, Joaquim; Garcia-López, Joan

2016-09-01

Multipotent mesenchymal stromal cells (MSC) have achieved a notable prominence in the field of regenerative medicine, despite the lack of common standards in the production processes and suitable quality controls compatible with Good Manufacturing Practice (GMP). Herein we describe the design of a bioprocess for bone marrow (BM)-derived MSC isolation and expansion, its validation and production of 48 consecutive batches for clinical use. BM samples were collected from the iliac crest of patients for autologous therapy. Manufacturing procedures included: (i) isolation of nucleated cells (NC) by automated density-gradient centrifugation and plating; (ii) trypsinization and expansion of secondary cultures; and (iii) harvest and formulation of a suspension containing 40 ± 10 × 10(6) viable cells. Quality controls were defined as: (i) cell count and viability assessment; (ii) immunophenotype; and (iii) sterility tests, Mycoplasma detection, endotoxin test and Gram staining. A 3-week manufacturing bioprocess was first designed and then validated in 3 consecutive mock productions, prior to producing 48 batches of BM-MSC for clinical use. Validation included the assessment of MSC identity and genetic stability. Regarding production, 139.0 ± 17.8 mL of BM containing 2.53 ± 0.92 × 10(9) viable NC were used as starting material, yielding 38.8 ± 5.3 × 10(6) viable cells in the final product. Surface antigen expression was consistent with the expected phenotype for MSC, displaying high levels of CD73, CD90 and CD105, lack of expression of CD31 and CD45 and low levels of HLA-DR. Tests for sterility, Mycoplasma, Gram staining and endotoxin had negative results in all cases. Herein we demonstrated the establishment of a feasible, consistent and reproducible bioprocess for the production of safe BM-derived MSC for clinical use. Copyright © 2016 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

A xenogeneic-free bioreactor system for the clinical-scale expansion of human mesenchymal stem/stromal cells.

PubMed

Dos Santos, Francisco; Campbell, Andrew; Fernandes-Platzgummer, Ana; Andrade, Pedro Z; Gimble, Jeffrey M; Wen, Yuan; Boucher, Shayne; Vemuri, Mohan C; da Silva, Cláudia L; Cabral, Joaquim M S

2014-06-01

The large cell doses (>1 × 10(6)  cells/kg) used in clinical trials with mesenchymal stem/stromal cells (MSC) will require an efficient production process. Moreover, monitoring and control of MSC ex-vivo expansion is critical to provide a safe and reliable cell product. Bioprocess engineering approaches, such as bioreactor technology, offer the adequate tools to develop and optimize a cost-effective culture system for the rapid expansion of human MSC for cellular therapy. Herein, a xenogeneic (xeno)-free microcarrier-based culture system was successfully established for bone marrow (BM) MSC and adipose tissue-derived stem/stromal cell (ASC) cultivation using a 1L-scale controlled stirred-tank bioreactor, allowing the production of (1.1 ± 0.1) × 10(8) and (4.5 ± 0.2) × 10(7) cells for BM MSC and ASC, respectively, after 7 days. Additionally, the effect of different percent air saturation values (%Airsat ) and feeding regime on the proliferation and metabolism of BM MSC was evaluated. No significant differences in cell growth and metabolic patterns were observed under 20% and 9%Airsat . Also, the three different feeding regimes studied-(i) 25% daily medium renewal, (ii) 25% medium renewal every 2 days, and (iii) fed-batch addition of concentrated nutrients and growth factors every 2 days-yielded similar cell numbers, and only slight metabolic differences were observed. Moreover, the immunophenotype (positive for CD73, CD90 and CD105 and negative for CD31, CD80 and HLA-DR) and multilineage differentiative potential of expanded cells were not affected upon bioreactor culture. These results demonstrated the feasibility of expanding human MSC from different sources in a clinically relevant expansion configuration in a controlled microcarrier-based stirred culture system under xeno-free conditions. The further optimization of this bioreactor culture system will represent a crucial step towards an efficient GMP-compliant clinical-scale MSC production system. © 2014 Wiley Periodicals, Inc.

Root iron plaque alleviates cadmium toxicity to rice (Oryza sativa) seedlings.

PubMed

Fu, Youqiang; Yang, Xujian; Shen, Hong

2018-06-18

Iron plaque (IP) on root surface can enhance the tolerance of plants to environmental stresses. However, it remains unclear the impact of Fe 2+ on cadmium (Cd) toxicity to rice (Oryza sativa) seedlings. In this study, the effects of different Fe 2+ and Cd 2+ concentration combinations on rice growth were examined hydroponically. Results indicated that Fe 2+ concentration up to 3.2 mM did not damage rice roots while induced IP formation obviously. Cd 2+ of 10 μM repressed rice growth significantly, while the addition of 0.2 mM Fe 2+ to 10 μM Cd 2+ solution (Cd+Fe) did not damage rice roots, indicating that Fe 2+ could ameliorate Cd toxicity to rice seedlings. Microstructure analysis showed Cd+Fe treatment induced the formation of IP with dense and intricate network structure, Cd adsorption on the root surface was reduced significantly. Cd concentration of rice roots and shoots and Cd translocation from roots to shoots with Fe+Cd treatment were reduced by 34.1%, 36.0% and 20.1%, respectively, in comparison to a single Cd treatment. Noteworthy, the removal of IP resulted in a larger loss of root biomass under Cd treatment. In addition, Cd+Fe treatment increased the activities of root superoxide dismutase and catalase by 105.5% and 177.4%, and decreased H 2 O 2 and O 2 · - accumulation of rice roots by 56.9% and 35.9%, and recovered Cd-triggered electrolyte leakage obviously, when compared with a single Cd treatment. The results from this experiment indicated that the formed dense IP on rice roots decreased Cd absorption and reactive oxygen species accumulation, and Fe 2+ supply alleviated Cd toxicity to rice seedlings. Copyright © 2018 Elsevier Inc. All rights reserved.

Isolation of a circulating CD45−, CD34dim cell population and validation of their endothelial phenotype

PubMed Central

Tropea, Margaret M.; Harper, Bonnie J. A.; Graninger, Grace M.; Phillips, Terry M.; Ferreyra, Gabriela; Mostowski, Howard S.; Danner, Robert L.; Suffredini, Anthony F.; Solomon, Michael A.

2016-01-01

Summary Accurately detecting circulating endothelial cells (CECs) is important since their enumeration has been proposed as a biomarker to measure injury to the vascular endothelium. However, there is no single methodology for determining CECs in blood, making comparison across studies difficult. Many methods for detecting CECs rely on characteristic cell surface markers and cell viability indicators, but lack secondary validation. Here, a CEC population in healthy adult human subjects was identified by flow cytometry as CD45−, CD34dim that is comparable to a previously described CD45−, CD31bright population. In addition, nuclear staining with 7-aminoactinomycin D (7-AAD) was employed as a standard technique to exclude dead cells. Unexpectedly, the CD45−, CD34dim, 7-AAD− CECs lacked surface detectable CD146, a commonly used marker of CECs. Furthermore, light microscopy revealed this cell population to be composed primarily of large cells without a clearly defined nucleus. Nevertheless, immunostains still demonstrated the presence of the lectin Ulex europaeus and van Willebrand factor. Ultramicro analytical immunochemistry assays for the endothelial cell proteins CD31, CD34, CD62E, CD105, CD141, CD144 and vWF indicated these cells possess an endothelial phenotype. However, only a small amount of RNA, which was mostly degraded, could be isolated from these cells. Thus the majority of CECs in healthy individuals as defined by CD45−, CD34dim, and 7-AAD− have shed their CD146 surface marker and are senescent cells without an identifiable nucleus and lacking RNA of sufficient quantity and quality for transcriptomal analysis. This study highlights the importance of secondary validation of CEC identification. PMID:25057108

An index with improved diagnostic accuracy for the diagnosis of Crohn's disease derived from the Lennard-Jones criteria.

PubMed

Reinisch, S; Schweiger, K; Pablik, E; Collet-Fenetrier, B; Peyrin-Biroulet, L; Alfaro, I; Panés, J; Moayyedi, P; Reinisch, W

2016-09-01

The Lennard-Jones criteria are considered the gold standard for diagnosing Crohn's disease (CD) and include the items granuloma, macroscopic discontinuity, transmural inflammation, fibrosis, lymphoid aggregates and discontinuous inflammation on histology. The criteria have never been subjected to a formal validation process. To develop a validated and improved diagnostic index based on the items of Lennard-Jones criteria. Included were 328 adult patients with long-standing CD (median disease duration 10 years) from three centres and classified as 'established', 'probable' or 'non-CD' by Lennard-Jones criteria at time of diagnosis. Controls were patients with ulcerative colitis (n = 170). The performance of each of the six diagnostic items of Lennard-Jones criteria was modelled by logistic regression and a new index based on stepwise backward selection and cut-offs was developed. The diagnostic value of the new index was analysed by comparing sensitivity, specificity and accuracy vs. Lennard-Jones criteria. By Lennard-Jones criteria 49% (n = 162) of CD patients would have been diagnosed as 'non-CD' at time of diagnosis (sensitivity/specificity/accuracy, 'established' CD: 0.34/0.99/0.67; 'probable' CD: 0.51/0.95/0.73). A new index was derived from granuloma, fibrosis, transmural inflammation and macroscopic discontinuity, but excluded lymphoid aggregates and discontinuous inflammation on histology. Our index provided improved diagnostic accuracy for 'established' and 'probable' CD (sensitivity/specificity/accuracy, 'established' CD: 0.45/1/0.72; 'probable' CD: 0.8/0.85/0.82), including the subgroup isolated colonic CD ('probable' CD, new index: 0.73/0.85/0.79; Lennard-Jones criteria: 0.43/0.95/0.69). We developed an index based on items of Lennard-Jones criteria providing improved diagnostic accuracy for the differential diagnosis between CD and UC. © 2016 John Wiley & Sons Ltd.

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.

A novel immunotoxin reveals a new role for CD321 in endothelial cells

PubMed Central

Kim, Jia; Hokaiwado, Shintaro; Nawa, Makiko; Okamoto, Hayato; Kogiso, Tomohiko; Watabe, Tetsuro; Hattori, Nobutaka

2017-01-01

There are currently several antibody therapies that directly target tumors, and antibody-drug conjugates represent a novel moiety as next generation therapeutics. Here, we used a unique screening probe, DT3C, to identify functional antibodies that recognized surface molecules and functional epitopes, and which provided toxin delivery capability. Accordingly, we generated the 90G4 antibody, which induced DT3C-dependent cytotoxicity in endothelial cells. Molecular analysis revealed that 90G4 recognized CD321, a protein localized at tight junctions. Although CD321 plays a pivotal role in inflammation and lymphocyte trans-endothelial migration, little is known about its mechanism of action in endothelial cells. Targeting of CD321 by the 90G4 immunotoxin induced cell death. Moreover, 90G4 immunotoxin caused cytotoxicity primarily in migratory endothelial cells, but not in those forming sheets, suggesting a critical role for CD321 in tumor angiogenesis. We also found that hypoxia triggered redistribution of CD321 to a punctate localization on the basal side of cells, resulting in functional impairment of tight junctions and increased motility. Thus, our findings raise the intriguing possibility that endothelial CD321 presented cellular localization in tight junction as well as multifunctional dynamics in several conditions, leading to illuminate the importance of widely-expressed CD321 as a potential target for antitumor therapy. PMID:29028806

[Hepatitis-associated aplastic anaemia: clinical characteristics and immunosuppressive therapy outcomes].

PubMed

Yang, W R; Jing, L P; Zhou, K; Peng, G X; Li, Y; Ye, L; Li, Y; Li, J P; Fan, H H; Song, L; Zhao, X; Yang, Y; Zhang, F K; Zhang, L

2016-05-14

To analyze the clinical characteristics and to evaluate immunosuppressive therapy (IST) response and survival in hepatitis-associated aplastic anemia (HAAA). We retrospectively analyzed clinical characteristics, IST response, long-term survival and clonal evolution in 41 HAAA patients, and compared those with age and bone marrow failure matched idiopathic aplastic anemia (IAA) patients. The prevalence of HAAA among cases of SAA was 4.34% (41/944). The proportion of VSAA in HAAA cases was significantly higher than IAA (65.9% vs 39.4%, P=0.001). There was no significant difference in the prevalence of hemorrhage and infections between HAAA and IAA patients, but the duration of infection persistence in HAAA group was much longer than IAA group [21 (4-100) d vs 13 (3-139) d, P=0.048]. The absolute counts of CD3(+) T-cell, CD3(+)CD4(+)T-cell, CD3(+)CD8(+)T-cell and ratio of CD4(+) T-cell/CD8(+) T-cell in HAAA were significant lower than that in IAA patients. However, the percentage of CD3(+)CD8(+)T-cell in HAAA was significant higher than that in IAA (P <0.05). The total response in HAAA and IAA patients treated with IST were 34.1% vs 34.1% (P=1.000), 56.1% vs 53.7% (P=0.787), and 73.2% vs 68.3% (P=0.558) at 3, 6, 12 months after IST, respectively. There were no significant difference in 5-year overall survival and event-free survival between HAAA and IAA patients (90% vs 87.1%, P=0.700; 71.9% vs 62.4%, P=0.450). HAAA was a rare distinct variant of aplastic anemia with more severe bone marrow failure and more severe imbalance of the T cell immune system than IAA. Treatment outcomes were comparable in patients with HAAA and IAA.

Optimization of individualized graft composition: CD3/CD19 depletion combined with CD34 selection for haploidentical transplantation.

PubMed

Huenecke, Sabine; Bremm, Melanie; Cappel, Claudia; Esser, Ruth; Quaiser, Andrea; Bonig, Halvard; Jarisch, Andrea; Soerensen, Jan; Klingebiel, Thomas; Bader, Peter; Koehl, Ulrike

2016-09-01

Excessive T-cell depletion (TCD) is a prerequisite for graft manufacturing in haploidentical stem cell (SC) transplantation by using either CD34 selection or direct TCD such as CD3/CD19 depletion. To optimize graft composition we compared 1) direct or indirect TCD only, 2) a combination of CD3/CD19-depleted with CD34-selected grafts, or 3) TCD twice for depletion improvement based on our 10-year experience with 320 separations in graft manufacturing and quality control. SC recovery was significantly higher (85%, n = 187 vs. 73%, n = 115; p < 0.0001), but TCD was inferior (median log depletion, -3.6 vs. -5.2) for CD3/CD19 depletion compared to CD34 selection, respectively. For end products with less than -2.5 log TCD, a second depletion step led to a successful improvement in TCD. Thawing of grafts showed a high viability and recovery of SCs, but low NK-cell yield. To optimize individualized graft engineering, a calculator was developed to estimate the results of the final graft based on the content of CD34+ and CD3+ cells in the leukapheresis product. Finally, calculated splitting of the starting product followed by CD3/19 depletion together with CD34+ graft manipulation may enable the composition of optimized grafts with high CD34+-cell and minimal T-cell content. © 2016 AABB.

CD147 silencing inhibits tumor growth by suppressing glucose transport in melanoma.

PubMed

Su, Juan; Gao, Tianyuan; Jiang, Minghao; Wu, Lisha; Zeng, Weiqi; Zhao, Shuang; Peng, Cong; Chen, Xiang

2016-10-04

Melanoma is a very malignant disease and there are still no effective treatments. CD147 participates in the carcinogenesis of multiple human cancers and GLUT-1, as a glucose transporter, is associated with tumor growth. However, the function of CD147 and GLUT-1 in melanoma have not been completely understood. Thus, in this study we investigated the expression of CD147 and GLUT-1 in melanoma tissue, which were overexpressed compared with that in nevus tissue. In addition, CD147 and GLUT-1 were co-localized in the cytoplasm of human melanoma A375 cells. Immunoprecipitation proved that CD147 interacted with GLUT-1 at D105-199. Silencing CD147 by specific siRNA could downregulate GLUT-1 level via inhibiting PI3K/Akt signaling and decrease glucose uptake in A375 cells. In vivo experiments also supported that CD147 knockdown suppressed the tumor growth in melanoma subcutaneous mice model, observed by micro PET/CT. Our results could help validate CD147 as a new therapeutic target for treating melanoma.

CD147 silencing inhibits tumor growth by suppressing glucose transport in melanoma

PubMed Central

Su, Juan; Gao, Tianyuan; Jiang, Minghao; Wu, Lisha; Zeng, Weiqi; Zhao, Shuang; Peng, Cong; Chen, Xiang

2016-01-01

Melanoma is a very malignant disease and there are still no effective treatments. CD147 participates in the carcinogenesis of multiple human cancers and GLUT-1, as a glucose transporter, is associated with tumor growth. However, the function of CD147 and GLUT-1 in melanoma have not been completely understood. Thus, in this study we investigated the expression of CD147 and GLUT-1 in melanoma tissue, which were overexpressed compared with that in nevus tissue. In addition, CD147 and GLUT-1 were co-localized in the cytoplasm of human melanoma A375 cells. Immunoprecipitation proved that CD147 interacted with GLUT-1 at D105-199. Silencing CD147 by specific siRNA could downregulate GLUT-1 level via inhibiting PI3K/Akt signaling and decrease glucose uptake in A375 cells. In vivo experiments also supported that CD147 knockdown suppressed the tumor growth in melanoma subcutaneous mice model, observed by micro PET/CT. Our results could help validate CD147 as a new therapeutic target for treating melanoma. PMID:27556188

Improvement in Thermal Stability of Sucralose by γ-Cyclodextrin Metal-Organic Frameworks.

PubMed

Lv, Nana; Guo, Tao; Liu, Botao; Wang, Caifen; Singh, Vikaramjeet; Xu, Xiaonan; Li, Xue; Chen, Dawei; Gref, Ruxandra; Zhang, Jiwen

2017-02-01

To explain thermal stability enhancement of an organic compound, sucralose, with cyclodextrin based metal organic frameworks. Micron and nanometer sized basic CD-MOFs were successfully synthesized by a modified vapor diffusion method and further neutralized with glacial acetic acid. Sucralose was loaded into CD-MOFs by incubating CD-MOFs with sucralose ethanol solutions. Thermal stabilities of sucralose-loaded basic CD-MOFs and neutralized CD-MOFs were investigated using thermogravimetric analysis (TGA), differential scanning calorimetry (DSC) and high performance liquid chromatography with evaporative light-scattering detection (HPLC-ELSD). Scanning electron microscopy (SEM) and powder X-ray diffraction (PXRD) results showed that basic CD-MOFs were cubic crystals with smooth surface and uniform sizes. The basic CD-MOFs maintained their crystalline structure after neutralization. HPLC-ELSD analysis indicated that the CD-MOF crystal size had significant influence on sucralose loading (SL). The maximal SL of micron CD-MOFs (CD-MOF-Micro) was 17.5 ± 0.9% (w/w). In contrast, 27.9 ± 1.4% of sucralose could be loaded in nanometer-sized basic CD-MOFs (CD-MOF-Nano). Molecular docking modeling showed that sucralose molecules preferentially located inside the cavities of γ-CDs pairs in CD-MOFs. Raw sucralose decomposed fast at 90°C, with 86.2 ± 0.2% of the compound degraded within only 1 h. Remarkably, sucralose stability was dramatically improved after loading in neutralized CD-MOFs, with only 13.7 ± 0.7% degradation at 90°C within 24 h. CD-MOFs efficiently incorporated sucralose and maintained its integrity upon heating at elevated temperatures.

Electrical characterization of the temperature dependence in CdTe/CdS heterojunctions deposited in-situ by pulsed laser deposition

NASA Astrophysics Data System (ADS)

Avila-Avendano, Jesus; Quevedo-Lopez, Manuel; Young, Chadwin

2018-02-01

The I-V and C-V characteristics of CdTe/CdS heterojunctions deposited in-situ by Pulsed Laser Deposition (PLD) were evaluated. In-situ deposition enables the study of the CdTe/CdS interface by avoiding potential impurities at the surface and interface as a consequence of exposure to air. The I-V and C-V characteristics of the resulting junctions were obtained at different temperatures, ranging from room temperature to 150 °C, where the saturation current (from 10-8 to 10-4 A/cm2), ideality factor (between 1 and 2), series resistance (from 102 to 105 Ω), built-in potential (0.66-0.7 V), rectification factor (˜106), and carrier concentration (˜1016 cm-3) were obtained. The current-voltage temperature dependence study indicates that thermionic emission is the main transport mechanism at the CdTe/CdS interface. This study also demonstrated that the built-in potential (Vbi) calculated using a thermionic emission model is more accurate than that calculated using C-V extrapolation since C-V plots showed a Vbi shift as a function of frequency. Although CdTe/CdS is widely used for photovoltaic applications, the parameters evaluated in this work indicate that CdTe/CdS heterojunctions could be used as rectifying diodes and junction field effect transistors (JFETs). JFETs require a low PN diode saturation current, as demonstrated for the CdTe/CdS junction studied here.

Preparation of alpha-cyclodextrin-terminated polyrotaxane consisting of beta-cyclodextrins and pluronic as a building block of a biodegradable network.

PubMed

Ooya, Tooru; Ito, Akihiro; Yui, Nobuhiko

2005-05-23

A beta-CD-based biodegradable polyrotaxane was prepared by capping both terminals of polypseudorotaxane consisting of hydrazide-terminated PEG-block-PPG-block-PEG (Pluronic P-105) and beta-CD-succinates with mono-aldehyde alpha-CDs. By decreasing pH, the fluorescent intensity of TNS was increased with time, indicating cleavage of the terminal hydrazone bonds followed by beta-CD-succinate release. The terminal alpha-CD moieties of the polyrotaxane are useful for self-assembled formation with some guest molecules. [Diagram: see text

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

The sensitivity of human mesenchymal stem cells to vibration and cold storage conditions representative of cold transportation

PubMed Central

Nikolaev, N. I.; Liu, Y.; Hussein, H.; Williams, D. J.

2012-01-01

In the current study, the mechanical and hypothermic damage induced by vibration and cold storage on human mesenchymal stem cells (hMSCs) stored at 2–8°C was quantified by measuring the total cell number and cell viability after exposure to vibration at 50 Hz (peak acceleration 140 m s−2 and peak displacement 1.4 mm), 25 Hz (peak acceleration 140 m s−2, peak displacement 5.7 mm), 10 Hz (peak acceleration 20 m s−2, peak displacement 5.1 mm) and cold storage for several durations. To quantify the viability of the cells, in addition to the trypan blue exclusion method, the combination of annexin V-FITC and propidium iodide was applied to understand the mode of cell death. Cell granularity and a panel of cell surface markers for stemness, including CD29, CD44, CD105 and CD166, were also evaluated for each condition. It was found that hMSCs were sensitive to vibration at 25 Hz, with moderate effects at 50 Hz and no effects at 10 Hz. Vibration at 25 Hz also increased CD29 and CD44 expression. The study further showed that cold storage alone caused a decrease in cell viability, especially after 48 h, and also increased CD29 and CD44 and attenuated CD105 expressions. Cell death would most likely be the consequence of membrane rupture, owing to necrosis induced by cold storage. The sensitivity of cells to different vibrations within the mechanical system is due to a combined effect of displacement and acceleration, and hMSCs with a longer cold storage duration were more susceptible to vibration damage, indicating a coupling between the effects of vibration and cold storage. PMID:22628214

Apigenin inhibited hypoxia induced stem cell marker expression in a head and neck squamous cell carcinoma cell line.

PubMed

Ketkaew, Yuwaporn; Osathanon, Thanaphum; Pavasant, Prasit; Sooampon, Sireerat

2017-02-01

Cancer stem cells contribute to tumor recurrence, and a hypoxic environment is critical for maintaining cancer stem cells. Apigenin is a natural product with anticancer activity. However, the effect of apigenin on cancer stem cells remains unclear. Our aim was to investigate the effect of apigenin on cancer stem cell marker expression in head and neck squamous cell carcinoma cells under hypoxia. We used three head and neck squamous cell carcinoma cell lines; HN-8, HN-30, and HSC-3. The mRNA expression of cancer stem cell markers was determined by semiquantitative RT-PCR and Real-time PCR. The cytotoxic effect of apigenin was determined by MTT colorimetric assay. Flow cytometry was used to reveal the number of cells expressing cancer stem cell surface markers. HN-30 cells, a cancer cell line from the pharynx, showed the greatest response to hypoxia by increasing their expression of CD44, CD105, NANOG, OCT-4, REX-1, and VEGF. Apigenin significantly decreased HN-30 cell viability in dose- and time-dependent manners. In addition, 40μM apigenin significantly down-regulated the mRNA expression of CD44, NANOG, and CD105. Consistent with these results, the hypoxia-induced increase in CD44 + cells, CD105 + cells, and STRO-1 + cells was significantly abolished by apigenin. Apigenin suppresses cancer stem cell marker expression and the number of cells expressing cell surface markers under hypoxia. Copyright © 2016 Elsevier Ltd. All rights reserved.

CD73 specific siRNA loaded chitosan lactate nanoparticles potentiate the antitumor effect of a dendritic cell vaccine in 4T1 breast cancer bearing mice.

PubMed

Jadidi-Niaragh, Farhad; Atyabi, Fatemeh; Rastegari, Ali; Kheshtchin, Nasim; Arab, Samaneh; Hassannia, Hadi; Ajami, Maryam; Mirsanei, Zahra; Habibi, Sima; Masoumi, Farimah; Noorbakhsh, Farshid; Shokri, Fazel; Hadjati, Jamshid

2017-01-28

The efficacy of conventional anti-tumor immunotherapeutic approaches is markedly affected by the immunosuppressive microenvironment of tumor. Since adenosine is one of the main orchestra leaders in immunosuppression symphony of tumor, targeting its producing molecules such as CD73 can help to achieve a better clinical outcome following conventional cancer immunotherapeutic approaches. In the present study, we evaluated the efficacy of CD73-specific siRNA-loaded chitosan-lactate nanoparticles (ChLa NPs) in combination with tumor lysate pulsed dendritic cells (DCs) vaccine in treatment of 4T1 (murine derived) breast cancer bearing mice. Our results showed that intravenous administration of CD73-specific siRNA-loaded NPs led to reduced expression of CD73 in tumor cells which was associated with decreased tumor growth and metastasis, and improved mice survival. Furthermore, we found that the mechanism by which combination therapy inhibits tumor growth is in part related to downregulation of regulatory T (Treg), myeloid derived suppressor cells (MDSCs), and tumor associated macrophages, an augmented CTL effector function, improved proliferation status of T cells, increased production of inflammatory cytokines interferon (IFN)-γ and interleukin (IL)-17 and reduced levels of IL-10. Moreover, this treatment protocol attenuated the expression and activities of matrix metalloproteinases (MMPs) 2 and 9 which could be associated to the prevention of lung metastasis. In conclusion, our findings indicate that the use of CD73-specific siRNA-loaded NPs provides an immune potentiating function, thereby improves the efficacy of DC based cancer immunotherapy. Copyright © 2016 Elsevier B.V. All rights reserved.

Cadmium dynamics in the rhizosphere and Cd uptake of different plant species evaluated by a mechanistic model.

PubMed

Stritsis, Christos; Steingrobe, Bernd; Claassen, Norbert

2014-01-01

Maize, sunflower,flax, and spinach differed in the accumulation of Cd when grown on a Cd contaminated soil. This was mainly due to the different Cd net influx, In, that varied among species by a factor of up to 30. The objective of this study was to find possible reasons for the different Cd In by using a mechanistic model. After 14 days of Cd uptake the model calculated only a small Cd depletion at the root surface, e.g. from 0.22 mumol L(-1) down to 0.19 mumol L(-1) for maize and from 0.48 mumol L(-1) down to 0.35 mumol L(-1)for spinach. Even so the model always overestimated the Cd I(n), for spinach by a factor of 1.5 and for maize by a factor of 10. Only simulating a decrease of C(Li) or the root absorbing power, alpha, by 40% to 90% gave an agreement of calculated and measured I(n),. This may be interpreted as that about 40% in the case of spinach and 90% in the case of maize of the Cd in soil solution were not accessible for plant uptake. The high sensitivity to alpha also shows that not the Cd transport to the root but alpha was limiting the step for Cd uptake.

CD34+CD38-CD123+ Cells Are Present in Virtually All Acute Myeloid Leukaemia Blasts: A Promising Single Unique Phenotype for Minimal Residual Disease Detection.

PubMed

Al-Mawali, Adhra; Pinto, Avinash Daniel; Al-Zadjali, Shoaib

In CD34-positive acute myeloid leukaemia (AML), the leukaemia-initiating event likely takes place in the CD34+CD38- cell compartment. CD123 has been shown to be a unique marker of leukaemic stem cells within the CD34+CD38- compartment. The aim of this study was to identify the percentage of CD34+CD38-CD123+ cells in AML blasts, AML CD34+CD38- stem cells, and normal and regenerating bone marrow CD34+CD38- stem cells from non-myeloid malignancies. Thirty-eight adult de novo AML patients with intention to treat were enrolled after the application of inclusion criteria from February 2012 to February 2017. The percentage of the CD34+CD38-CD123+ phenotype in the blast population at diagnosis was determined using a CD45-gating strategy and CD34+ backgating by flow cytometry. We studied the CD34+CD38-CD123+ fraction in AML blasts at diagnosis, and its utility as a unique phenotype for minimal residual disease (MRD) of AML patients. CD123+ cells were present in 97% of AML blasts in patients at diagnosis (median 90%; range 21-99%). CD123+ cells were also present in 97% of the CD34+CD38- compartment (median 0.8164%, range 0.0262-39.7%). Interestingly, CD123 was not present in normal and regenerating CD34+CD38- bone marrow stem cells (range 0.002- 0.067 and 0.004-0.086, respectively). The CD34+CD38-CD123+ phenotype is present in virtually all AML blasts and it may be used as a unique single phenotype for MRD detection in AML patients. © 2017 The Author(s) Published by S. Karger AG, Basel.

Theranostic Unimolecular Micelles Based on Brush-Shaped Amphiphilic Block Copolymers for Tumor-Targeted Drug Delivery and Positron Emission Tomography Imaging

PubMed Central

2015-01-01

Brush-shaped amphiphilic block copolymers were conjugated with a monoclonal antibody against CD105 (i.e., TRC105) and a macrocyclic chelator for 64Cu-labeling to generate multifunctional theranostic unimolecular micelles. The backbone of the brush-shaped amphiphilic block copolymer was poly(2-hydroxyethyl methacrylate) (PHEMA) and the side chains were poly(l-lactide)-poly(ethylene glycol) (PLLA-PEG). The doxorubicin (DOX)-loaded unimolecular micelles showed a pH-dependent drug release profile and a uniform size distribution. A significantly higher cellular uptake of TRC105-conjugated micelles was observed in CD105-positive human umbilical vein endothelial cells (HUVEC) than nontargeted micelles due to CD105-mediated endocytosis. In contrast, similar and extremely low cellular uptake of both targeted and nontargeted micelles was observed in MCF-7 human breast cancer cells (CD105-negative). The difference between the in vivo tumor accumulation of 64Cu-labeled TRC105-conjugated micelles and that of nontargeted micelles was studied in 4T1 murine breast tumor-bearing mice, by serial positron emission tomography (PET) imaging and validated by biodistribution studies. These multifunctional unimolecular micelles offer pH-responsive drug release, noninvasive PET imaging capability, together with both passive and active tumor-targeting abilities, thus making them a desirable nanoplatform for cancer theranostics. PMID:24628452

Transplantation of mesenchymal stem cells cultured on biomatrix support induces repairing of digestive tract defects, in animal model.

PubMed

Sîrbu-Boeţi, Mirela-Patricia; Chivu, Mihaela; Pâslaru, Liliana Livia; Efrimescu, C; Herlea, V; Pecheanu, C; Moldovan, Lucia; Dragomir, Laura; Bleotu, Coralia; Ciucur, Elena; Vidulescu, Cristina; Vasilescu, Mihaela; Boicea, Anişoara; Mănoiu, S; Ionescu, M I; Popescu, I

2009-01-01

Transplanted mesenchymal stem cells (MSCs) appear to play a significant role in adult tissue repair. The aim of this research was to obtain MSCs enriched, three dimensional (3D) patches for transplant, and to test their ability to induce repair of iatrogenic digestive tract defects in rats. MSCs were obtained from human and rat bone marrow, cultured in vitro, and seeded in a collagen-agarose scaffold, where they showed enhanced viability and proliferation. The phenotype of the cultured cells was representative for MSCs (CD105+, CD90+, and CD34-, CD45-, CD3-, CD14-). The 3D patch was obtained by laying the MSCs enriched collagen-agarose scaffold on a human or swine aortic fragment. After excision of small portions of the rat digestive tract, the 3D patches were sutured at the edge of the defect using micro-surgical techniques. The rats were sacrificed at time-points and the regeneration of the digestive wall was investigated by immunofluorescence, light and electron microscopy. The MSCs enriched 3D patches were biocompatible, biodegradable, and prompted the regeneration of the four layers of the stomach and intestine wall in rats. Human cells were identified in the rat regenerated digestive wall as a hallmark of the transplanted MSCs. For the first time we constructed 3D patches made of cultured bone marrow MSCs, embedded into a collagen-rich biomatrix, on vascular bio-material support, and transplanted them in order to repair iatrogenic digestive tract defects. The result was a complete repair with preservation of the four layered structure of the digestive wall.

[Aging and influence of inversion of the CD4:CD8 ratio in the incidence of co-morbidities and mortality in a cohort of patients infected with human immunodeficiency virus].

PubMed

Cervero, Miguel; Torres, Rafael; Agud, Jose Luis; Pastor, Susana; Jusdado, Juan José

2016-03-04

It has been postulated that the inversion of the CD4:CD8 ratio as a hallmark of immunosenescence can be an independent factor that can herald the risk of co-morbidities. We studied the influence of aging and inversion of the CD4:CD8 ratio in the incidence of comorbidities and mortality in the cohort of Hosptital Severo Ochoa. We analyzed the differences in the incidence rates of age-adjusted morbidities and evaluated the inversion of the CD4:CD8 ratio as predictor of mortality and development of comorbidities. Age was associated with an increased incidence rate of diabetes mellitus, fractures, COPD and non-AIDS malignancies. We found an increased incidence rate of non-AIDS clinical events (OR 2.25; 95% CI 1.025-4.94) and AIDS events (OR 3.48; 95% CI 1.58-7.64) in individuals with CD4:CD8 ratio<0.7. Moreover, patients with a CD4:CD8 ratio<0.7 ratio had a higher risk of mortality (OR 5.96; 95% CI 0.73 to 48.40). It is important to detect and prevent non-AIDS comorbidities in the presence of a CD4:CD8 ratio<0.7. Copyright © 2015 Elsevier España, S.L.U. All rights reserved.

Clinical features of Crohn disease concomitant with ankylosing spondylitis

PubMed Central

Liu, Song; Ding, Jie; Wang, Meng; Zhou, Wanqing; Feng, Min; Guan, Wenxian

2016-01-01

Abstract Extraintestinal manifestations (EIMs) cause increased morbidity and decreased quality of life in Crohn disease (CD). Ankylosing spondylitis (AS) belongs to EIMs. Very little is known on the clinical features of CD concomitant with AS. This study is to investigate the clinical features of CD patients with AS. We retrospectively collected all CD patients with AS in our hospital, and established a comparison group (CD without AS) with age, sex, and duration of Crohn disease matched. Clinical information was retrieved for comparison. Eight CD + AS patients were identified from 195 CD patients. Sixteen CD patients were randomly selected into comparison group. All CD + AS patients were male, HLA-B27 (+), and rheumatoid factor (−) with an average age of 40.8 ± 4.52 years. Significant correlation between disease activity of CD and AS was revealed (r = 0.857, P = 0.011). Significant correlation between disease activity of CD and functional limitation associated with AS was identified (r = 0.881, P < 0.01). C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), and globulin were positively correlated to Crohn disease activity index (CDAI), Bath AS disease activity index, and Bath AS functional index(BASFI) scores (r = 0.73–0.93, P < 0.05). Albumin was negatively associated with CDAI and BASFI (r = −0.73 to −0.91, P < 0.05). The ratio of albumin to globulin (Alb/Glo) was significantly related to all 3 scores (r = −0.81 to −0.91, P < 0.05). Male predominance with a 4.12% concomitant incidence of AS is observed in CD patients. Disease activity of CD correlates with disease activity of AS and functional limitation caused by AS. CRP, ESR, and Alb/Glo may serve as biomarkers for disease activity and functional limitation in CD patients concomitant with AS, although future studies are expected. PMID:27428240

Clinical features of Crohn disease concomitant with ankylosing spondylitis: A preliminary single-center study.

PubMed

Liu, Song; Ding, Jie; Wang, Meng; Zhou, Wanqing; Feng, Min; Guan, Wenxian

2016-07-01

Extraintestinal manifestations (EIMs) cause increased morbidity and decreased quality of life in Crohn disease (CD). Ankylosing spondylitis (AS) belongs to EIMs. Very little is known on the clinical features of CD concomitant with AS. This study is to investigate the clinical features of CD patients with AS.We retrospectively collected all CD patients with AS in our hospital, and established a comparison group (CD without AS) with age, sex, and duration of Crohn disease matched. Clinical information was retrieved for comparison.Eight CD + AS patients were identified from 195 CD patients. Sixteen CD patients were randomly selected into comparison group. All CD + AS patients were male, HLA-B27 (+), and rheumatoid factor (-) with an average age of 40.8 ± 4.52 years. Significant correlation between disease activity of CD and AS was revealed (r = 0.857, P = 0.011). Significant correlation between disease activity of CD and functional limitation associated with AS was identified (r = 0.881, P < 0.01). C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), and globulin were positively correlated to Crohn disease activity index (CDAI), Bath AS disease activity index, and Bath AS functional index(BASFI) scores (r = 0.73-0.93, P < 0.05). Albumin was negatively associated with CDAI and BASFI (r = -0.73 to -0.91, P < 0.05). The ratio of albumin to globulin (Alb/Glo) was significantly related to all 3 scores (r = -0.81 to -0.91, P < 0.05).Male predominance with a 4.12% concomitant incidence of AS is observed in CD patients. Disease activity of CD correlates with disease activity of AS and functional limitation caused by AS. CRP, ESR, and Alb/Glo may serve as biomarkers for disease activity and functional limitation in CD patients concomitant with AS, although future studies are expected.

Selective Expansion of Memory CD4+ T cells By Mitogenic Human CD28 Generates Inflammatory Cytokines and Regulatory T cells

PubMed Central

Singh, Manisha; Basu, Sreemanti; Camell, Christina; Couturier, Jacob; Nudelman, Rodolfo J.; Medina, Miguel A.; Rodgers, John R.; Lewis, Dorothy E.

2009-01-01

Co-stimulatory signals are important for development of effector and regulatory T cells. In this case, CD28 signaling is usually considered inert in the absence of signaling through the TCR. By contrast, mitogenic rat CD28 mAbs reportedly expand regulatory T cells without TCR stimulation. We found that a commercially available human CD28 mAb (ANC28) stimulated PBMCs without TCR co-ligation or cross-linking; ANC28 selectively expanded CD4+CD25+FoxP3−(T effector) and CD4+CD25+FoxP3+ (Treg) cells. ANC28 stimulated the CD45RO+ CD4+ (memory) population whereas CD45RA+CD4+ (naïve) cells did not respond. ANC28 also induced inflammatory cytokines. Treg induced by ANC28 retain the Treg phenotype longer than did co-stimulated Treg. Treg induced by ANC28 suppressed CD25− T cells through a contact-dependent mechanism. Purity influenced the response of CD4+CD25+ cells because bead-purified CD4+CD25+ cells (85–90% pure) responded strongly to ANC28, whereas 98% pure FACS-sorted CD4+CD25 bright (T-reg) did not respond. Purified CD4+CD25int cells responded similarly to the bead-purified CD4+CD25+ cells. Thus, pre-activated CD4+ T cells (CD25int) respond to ANC28 rather than Treg (CD25bright). The ability of ANC28 to expand both effectors producing inflammatory cytokines as well as suppressive regulatory T cells might be useful for ex vivo expansion of therapeutic T cells. PMID:18446791

Evaluation of automated repetitive-sequence-based PCR (DiversiLab) compared to PCR ribotyping for rapid molecular typing of community- and nosocomial-acquired Clostridium difficile.

PubMed

Church, Deirdre L; Chow, Barbara L; Lloyd, Tracie; Gregson, Daniel B

2011-06-01

Automated repetitive PCR (rep-PCR; DiversiLab) was compared to PCR ribotyping of the 16S-23S RNA intergenic spacer of Clostridium difficile (CD) as the "gold standard" method for CD typing. PCR products were separated on DiversiLab LabChips (bioMérieux, St. Laurent, Quebec, Canada) utilizing a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA) operating the DiversiLab v1.4 assay. Bioanalyzer data were exported to a secure DiversiLab website and analyzed with DiversiLab v3.4 software. Replicability of each method was verified by confirming that the 5 CD reference strains (RS) formed distinct clusters (CD4, CD6, VL0047, VL0013 [ribotype 027], VL0018 [ribotype 001]) by both typing methods. Ninety randomly selected clinical isolates (CS) were analyzed by both methods: 49 from community-acquired and 41 from hospital-acquired cases. A similarity index (SI) of ≥90% was used to define clusters when comparing the known RS cluster to the PCR ribotyping and rep-PCR patterns of CS. Fourteen different PCR-ribotype clusters were identified, but most CS formed 4 major clusters (i.e., CD4 [15/90; 17%], CD6 [17%], 027 [12%], and 001 [9%]). A total of 7 rep-PCR types were identified, but most CS formed 2 major rep-PCR clusters (i.e., CD4 [29/90; 32%] and CD6 [23%]); several PCR ribotypes occurred within a single rep-PCR cluster. Rep-PCR did not distinguish 027 or 001 isolates; i) 027 RS strain did not cluster, ii) eleven 027 CS strains clustered as CD4, iii) no 027 CS strains clustered with the 027 RS, and iv) only 2 001 CS clustered with the RS. Agreement between the PCR-ribotype and rep-PCR clusters only occurred for 35/90 (39%) of the CS using a rep-PCR SI of ≥90%. Rep-PCR time to results was similar, but the annual costs of routinely using this method are 32% higher than PCR ribotyping. Routine use of rep-PCR for CD typing is limited by its lack of definitive separation of the hypertoxigenic 027 or 001 outbreak CD strains. Copyright © 2011 Elsevier Inc. All rights reserved.

CdO nanorods and Cd(OH)2/Ag core/satellite nanorods: Rapid and efficient sonochemical synthesis, characterization and their magnetic properties.

PubMed

Abbas, Mohamed; Tawfik, Wael; Chen, Jiangang

2018-01-01

We have designed an efficient and direct sonochemical method for the facile synthesis of Cd(OH) 2 , CdO, and Cd(OH) 2 /Ag core/satellite nanorods. A Cd(OH) 2 nanorod was synthesized with a one-pot, environmentally-friendly aqueous sonochemical reaction, followed by calcination at 500°C to produce CdO nanorods. Thirty minutes of re-ultrasonicated CdO nanorods in the presence of the Ag precursor was sufficient for phase transformation from the cubic structure of CdO to the monoclinic crystalline structure of Cd(OH) 2 , accompanied by deposition of Ag nanodots on the surface to form Cd(OH) 2 /Ag core/satellite nanorods. X-ray diffraction, transmission electron microscopy, high-resolution transmission electron microscopy, energy-dispersive spectroscopy, N 2 Brunauer-Emmett-Teller adsorption-desorption, and Fourier-transform infrared spectroscopy measurements confirmed the successful formation of the various phases and the unique morphology of the nanorods/satellites. We also measured the magnetic properties using a vibrating sample magnetometer at room temperature, and the produced nanorods showed weak unsaturated ferromagnetic properties with a magnetic moment values of 0.105 and 0.076emu/g for CdO and Cd(OH) 2 /Ag NRs, respectively. In conclusion, our one-pot, cost-effective, sonochemical approach holds promise for the synthesis of various oxides and core/satellite nanoparticles. Copyright © 2017 Elsevier B.V. All rights reserved.

High Aldehyde Dehydrogenase Activity Identifies a Subset of Human Mesenchymal Stromal Cells with Vascular Regenerative Potential.

PubMed

Sherman, Stephen E; Kuljanin, Miljan; Cooper, Tyler T; Putman, David M; Lajoie, Gilles A; Hess, David A

2017-06-01

During culture expansion, multipotent mesenchymal stromal cells (MSCs) differentially express aldehyde dehydrogenase (ALDH), an intracellular detoxification enzyme that protects long-lived cells against oxidative stress. Thus, MSC selection based on ALDH-activity may be used to reduce heterogeneity and distinguish MSC subsets with improved regenerative potency. After expansion of human bone marrow-derived MSCs, cell progeny was purified based on low versus high ALDH-activity (ALDH hi ) by fluorescence-activated cell sorting, and each subset was compared for multipotent stromal and provascular regenerative functions. Both ALDH l ° and ALDH hi MSC subsets demonstrated similar expression of stromal cell (>95% CD73 + , CD90 + , CD105 + ) and pericyte (>95% CD146 + ) surface markers and showed multipotent differentiation into bone, cartilage, and adipose cells in vitro. Conditioned media (CDM) generated by ALDH hi MSCs demonstrated a potent proliferative and prosurvival effect on human microvascular endothelial cells (HMVECs) under serum-free conditions and augmented HMVEC tube-forming capacity in growth factor-reduced matrices. After subcutaneous transplantation within directed in vivo angiogenesis assay implants into immunodeficient mice, ALDH hi MSC or CDM produced by ALDH hi MSC significantly augmented murine vascular cell recruitment and perfused vessel infiltration compared with ALDH l ° MSC. Although both subsets demonstrated strikingly similar mRNA expression patterns, quantitative proteomic analyses performed on subset-specific CDM revealed the ALDH hi MSC subset uniquely secreted multiple proangiogenic cytokines (vascular endothelial growth factor beta, platelet derived growth factor alpha, and angiogenin) and actively produced multiple factors with chemoattractant (transforming growth factor-β, C-X-C motif chemokine ligand 1, 2, and 3 (GRO), C-C motif chemokine ligand 5 (RANTES), monocyte chemotactic protein 1 (MCP-1), interleukin [IL]-6, IL-8) and matrix-modifying functions (tissue inhibitor of metalloprotinase 1 & 2 (TIMP1/2)). Collectively, MSCs selected for ALDH hi demonstrated enhanced proangiogenic secretory functions and represent a purified MSC subset amenable for vascular regenerative applications. Stem Cells 2017;35:1542-1553. © 2017 AlphaMed Press.

Immune activation underlies a sustained clinical response to Yttrium-90 radioembolisation in hepatocellular carcinoma.

PubMed

Chew, Valerie; Lee, Yun Hua; Pan, Lu; Nasir, Nurul J M; Lim, Chun Jye; Chua, Camillus; Lai, Liyun; Hazirah, Sharifah Nur; Lim, Tony Kiat Hon; Goh, Brian K P; Chung, Alexander; Lo, Richard H G; Ng, David; Filarca, Rene L F; Albani, Salvatore; Chow, Pierce K H

2018-02-13

Yttrium-90 (Y90)-radioembolisation (RE) significantly regresses locally advanced hepatocellular carcinoma and delays disease progression. The current study is designed to deeply interrogate the immunological impact of Y90-RE, which elicits a sustained therapeutic response. Time-of-flight mass cytometry and next-generation sequencing (NGS) were used to analyse the immune landscapes of tumour-infiltrating leucocytes (TILs), tumour tissues and peripheral blood mononuclear cells (PBMCs) at different time points before and after Y90-RE. TILs isolated after Y90-RE exhibited signs of local immune activation: higher expression of granzyme B (GB) and infiltration of CD8 + T cells, CD56 + NK cells and CD8 + CD56 + NKT cells. NGS confirmed the upregulation of genes involved in innate and adaptive immune activation in Y90-RE-treated tumours. Chemotactic pathways involving CCL5 and CXCL16 correlated with the recruitment of activated GB + CD8 + T cells to the Y90-RE-treated tumours. When comparing PBMCs before and after Y90-RE, we observed an increase in tumour necrosis factor-α on both the CD8 + and CD4 + T cells as well as an increase in percentage of antigen-presenting cells after Y90-RE, implying a systemic immune activation. Interestingly, a high percentage of PD-1 + /Tim-3 + CD8 + T cells coexpressing the homing receptors CCR5 and CXCR6 denoted Y90-RE responders. A prediction model was also built to identify sustained responders to Y90-RE based on the immune profiles from pretreatment PBMCs. High-dimensional analysis of tumour and systemic immune landscapes identified local and systemic immune activation that corresponded to the sustained response to Y90-RE. Potential biomarkers associated with a positive clinical response were identified and a prediction model was built to identify sustained responders prior to treatment. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

The effect of Clostridium difficile infection on cardiac surgery outcomes.

PubMed

Lemaire, Anthony; Dombrovskiy, Viktor; Batsides, George; Scholz, Peter; Solina, Al; Brownstone, Nicholas; Spotnitz, Alan; Lee, Leonard Y

2015-02-01

Clostridium difficile (CD) is a common cause of healthcare-associated infectious colitis that complicates about 1% of all hospital stays in the U.S. The impact of CD on outcomes after coronary artery bypass grafting (CABG) and valvular surgery (VS) is not well known. The Nationwide Inpatient Sample (2002-2009) was queried to identify CABG and VS patients utilizing International Classification of Diseases, Ninth Revision, Clinical Modification (ICD-9-CM) diagnosis codes. Rates of CD, post-operative endocarditis and mediastinitis, hospital mortality rate, and resource utilization were evaluated. We identified 421,294 and 90,923 patients of age 40 yrs and older who underwent CABG and VS, respectively. The CD infection was more likely to develop in patients undergoing VS than in those having CABG (odds ratio [OR] 1.8; 95% confidence interval [CI] 1.64-1.92) and was more likely after urgent or emergency admission than after elective admission (OR 1.8; 95% CI 1.68-1.94). There was a greater likelihood of mediastinitis in patients with CD after CABG than in non-complicated cases without CD, both by univariable (OR 6.0; 95% CI 3.07-11.62) and multivariable analysis with adjustment for patient age, gender, race, type of admission, and co-morbidities (OR 3.1; 95% CI 1.49-6.51). The infection thus was most likely a result of the antibiotics used to treat mediastinitis, as the patients treated for mediastinitis were most likely to develop CD. There was a significant association in patients with CD and endocarditis who underwent VS but not in patients who did not have CD. The CD infection in these patients thus was most likely a result of the antibiotics used to treat endocarditis. Endocarditis and CD developed 3.2 times (95% CI 2.65-3.97) as often as in patients without CD, a finding that was confirmed by multivariable analysis (OR 2.2; 95% CI 1.70-2.84). At the same time, in patients having VS, there was no significant association of CD and mediastinitis. Clostridium difficile infection affected the hospital mortality rate significantly after both CABG (OR 2.0; 95% CI 1.65-2.35) and VS (OR 1.9; 95% CI 1.51-2.39). Development of CD increased median hospital length of stay and cost dramatically after both CABG (from 7 d to 19 d and from $33,105 to $65,535, respectively; p<0.0001 for both) and VS (from 8 d to 24 d and from $41,876 to $95,699, respectively; p<0.0001 for both). The development of CD worsened significantly the outcomes of adult patients undergoing cardiac surgery. There was a greater risk of CD in patients with either mediastinitis or endocarditis. The infection was associated with a higher hospital mortality rate, longer hospital stays, and greater cost after both CABG and VS.

Improved Therapy by Pretargeted Radioimmunotherapy of Non-Hodgkin Lymphoma with a New Recombinant, Trivalent, Anti-CD20, Bispecific Antibody

PubMed Central

Sharkey, Robert M.; Karacay, Habibe; Litwin, Samuel; Rossi, Edmund A.; McBride, William J.; Chang, Chein-Hsing; Goldenberg, David M.

2008-01-01

We examined whether a pretargeting method using a new, recombinant anti-CD20 bispecific antibody (bsMAb) followed by 90Y-DOTA-peptide could reduce hematological toxicity yet improve therapeutic responses, compared to conventional 90Y-anti-CD20 IgG and a chemically-conjugated bsMAb. TF4, a humanized, tri-Fab, bsMAb with 2 Fabs binding CD20 and 1 Fab binding HSG (histamine-succinyl-glycine), developed by the Dock-and-Lock (DNL) method, was tested in nude mice with Ramos B-cell lymphomas. Optimal pretargeting required a 29-h interval between TF4 and 90Y-DOTA-HSG, and 20-fold more moles of TF4. TF4 cleared more rapidly from the blood than anti-CD20 IgG, with early processing in the liver, spleen and kidneys. At 24 h, TF4 improved tumor uptake of 111In-HSG-peptide 2.6-fold (13% vs 5% injected-dose/g) and enhanced tumor/blood ratios more than 45-fold (770 vs 17), compared to an anti-CD20 Fab x anti-HSG Fab chemical conjugate, and by 1.6-fold (9.0% vs 5.6% injected-dose/g) and 1600-fold (522 vs 0.32), respectively, compared to radiolabeled anti-CD20 IgG. A severe (≥90%) and prolonged reduction of white blood cells was observed at the maximum dose of 90Y-anti-CD20 IgG, whereas pretargeting resulted in a ≤60% transient drop. TF4-pretargeting resulted in highly significant improvement in survival, curing 33-90% of the animals, even at relatively low doses, while most tumors progressed quickly without cures with 90Y-anti-CD20 IgG. These results indicate an improved therapeutic index with pretargeting radioimmunotherapy using a DNL-constructed tri-Fab, bsMAb, as compared to conventional therapy with directly-radiolabeled antibody or with a chemically-conjugated bsMAb. These encouraging results prompt testing of these constructs for pretargeting radioimmunotherapy in patients. PMID:18593929

Comparative magnitude and kinetics of human cytomegalovirus-specific CD4⁺ and CD8⁺ T-cell responses in pregnant women with primary versus remote infection and in transmitting versus non-transmitting mothers: Its utility for dating primary infection in pregnancy.

PubMed

Fornara, Chiara; Furione, Milena; Arossa, Alessia; Gerna, Giuseppe; Lilleri, Daniele

2016-07-01

To discriminate between primary (PI) and remote (RI) human cytomegalovirus (HCMV) infection, several immunological parameters were monitored for a 2-year period in 53 pregnant women with PI, and 33 pregnant women experiencing HCMV PI at least 5 years prior. Cytokine (IFN-γ and IL-2) production by and phenotype (effector/memory CD45RA(+)) of HCMV-specific CD4(+) and CD8(+) T-cells as well as the lymphoproliferative responses (LPR) were evaluated, with special reference to the comparison between a group of women transmitting (T) and a group of non-transmitting (NT) the infection to fetus. While HCMV-specific CD4(+) T-cells reached at 90 days post-infection (p.i.) values comparable to RI, CD8(+) T-cells reached at 60 days p.i. levels significantly higher and persisting throughout the entire follow-up. Instead, IL-2 production and lymphoproliferative responses were lower in PI than RI for the entire follow-up period. Effector memory CD45RA(+) CD4(+) and CD8(+) HCMV-specific T-cells increased until 90 days p.i., reaching and maintaining levels higher than RI. The comparison between T and NT women showed that, at 30 days p.i., in NT women there was a significantly higher IL-2 production by HCMV-specific CD4(+) T-cells, and at 60 days p.i. a significantly higher frequency of both specific CD4(+) and CD8(+) CD45RA(+) T-cells. HCMV T-cell response appears to correlate with virus transmission to fetus and some parameters (CD4(+) lymphoproliferation, and frequency of HCMV-specific CD8(+) IL2(+) T-cells) may help in dating PI during pregnancy. © 2015 Wiley Periodicals, Inc.

Spectrum of benzo[a]pyrene-induced mutations in the Pig-a gene of L5178YTk+/- cells identified with next generation sequencing.

PubMed

Revollo, Javier; Wang, Yiying; McKinzie, Page; Dad, Azra; Pearce, Mason; Heflich, Robert H; Dobrovolsky, Vasily N

2017-12-01

We used Sanger sequencing and next generation sequencing (NGS) for analysis of mutations in the endogenous X-linked Pig-a gene of clonally expanded L5178YTk +/- cells. The clones developed from single cells that were sorted on a flow cytometer based upon the expression pattern of the GPI-anchored marker, CD90, on their surface. CD90-deficient and CD90-proficient cells were sorted from untreated cultures and CD90-deficient cells were sorted from cultures treated with benzo[a]pyrene (B[a]P). Pig-a mutations were identified in all clones developed from CD90-deficient cells; no Pig-a mutations were found in clones of CD90-proficient cells. The spectrum of B[a]P-induced Pig-a mutations was dominated by basepair substitutions, small insertions and deletions at G:C, or at sequences rich in G:C content. We observed high concordance between Pig-a mutations determined by Sanger sequencing and by NGS, but NGS was able to identify mutations in samples that were difficult to analyze by Sanger sequencing (e.g., mixtures of two mutant clones). Overall, the NGS method is a cost and labor efficient high throughput approach for analysis of a large number of mutant clones. Published by Elsevier B.V.

Functional characteristics of mesenchymal stem cells derived from the adipose tissue of a patient with achondroplasia.

PubMed

Park, Jeong-Ran; Lee, Hanbyeol; Kim, Chung-Hyo; Hong, Seok-Ho; Ha, Kwon-Soo; Yang, Se-Ran

2016-05-01

Mesenchymal stem cells (MSCs) can be isolated from various tissues including bone marrow, adipose tissue, skin dermis, and umbilical Wharton's jelly as well as injured tissues. MSCs possess the capacity for self-renewal and the potential for differentiation into adipogenic, osteogenic, and chondrogenic lineages. However, the characteristics of MSCs in injured tissues, such as achondroplasia (ACH), are not well known. In this study, we isolated MSCs from human subcutaneous adipose (ACH-SAMSCs) tissue and circumjacent human adipose tissue of the cartilage (ACH-CAMSCs) from a patient with ACH. We then analyzed the characterization of ACH-SAMSCs and ACH-CAMSCs, compared with normal human dermis-derived MSCs (hDMSCs). In flow cytometry analysis, the isolated ACH-MSCs expressed low levels of CD73, CD90, and CD105, compared with hDMSCs. Moreover, both ACH- SAMSCs and ACH-CAMSCs had constitutionally overactive fibroblast growth factor receptor 3 (FGFR3) and exhibited significantly reduced osteogenic differentiation, compared to enhanced adipogenic differentiation. The activity of extracellular signal-regulated kinases 1/2 (ERK1/2) and p38 mitogen-activated protein kinases (p38 MAPK) was increased in ACH-MSCs. In addition, the efficacy of osteogenic differentiation was slightly restored in osteogenic differentiation medium with MAPKs inhibitors. These results suggest that they play essential roles in MSC differentiation toward adipogenesis in ACH pathology. In conclusion, the identification of the characteristics of ACH-MSCs and the favoring of adipogenic differentiation via the FGFR3/MAPK axis might help to elucidate the pathogenic mechanisms relevant to other skeletal diseases and could provide targets for therapeutic interventions.

Mesenchymal stem cells as a vector for the inflammatory prostate microenvironment

PubMed Central

Brennen, W Nathaniel; Denmeade, Samuel R; Isaacs, John T

2014-01-01

Mesenchymal stem cells (MSCs) have an inherent tropism for sites of inflammation, which are frequently present in sites of cancer, including prostatic lesions. MSCs have been defined as CD73/CD90/CD105 triple-positive cells in the absence of hematopoietic lineage markers with the ability to differentiate into multiple mesodermal lineages, including osteoblasts, adipocytes, and chondrocytes. Our group has previously demonstrated that MSCs represent between 0.01 and 1.1% of the total cells present in human prostatectomy tissue. In addition to their multi-lineage differentiation potential, MSCs are immunoprivileged in nature and have a range of immunomodulatory effects on both the innate and adaptive arms of the immune system. MSCs have been detected in an increasing array of tissues, and evidence suggests that they are likely present in perivascular niches throughout the body. These observations suggest that MSCs represent critical mediators of the overall immune response during physiological homeostasis and likely contribute to pathophysiological conditions as well. Chronic inflammation has been suggested as an initiating event and progression factor in prostate carcinogenesis, a process in which the immunosuppressive properties of MSCs may play a role. MSCs have also been shown to influence malignant progression through a variety of other mechanisms, including effects on tumor proliferation, angiogenesis, survival, and metastasis. Additionally, human bone marrow-derived MSCs have been shown to traffic to human prostate cancer xenografts in immunocompromised murine hosts. The trafficking properties and immunoprivileged status of MSCs suggest that they can be exploited as an allogeneic cell-based vector to deliver cytotoxic or diagnostic agents for therapy. PMID:23975882

Behavior of Human Bone Marrow-Derived Mesenchymal Stem Cells on Various Titanium-Based Coatings

PubMed Central

Qu, Chengjuan; Kaitainen, Salla; Kröger, Heikki; Lappalainen, Reijo; Lammi, Mikko J.

2016-01-01

The chemical composition and texture of titanium coatings can influence the growth characteristics of the adhered cells. An enhanced proliferation of the human mesenchymal stem cells (hMSCs) would be beneficial. The present study was aimed to investigate whether titanium deposited at different atmospheres would affect the cell growth properties, cellular morphology, and expression of surface markers of hMSCs. Titanium-based coatings were deposited on silicon wafers under oxygen, nitrogen, or argon atmospheres by ultra-short pulsed laser deposition using two different gas pressures followed by heating at 400 °C for 2 h. The characteristics of the coated surfaces were determined via contact angle, zeta potential, and scanning electron microscopy (SEM) techniques. Human MSCs were cultivated on differently coated silicon wafers for 48 h. Subsequently, the cell proliferation rates were analyzed with an MTT assay. The phenotype of hMSCs was checked via immunocytochemical stainings of MSC-associated markers CD73, CD90, and CD105, and the adhesion, spreading, and morphology of hMSCs on coated materials via SEM. The cell proliferation rates of the hMSCs were similar on all coated silicon wafers. The hMSCs retained the MSC phenotype by expressing MSC-associated markers and fibroblast-like morphology with cellular projections. Furthermore, no significant differences could be found in the size of the cells when cultured on all various coated surfaces. In conclusion, despite certain differences in the contact angles and the zeta potentials of various titanium-based coatings, no single coating markedly improved the growth characteristics of hMSCs. PMID:28773947

Office-Based Point of Care Testing (IgA/IgG-Deamidated Gliadin Peptide) for Celiac Disease.

PubMed

Lau, Michelle S; Mooney, Peter D; White, William L; Rees, Michael A; Wong, Simon H; Hadjivassiliou, Marios; Green, Peter H R; Lebwohl, Benjamin; Sanders, David S

2018-06-19

Celiac disease (CD) is common yet under-detected. A point of care test (POCT) may improve CD detection. We aimed to assess the diagnostic performance of an IgA/IgG-deamidated gliadin peptide (DGP)-based POCT for CD detection, patient acceptability, and inter-observer variability of the POCT results. From 2013-2017, we prospectively recruited patients referred to secondary care with gastrointestinal symptoms, anemia and/or weight loss (group 1); and patients with self-reported gluten sensitivity with unknown CD status (group 2). All patients had concurrent POCT, IgA-tissue transglutaminase (IgA-TTG), IgA-endomysial antibodies (IgA-EMA), total IgA levels, and duodenal biopsies. Five hundred patients completed acceptability questionnaires, and inter-observer variability of the POCT results was compared among five clinical staff for 400 cases. Group 1: 1000 patients, 58.5% female, age 16-91, median age 57. Forty-one patients (4.1%) were diagnosed with CD. The sensitivities of the POCT, IgA-TTG, and IgA-EMA were 82.9, 78.1, and 70.7%; the specificities were 85.4, 96.3, and 99.8%. Group 2: 61 patients, 83% female; age 17-73, median age 35. The POCT had 100% sensitivity and negative predictive value in detecting CD in group 2. Most patients preferred the POCT to venepuncture (90.4% vs. 2.8%). There was good inter-observer agreement on the POCT results with a Fleiss Kappa coefficient of 0.895. The POCT had comparable sensitivities to serology, and correctly identified all CD cases in a gluten sensitive cohort. However, its low specificity may increase unnecessary investigations. Despite its advantage of convenience and rapid results, it may not add significant value to case finding in an office-based setting.

In utero and intra-partum HIV-1 transmission and acute HIV-1 infection during pregnancy: using the BED capture enzyme-immunoassay as a surrogate marker for acute infection.

PubMed

Marinda, Edmore T; Moulton, Lawrence H; Humphrey, Jean H; Hargrove, John W; Ntozini, Robert; Mutasa, Kuda; Levin, Jonathan

2011-08-01

The BED assay was developed to estimate the proportion of recent HIV infections in a population. We used the BED assay as a proxy for acute infection to quantify the associated risk of mother-to-child-transmission (MTCT) during pregnancy and delivery. Design A total of 3773 HIV-1 sero-positive women were tested within 96 h of delivery using the BED assay, and CD4 cell count measurements were taken. Mothers were classified according to their likelihood of having recently seroconverted. The risk of MTCT in utero and intra-partum was assessed comparing different groups defined by BED and CD4 cell count, adjusting for background factors using multinomial logistic models. Compared with women with BED ≥ 0.8/CD4 ≥ 350 (typical of HIV-1 chronic patients) there was insufficient evidence to conclude that women presenting with BED < 0.8/CD4 ≥ 350 (typical of recent infections) were more likely to transmit in utero [adjusted odds ratio (aOR) = 1.37, 96% confidence interval (CI) 0.90-2.08, P = 0.14], whereas women with BED < 0.8/CD4 200-349 (possibly recently infected patients) had a 2.57 (95% CI 1.39-4.77, P-value < 0.01) odds of transmitting in utero. Women who had BED < 0.8/CD4 < 200 were most likely to transmit in utero (aOR 3.73, 95% CI 1.27-10.96, P = 0.02). BED and CD4 cell count were not predictive of intra-partum infections. These data provide evidence that in utero transmission of HIV might be higher among women who seroconvert during pregnancy.

Basophil Activation Test is a Relevant Biomarker of the Outcome of Rapid Desensitization in Platinum Compounds-Allergy.

PubMed

Giavina-Bianchi, Pedro; Galvão, Violeta Régnier; Picard, Matthieu; Caiado, Joana; Castells, Mariana C

Rapid drug desensitization (RDD) has become a cornerstone in the management of immediate drug hypersensitivity reactions (DHRs) to chemotherapeutic agents. Because of the inherent risk of anaphylaxis during RDD, biomarkers to predict patients at risk of developing such severe reactions are needed. The basophil activation test (BAT) has been used in DHRs as a diagnostic tool. We evaluated basophil CD63 and CD203c expression (BAT) as a biomarker to assess the safety and effectiveness of RDD in platinum compounds-allergic patients. Patients allergic to platinum compounds (n = 15) undergoing RDD were assessed through clinical history, skin testing, serum tryptase levels, and BAT. BAT was performed immediately before RDD, assessing CD203c and CD63 expression on basophils. BAT was also performed in 6 patients tolerant to platinum compounds and in 6 healthy volunteers. BAT was positive to CD203c or CD63 in 11 out of 15 patients allergic to platinum compounds (73%), with increased expression of CD203c and CD63 in 11 (73%) and 6 (40%) patients, respectively. Increased CD63 expression tended to be associated with more severe initial reactions. All controls had negative test results. Reactions during RDD were associated with BAT positivity and increased tryptase levels. Only 1 of 4 patients with negative BAT had a mild reaction during RDD. BAT remained positive in multiple sequential RDD. BAT identified patients allergic to platinum compounds with an increased risk of reactions during desensitization and higher CD63 expression was observed in severe reactions. Multiple RDDs to platinum compounds did not induce persistent hyporesponsiveness on basophils. BAT is a potential biomarker for RDD. Copyright © 2016 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

Phytochelatin accumulation and cadmium tolerance in selected tomato cell lines.

PubMed

Gupta, S C; Goldsbrough, P B

1991-09-01

Four cell lines of tomato, Lycopersicon esculentum Mill. cv VFNT-Cherry, were selected for their ability to grow in the presence of up to 6 millimolar CdCl(2). The intracellular Cd concentration in these cells was at least 2.3 times higher than in the medium. Growth in media containing higher concentrations of Cd was accompanied by increased production of Cd-binding phytochelatins and a trend toward accumulation of higher molecular weight phytochelatins. At least 90% of the Cd in the most tolerant cells was associated with Cd-phytochelatin complexes. Cell lines maintained an increased tolerance of Cd in the absence of continuous selection pressure.

Microvesicles released from human renal cancer stem cells stimulate angiogenesis and formation of lung premetastatic niche.

PubMed

Grange, Cristina; Tapparo, Marta; Collino, Federica; Vitillo, Loriana; Damasco, Christian; Deregibus, Maria Chiara; Tetta, Ciro; Bussolati, Benedetta; Camussi, Giovanni

2011-08-01

Recent studies suggest that tumor-derived microvesicles (MV) act as a vehicle for exchange of genetic information between tumor and stromal cells, engendering a favorable microenvironment for cancer development. Within the tumor mass, all cell types may contribute to MV shedding, but specific contributions to tumor progression have yet to be established. Here we report that a subset of tumor-initiating cells expressing the mesenchymal stem cell marker CD105 in human renal cell carcinoma releases MVs that trigger angiogenesis and promote the formation of a premetastatic niche. MVs derived only from CD105-positive cancer stem cells conferred an activated angiogenic phenotype to normal human endothelial cells, stimulating their growth and vessel formation after in vivo implantation in immunocompromised severe combined immunodeficient (SCID) mice. Furthermore, treating SCID mice with MVs shed from CD105-positive cells greatly enhanced lung metastases induced by i.v. injection of renal carcinoma cells. Molecular characterization of CD105-positive MVs defines a set of proangiogenic mRNAs and microRNAs implicated in tumor progression and metastases. Our results define a specific source of cancer stem cell-derived MVs that contribute to triggering the angiogenic switch and coordinating metastatic diffusion during tumor progression.

Peripheral T-cell lymphomas of follicular helper T-cell type frequently display an aberrant CD3(-/dim)CD4(+) population by flow cytometry: an important clue to the diagnosis of a Hodgkin lymphoma mimic.

PubMed

Alikhan, Mir; Song, Joo Y; Sohani, Aliyah R; Moroch, Julien; Plonquet, Anne; Duffield, Amy S; Borowitz, Michael J; Jiang, Liuyan; Bueso-Ramos, Carlos; Inamdar, Kedar; Menon, Madhu P; Gurbuxani, Sandeep; Chan, Ernest; Smith, Sonali M; Nicolae, Alina; Jaffe, Elaine S; Gaulard, Philippe; Venkataraman, Girish

2016-10-01

Nodal follicular helper T-cell-derived lymphoproliferations (specifically the less common peripheral T-cell lymphomas of follicular type) exhibit a spectrum of histologic features that may mimic reactive hyperplasia or Hodgkin lymphoma. Even though angioimmunoblastic T-cell lymphoma and peripheral T-cell lymphoma of follicular type share a common biologic origin from follicular helper T-cells and their morphology has been well characterized, flow cytometry of peripheral T-cell lymphomas of follicular type has not been widely discussed as a tool for identifying this reactive hyperplasia/Hodgkin lymphoma mimic. We identified 10 peripheral T-cell lymphomas of follicular type with available flow cytometry data from five different institutions, including two cases with peripheral blood evaluation. For comparison, we examined flow cytometry data for 8 classical Hodgkin lymphomas (including 1 lymphocyte-rich classical Hodgkin lymphoma), 15 nodular lymphocyte predominant Hodgkin lymphomas, 15 angioimmunoblastic T-cell lymphomas, and 26 reactive nodes. Lymph node histology and flow cytometry data were reviewed, specifically for the presence of a CD3(-/dim)CD4(+) aberrant T-cell population (described in angioimmunoblastic T-cell lymphomas), besides other T-cell aberrancies. Nine of 10 (90%) peripheral T-cell lymphomas of follicular type showed a CD3(-/dim)CD4(+) T-cell population constituting 29.3% (range 7.9-62%) of all lymphocytes. Five of 10 (50%) had nodular lymphocyte predominant Hodgkin lymphoma or lymphocyte-rich classical Hodgkin lymphoma-like morphology with scattered Hodgkin-like cells that expressed CD20, CD30, CD15, and MUM1. Three cases had a nodular growth pattern and three others exhibited a perifollicular growth pattern without Hodgkin-like cells. Epstein-Barr virus was positive in 1 of 10 cases (10%). PCR analysis showed clonal T-cell receptor gamma gene rearrangement in all 10 peripheral T-cell lymphomas of follicular type. By flow cytometry, 11 of 15 (73.3%) angioimmunoblastic T-cell lymphomas showed the CD3(-/dim)CD4(+) population (mean: 19.5%, range: 3-71.8%). Using a threshold of 3% for CD3(-/dim)CD4(+) T cells, all 15 nodular lymphocyte predominant Hodgkin lymphoma controls and 8 classical Hodgkin lymphomas were negative (Mann-Whitney P=0.01, F-PTCL vs Hodgkin lymphomas), as were 25 of 26 reactive lymph nodes. The high frequency of CD3(-/dim)CD4(+) aberrant T cells is similar in angioimmunoblastic T-cell lymphomas and peripheral T-cell lymphomas of follicular type, and is a useful feature in distinguishing peripheral T-cell lymphomas of follicular type from morphologic mimics such as reactive hyperplasia or Hodgkin lymphoma.

Roles of the Adenosine Receptor and CD73 in the Regulatory Effect of γδ T Cells

PubMed Central

Liang, Dongchun; Zuo, Aijun; Shao, Hui; Chen, Mingjiazi; Kaplan, Henry J.; Sun, Deming

2014-01-01

The adenosine A2A receptor (A2AR), the main functional adenosine receptor on murine T cells, plays a unique role in the attenuation of inflammation and tissue damage in vivo. Here, we showed that, of the immune cell types tested, activated γδ T cells expressed the highest levels of A2AR mRNA and that A2AR ligation inhibited αβ T cell activation, but enhanced γδ T cell activation. We also showed that the inhibitory effect of an adenosine receptor agonist on autoreactive T cells was prevented by addition of a low percentage of activated γδ T cells. Furthermore, compared to resting cells, activated γδ T cells expressed significantly lower levels of CD73, an enzyme involved in the generation of extracellular adenosine. Exogenous AMP had a significant inhibitory effect on autoreactive T cell responses, but only in the presence of CD73+ γδ T cells, and this effect was abolished by a CD73 inhibitor. Our results show that expression of increased amounts of A2AR allows γδ T cells to bind adenosine and thereby attenuate its suppressive effect, while decreased expression of CD73 results in less generation of adenosine in the inflammatory site. Together, these events allow activated γδ T cells to acquire increased proinflammatory activity, leading to augmented autoimmune responses. PMID:25268760

Possible role of CD22, CD79b and CD20 expression in distinguishing small lymphocytic lymphoma from chronic lymphocytic leukemia.

PubMed

Jovanovic, Danijela; Djurdjevic, Predrag; Andjelkovic, Nebojsa; Zivic, Ljubica

2014-01-01

Flow cytometry has an important role in diagnosis and classification of B-cell lymphoproliferative disorders (BCLPDs). However, in distinguishing chronic lymphocytic leukemia (CLL) from small lymphocytic lymphoma (SLL) only clinical criteria are available so far. Aim of the study was to determine differences in the expression of common B cell markers (CD22, CD79b and CD20) on the malignant lymphocytes in the peripheral blood samples of CLL and SLL patients. Peripheral blood samples of 56 CLL and 11 SLL patients were analyzed by 5-color flow cytometry on the CD45/CD19/CD5 gate for CD22, CD79b and CD20. In the samples collected from the CLL patients, CD22 expression was detected in only 20% of patients in the low pattern, while in SLL patients the expression was medium and present in 90.9% of patients (p < 0.0001). For CD79b expression, statistical significance is reached both in the expression pattern, which was low/medium for CLL and high for SLL, and expression level (p = 0.006). The expression of CD20 was counted as the CD20/CD19 ratio. The average ratio was 0.512 in the CLL patients vs. 0.931 in the SLL patients (p = 0.0001). The pattern of expression and expression level of CD22, CD79b and CD20 in peripheral blood could be used for distinguishing SLL from CLL patients.

Magnesium and cadmium containing Heusler phases REPd2Mg, REPd2Cd, REAg2Mg, REAu2Mg and REAu2Cd

NASA Astrophysics Data System (ADS)

Johnscher, Michael; Stein, Sebastian; Niehaus, Oliver; Benndorf, Christopher; Heletta, Lukas; Kersting, Marcel; Höting, Christoph; Eckert, Hellmut; Pöttgen, Rainer

2016-02-01

Twenty-eight new Heusler phases REPd2Mg, REPd2Cd, REAg2Mg, REAu2Mg and REAu2Cd with different rare earth elements were synthesized from the elements in sealed niobium ampoules in a water-cooled sample chamber of an induction furnace. The samples were characterized by powder X-ray diffraction. The cell volumes show the expected lanthanide contraction. The structures of YPd2Cd, GdPd2Cd, GdAu2Cd, Y1.12Ag2Mg0.88 and GdAg2Mg were refined based on single crystal diffractometer data. The magnetic properties were determined for fifteen phase pure samples. LuAu2Mg is a weak Pauli paramagnet with a susceptibility of 1.0(2) × 10-5 emu mol-1 at room temperature. The remaining samples show stable trivalent rare earth ions and most of them order magnetically at low temperatures. The ferromagnet GdAg2Mg shows the highest ordering temperature of TC = 98.3 K. 113Cd and 89Y MAS NMR spectra of YAu2Cd and YPd2Cd confirm the presence of unique crystallographic sites. The resonances are characterized by large Knight shifts, whose magnitude can be correlated with electronegativity trends.

Endogenous Production of Extracellular Adenosine by Trabecular Meshwork Cells: Potential Role in Outflow Regulation

PubMed Central

Wu, Jing; Li, Guorong; Luna, Coralia; Spasojevic, Ivan; Epstein, David L.; Gonzalez, Pedro

2012-01-01

Purpose. To investigate the mechanisms for endogenous production of extracellular adenosine in trabecular meshwork (TM) cells and evaluate its physiological relevance to the regulation of aqueous humor outflow facility. Methods. Extra-cellular levels of adenosine monophosphate (AMP) and adenosine in porcine trabecular meshwork (PTM) cells treated with adenosine triphosphate (ATP), AMP, cAMP or forskolin with or without specific inhibitors of phosphodiesterases (IBMX) and CD73 (AMPCP) were determined by high-pressure liquid chromatography fluorometry. Extracellular adenosine was also evaluated in cell cultures subjected to cyclic mechanical stress (CMS) (20% stretching; 1 Hz) and after disruption of lipid rafts with methyl-β-cyclodextrin. Expression of CD39 and CD73 in porcine TM cells and tissue were examined by Q-PCR and Western blot. The effect of inhibition of CD73 on outflow facility was evaluated in perfused living mouse eyes. Results. PTM cells generated extracellular adenosine from extracellular ATP and AMP but not from extracellular cAMP. Increased intracellular cAMP mediated by forskolin led to a significant increase in extracellular adenosine production that was not prevented by IBMX. Inhibition of CD73 resulted, in all cases, in a significant decrease in extracellular adenosine. CMS induced a significant activation of extracellular adenosine production. Inhibition of CD73 activity with AMPCP in living mouse eyes resulted in a significant decrease in outflow facility. Conclusions. These results support the concept that the extracellular adenosine pathway might play an important role in the homeostatic regulation of outflow resistance in the TM, and suggest a novel mechanism by which pathologic alteration of the TM, such as increased tissue rigidity, could lead to abnormal elevation of IOP in glaucoma. PMID:22997289

Adenosine production by human B cells and B cell–mediated suppression of activated T cells

PubMed Central

Saze, Zenichiro; Schuler, Patrick J.; Hong, Chang-Sook; Cheng, Dongmei; Jackson, Edwin K.

2013-01-01

Antibody-independent role of B cells in modulating T-cell responses is incompletely understood. Freshly isolated or cultured B cells isolated from the peripheral blood of 30 normal donors were evaluated for CD39 and CD73 coexpression, the ability to produce adenosine 5′-monophosphate (AMP) and adenosine (ADO) in the presence of exogenous adenosine triphosphate (ATP) as well as A1, A2A, A2B, and A3 adenosine receptor (ADOR) expression. Human circulating B cells coexpress ectonucleotidases CD39 and CD73, hydrolyze exogenous ATP to 5′-AMP and ADO, and express messenger RNA for A1R, A2AR, and A3R. 2-chloroadenosine inhibited B-cell proliferation and cytokine expression, and only A3R selective antagonist restored B-cell functions. This suggested that B cells use the A3R for autocrine signaling and self-regulation. Mediated effects on B-cell growth ± ADOR antagonists or agonists were tested in carboxyfluorescein diacetate succinimidyl ester assays. In cocultures, resting B cells upregulated functions of CD4+ and CD8+ T cells. However, in vitro–activated B cells downregulated CD73 expression, mainly produced 5′-AMP, and inhibited T-cell proliferation and cytokine production. These B cells acquire the ability to restrict potentially harmful effects of activated T cells. Thus, B cells emerge as a key regulatory component of T cell–B cell interactions, and their dual regulatory activity is mediated by the products of ATP hydrolysis, 5′-AMP, and ADO. PMID:23678003

Semi-automated extraction and characterization of Stromal Vascular Fraction using a new medical device.

PubMed

Hanke, Alexander; Prantl, Lukas; Wenzel, Carina; Nerlich, Michael; Brockhoff, Gero; Loibl, Markus; Gehmert, Sebastian

2016-01-01

The stem cell rich Stromal Vascular Fraction (SVF) can be harvested by processing lipo-aspirate or fat tissue with an enzymatic digestion followed by centrifugation. To date neither a standardised extraction method for SVF nor a generally admitted protocol for cell application in patients exists. A novel commercially available semi-automated device for the extraction of SVF promises sterility, consistent results and usability in the clinical routine. The aim of this work was to compare the quantity and quality of the SVF between the new system and an established manual laboratory method. SVF was extracted from lipo-aspirate both by a prototype of the semi-automated UNiStation™ (NeoGenesis, Seoul, Korea) and by hand preparation with common laboratory equipment. Cell composition of the SVF was characterized by multi-parametric flow-cytometry (FACSCanto-II, BD Biosciences). The total cell number (quantity) of the SVF was determined as well the percentage of cells expressing the stem cell marker CD34, the leucocyte marker CD45 and the marker CD271 for highly proliferative stem cells (quality). Lipo-aspirate obtained from six patients was processed with both the novel device (d) and the hand preparation (h) which always resulted in a macroscopically visible SVF. However, there was a tendency of a fewer cell yield per gram of used lipo-aspirate with the device (d: 1.1×105±1.1×105 vs. h: 2.0×105±1.7×105; p = 0.06). Noteworthy, the percentage of CD34+ cells was significantly lower when using the device (d: 57.3% ±23.8% vs. h: 74.1% ±13.4%; p = 0.02) and CD45+ leukocyte counts tend to be higher when compared to the hand preparation (d: 20.7% ±15.8% vs. h: 9.8% ±7.1%; p = 0.07). The percentage of highly proliferative CD271+ cells was similar for both methods (d:12.9% ±9.6% vs. h: 13.4% ±11.6%; p = 0.74) and no differences were found for double positive cells of CD34+/CD45+ (d: 5.9% ±1.7% vs. h: 1.7% ±1.1%; p = 0.13), CD34+/CD271+ (d: 24.1% ±12.0% vs. h: 14.2% ±8.5%; p = 0.07). The semi-automated closed system provides a considerable amount of sterile SVF with high reproducibility. Furthermore, the SVF extracted by both methods showed a similar cell composition which is in accordance with the data from literature. This semi-automated device offers an opportunity to take research and application of the SVF one step further to the clinic.

IgG1 memory B cells keep the memory of IgE responses.

PubMed

He, Jin-Shu; Subramaniam, Sharrada; Narang, Vipin; Srinivasan, Kandhadayar; Saunders, Sean P; Carbajo, Daniel; Wen-Shan, Tsao; Hidayah Hamadee, Nur; Lum, Josephine; Lee, Andrea; Chen, Jinmiao; Poidinger, Michael; Zolezzi, Francesca; Lafaille, Juan J; Curotto de Lafaille, Maria A

2017-09-21

The unique differentiation of IgE cells suggests unconventional mechanisms of IgE memory. IgE germinal centre cells are transient, most IgE cells are plasma cells, and high affinity IgE is produced by the switching of IgG1 cells to IgE. Here we investigate the function of subsets of IgG1 memory B cells in IgE production and find that two subsets of IgG1 memory B cells, CD80 + CD73 + and CD80 - CD73 - , contribute distinctively to the repertoires of high affinity pathogenic IgE and low affinity non-pathogenic IgE. Furthermore, repertoire analysis indicates that high affinity IgE and IgG1 plasma cells differentiate from rare CD80 + CD73 + high affinity memory clones without undergoing further mutagenesis. By identifying the cellular origin of high affinity IgE and the clonal selection of high affinity memory B cells into the plasma cell fate, our findings provide fundamental insights into the pathogenesis of allergies, and on the mechanisms of antibody production in memory B cell responses.IgE is an important mediator of protective immunity as well as allergic reaction, but how high affinity IgE antibodies are produced in memory responses is not clear. Here the authors show that IgE can be generated via class-switch recombination in IgG1 memory B cells without additional somatic hypermutation.

Alterations in the adenosine metabolism and CD39/CD73 adenosinergic machinery cause loss of Treg cell function and autoimmunity in ADA-deficient SCID.

PubMed

Sauer, Aisha V; Brigida, Immacolata; Carriglio, Nicola; Hernandez, Raisa Jofra; Scaramuzza, Samantha; Clavenna, Daniela; Sanvito, Francesca; Poliani, Pietro L; Gagliani, Nicola; Carlucci, Filippo; Tabucchi, Antonella; Roncarolo, Maria Grazia; Traggiai, Elisabetta; Villa, Anna; Aiuti, Alessandro

2012-02-09

Adenosine acts as anti-inflammatory mediator on the immune system and has been described in regulatory T cell (Treg)-mediated suppression. In the absence of adenosine deaminase (ADA), adenosine and other purine metabolites accumulate, leading to severe immunodeficiency with recurrent infections (ADA-SCID). Particularly ADA-deficient patients with late-onset forms and after enzyme replacement therapy (PEG-ADA) are known to manifest immune dysregulation. Herein we provide evidence that alterations in the purine metabolism interfere with Treg function, thereby contributing to autoimmune manifestations in ADA deficiency. Tregs isolated from PEG-ADA-treated patients are reduced in number and show decreased suppressive activity, whereas they are corrected after gene therapy. Untreated murine ADA(-/-) Tregs show alterations in the plasma membrane CD39/CD73 ectonucleotidase machinery and limited suppressive activity via extracellular adenosine. PEG-ADA-treated mice developed multiple autoantibodies and hypothyroidism in contrast to mice treated with bone marrow transplantation or gene therapy. Tregs isolated from PEG-ADA-treated mice lacked suppressive activity, suggesting that this treatment interferes with Treg functionality. The alterations in the CD39/CD73 adenosinergic machinery and loss of function in ADA-deficient Tregs provide new insights into a predisposition to autoimmunity and the underlying mechanisms causing defective peripheral tolerance in ADA-SCID.

REGULATION OF THE T-CELL RESPONSE BY CD39

PubMed Central

Takenaka, Maisa C.; Robson, Simon; Quintana, Francisco J.

2016-01-01

The ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1, or CD39) catalyzes the phosphohydrolysis of extracellular adenosine triphosphate (eATP) and diphosphate (eADP) released under conditions of inflammatory stress and cell injury. CD39 generates adenosine monophosphate (AMP), which is in turn used by the ecto-5’-nucleotidase CD73 to synthesize adenosine. These ectonucleotidases have major impacts on the dynamic equilibrium of pro-inflammatory eATP and ADP nucleotides vs. immunosuppressive adenosine nucleosides. Indeed, CD39 plays a dominant role in the purinergic regulation of inflammation and the immune response because its expression is influenced by genetic and environmental factors. Here, we review the specific role of CD39 in the kinetic regulation of cellular immune responses in the evolution of disease. We focus on the effects of CD39 on T cells and explore potential clinical applications in autoimmunity, chronic infections and cancer. PMID:27236363

TNF-α enhancement of CD62E mediates adhesion of non-small cell lung cancer cells to brain endothelium via CD15 in lung-brain metastasis.

PubMed

Jassam, Samah A; Maherally, Zaynah; Smith, James R; Ashkan, Keyoumars; Roncaroli, Federico; Fillmore, Helen L; Pilkington, Geoffrey J

2016-05-01

CD15, which is overexpressed on various cancers, has been reported as a cell adhesion molecule that plays a key role in non-CNS metastasis. However, the role of CD15 in brain metastasis is largely unexplored. This study provides a better understanding of CD15/CD62E interaction, enhanced by tumor necrosis factor-α (TNF-α), and its correlation with brain metastasis in non-small cell lung cancer (NSCLC). CD15 and E-selectin (CD62E) expression was demonstrated in both human primary and metastatic NSCLC cells using flow cytometry, immunofluorescence, and Western blotting. The role of CD15 was investigated using an adhesion assay under static and physiological flow live-cell conditions. Human tissue sections were examined using immunohistochemistry. CD15, which was weakly expressed on hCMEC/D3 human brain endothelial cells, was expressed at high levels on metastatic NSCLC cells (NCI-H1299, SEBTA-001, and SEBTA-005) and at lower levels on primary NSCLC (COR-L105 and A549) cells (P < .001). The highest expression of CD62E was observed on hCMEC/D3 cells activated with TNF-α, with lower levels on metastatic NSCLC cells followed by primary NSCLC cells. Metastatic NSCLC cells adhered most strongly to hCMEC/D3 compared with primary NSCLC cells. CD15 immunoblocking decreased cancer cell adhesion to brain endothelium under static and shear stress conditions (P < .0001), confirming a correlation between CD15 and cerebral metastasis. Both CD15 and CD62E expression were detected in lung metastatic brain biopsies. This study enhances the understanding of cancer cell-brain endothelial adhesion and confirms that CD15 plays a crucial role in adhesion in concert with TNF-α activation of its binding partner, CD62E. © The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Neuro-Oncology.

TNF-α enhancement of CD62E mediates adhesion of non–small cell lung cancer cells to brain endothelium via CD15 in lung-brain metastasis

PubMed Central

Jassam, Samah A.; Maherally, Zaynah; Smith, James R.; Ashkan, Keyoumars; Roncaroli, Federico; Fillmore, Helen L.; Pilkington, Geoffrey J.

2016-01-01

Background CD15, which is overexpressed on various cancers, has been reported as a cell adhesion molecule that plays a key role in non-CNS metastasis. However, the role of CD15 in brain metastasis is largely unexplored. This study provides a better understanding of CD15/CD62E interaction, enhanced by tumor necrosis factor-α (TNF-α), and its correlation with brain metastasis in non–small cell lung cancer (NSCLC). Methods CD15 and E-selectin (CD62E) expression was demonstrated in both human primary and metastatic NSCLC cells using flow cytometry, immunofluorescence, and Western blotting. The role of CD15 was investigated using an adhesion assay under static and physiological flow live-cell conditions. Human tissue sections were examined using immunohistochemistry. Results CD15, which was weakly expressed on hCMEC/D3 human brain endothelial cells, was expressed at high levels on metastatic NSCLC cells (NCI-H1299, SEBTA-001, and SEBTA-005) and at lower levels on primary NSCLC (COR-L105 and A549) cells (P < .001). The highest expression of CD62E was observed on hCMEC/D3 cells activated with TNF-α, with lower levels on metastatic NSCLC cells followed by primary NSCLC cells. Metastatic NSCLC cells adhered most strongly to hCMEC/D3 compared with primary NSCLC cells. CD15 immunoblocking decreased cancer cell adhesion to brain endothelium under static and shear stress conditions (P < .0001), confirming a correlation between CD15 and cerebral metastasis. Both CD15 and CD62E expression were detected in lung metastatic brain biopsies. Conclusion This study enhances the understanding of cancer cell-brain endothelial adhesion and confirms that CD15 plays a crucial role in adhesion in concert with TNF-α activation of its binding partner, CD62E. PMID:26472821

Various types of stem cells, including a population of very small embryonic-like stem cells, are mobilized into peripheral blood in patients with Crohn's disease.

PubMed

Marlicz, Wojciech; Zuba-Surma, Ewa; Kucia, Magda; Blogowski, Wojciech; Starzynska, Teresa; Ratajczak, Mariusz Z

2012-09-01

Developmentally early cells, including hematopoietic stem progenitor cells (HSPCs), mesenchymal stem cells (MSCs), endothelial progenitor cells (EPCs), and very small embryonic-like stem cells (VSELs), are mobilized into peripheral blood (PB) in response to tissue/organ injury. We sought to determine whether these cells are mobilized into PB in patients with Crohn's disease (CD). Twenty-five patients with active CD, 20 patients in clinical remission, and 25 age-matched controls were recruited and PB samples harvested. The circulating CD133+/Lin-/CD45+ and CD34+/Lin-/CD45+ cells enriched for HSPCs, CD105+/STRO-1+/CD45- cells enriched for MSCs, CD34+/KDR+/CD31+/CD45-cells enriched for EPCs, and small CXCR4+CD34+CD133+ subsets of Lin-CD45- cells that correspond to the population of VSELs were counted by fluorescence-activated cell sorting (FACS) and evaluated by direct immunofluorescence staining for pluripotency embryonic markers and by reverse-transcription polymerase chain reaction (RT-PCR) for expression of messenger (m)RNAs for a panel of genes expressed in intestine epithelial stem cells. The serum concentration of factors involved in stem cell trafficking, such as stromal derived factor-1 (SDF-1), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF) were measured by enzyme-linked immunosorbent assay (ELISA). Our data indicate that cells expressing markers for MSCs, EPCs, and small Oct-4+Nanog+SSEA-4+CXCR4+lin-CD45- VSELs are mobilized into PB in CD. The mobilized cells also expressed at the mRNA level genes playing a role in development and regeneration of gastrointestinal epithelium. All these changes were accompanied by increased serum concentrations of VEGF and HGF. CD triggers the mobilization of MSCs, EPCs, and VSELs, while the significance and precise role of these mobilized cells in repair of damaged intestine requires further study. Copyright © 2012 Crohn's & Colitis Foundation of America, Inc.

PTEN and PI-3 kinase inhibitors control LPS signaling and the lymphoproliferative response in the CD19+ B cell compartment

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

Singh, Alok R.; Peirce, Susan K.; Joshi, Shweta

Pattern recognition receptors (PRRs), e.g. toll receptors (TLRs) that bind ligands within the microbiome have been implicated in the pathogenesis of cancer. LPS is a ligand for two TLR family members, TLR4 and RP105 which mediate LPS signaling in B cell proliferation and migration. Although LPS/TLR/RP105 signaling is well-studied; our understanding of the underlying molecular mechanisms controlling these PRR signaling pathways remains incomplete. Previous studies have demonstrated a role for PTEN/PI-3K signaling in B cell selection and survival, however a role for PTEN/PI-3K in TLR4/RP105/LPS signaling in the B cell compartment has not been reported. Herein, we crossed a CD19cremore » and PTEN{sup fl/fl} mouse to generate a conditional PTEN knockout mouse in the CD19+ B cell compartment. These mice were further crossed with an IL-14α transgenic mouse to study the combined effect of PTEN deletion, PI-3K inhibition and expression of IL-14α (a cytokine originally identified as a B cell growth factor) in CD19+ B cell lymphoproliferation and response to LPS stimulation. Targeted deletion of PTEN and directed expression of IL-14α in the CD19+ B cell compartment (IL-14+PTEN-/-) lead to marked splenomegaly and altered spleen morphology at baseline due to expansion of marginal zone B cells, a phenotype that was exaggerated by treatment with the B cell mitogen and TLR4/RP105 ligand, LPS. Moreover, LPS stimulation of CD19+ cells isolated from these mice display increased proliferation, augmented AKT and NFκB activation as well as increased expression of c-myc and cyclinD1. Interestingly, treatment of LPS treated IL-14+PTEN-/- mice with a pan PI-3K inhibitor, SF1126, reduced splenomegaly, cell proliferation, c-myc and cyclin D1 expression in the CD19+ B cell compartment and normalized the splenic histopathologic architecture. These findings provide the direct evidence that PTEN and PI-3K inhibitors control TLR4/RP105/LPS signaling in the CD19+ B cell compartment and that pan PI-3 kinase inhibitors reverse the lymphoproliferative phenotype in vivo. - Highlights: • First genetic evidence that PTEN controls LPS/TLR4 signaling in B lymphocytes. • Evidence that PTEN regulates LPS induced lymphoproliferation in vivo. • PI-3 kinase inhibitors block LPS induced lymphoproliferation in vivo.« less

CD73 regulates anti-inflammatory signaling between apoptotic cells and endotoxin-conditioned tissue macrophages

PubMed Central

Murphy, Patrick S; Wang, Jing; Bhagwat, Samir P; Munger, Joshua C; Janssen, William J; Wright, Terry W; Elliott, Michael R

2017-01-01

The phagocytosis of apoptotic cells (efferocytosis) shifts macrophages to an anti-inflammatory state through a set of still poorly understood soluble and cell-bound signals. Apoptosis is a common feature of inflamed tissues, and efferocytosis by tissue macrophages is thought to promote the resolution of inflammation. However, it is not clear how the exposure of tissue macrophages to inflammatory cues (e.g., PAMPs, DAMPs) in the early stages of inflammation affects immune outcomes of macrophage-apoptotic cell interactions occurring at later stages of inflammation. To address this, we used low-dose endotoxin conditioning (LEC, 1 ng/ml LPS 18 h) of mouse resident peritoneal macrophages (RPMФ) to model the effects of suboptimal (i.e., non-tolerizing), antecedent TLR activation on macrophage inflammatory responses to apoptotic cells. Compared with unconditioned macrophages (MФ), LEC-MФ showed a significant enhancement of apoptotic cell-driven suppression of many inflammatory cytokines (e.g., TNF, MIP-1β, MCP-1). We then found that enzymatic depletion of adenosine or inhibition of the adenosine receptor A2a on LEC-MФ abrogated apoptotic cell suppression of TNF, and this suppression was entirely dependent on the ecto-enzyme CD73 (AMP→adenosine) but not CD39 (ATP→AMP), both of which are highly expressed on RPMФ. In addition to a requirement for CD73, we also show that Adora2a levels in macrophages are a critical determinant of TNF suppression by apoptotic cells. LEC treatment of RPMФ led to a ~3-fold increase in Adora2a and a ~28-fold increase in adenosine sensitivity. Moreover, in RAW264.7 cells, ectopic expression of both A2a and CD73 was required for TNF suppression by apoptotic cells. In mice, mild, TLR4-dependent inflammation in the lungs and peritoneum caused a rapid increase in macrophage Adora2a and Adora2b levels, and CD73 was required to limit neutrophil influx in this peritonitis model. Thus immune signaling via the CD73–A2a axis in macrophages links early inflammatory events to subsequent immune responses to apoptotic cells. PMID:28060378

Predicting renal graft failure by sCD30 levels and de novo HLA antibodies at 1year post-transplantation.

PubMed

Wang, Dong; Wu, Guojun; Chen, Jinhua; Yu, Ziqiang; Wu, Weizhen; Yang, Shunliang; Tan, Jianming

2012-06-01

HLA antibodies and sCD30 levels were detected in the serum sampled from 620 renal graft recipients at 1 year post-transplantation, which were followed up for 5 years. Six-year graft and patient survivals were 81.6% and 91.0%. HLA antibodies were detected in 45 recipients (7.3%), of whom there were 14 cases with class I antibodies, 26 cases with class II, and 5 cases with both class I and II. Much more graft loss was record in recipients with HLA antibodies than those without antibodies (60% vs. 15.1%, p<0.001). Significantly higher sCD30 levels were recorded in recipients suffering graft loss than the others (73.9±48.8 U/mL vs. 37.3±14.6 U/mL, p<0.001). Compared with those with high sCD30 levels, recipients with low sCD30 levels (<50 U/mL) had much better 6-year graft survival (92.4% vs. 46.6%, p<0.001). Further statistical analysis showed that detrimental effect of de novo HLA antibodies and high sCD30 on graft survival was not only independent but also additive. Therefore, post-transplantation monitoring of HLA antibodies and sCD30 levels is necessary and recipients with elevated sCD30 level and/or de novo HLA antibody should be paid more attention in order to achieve better graft survival. Copyright © 2012 Elsevier B.V. All rights reserved.

Identification of SIV Nef CD8(+) T cell epitopes restricted by a MHC class I haplotype associated with lower viral loads in a macaque AIDS model.

PubMed

Nomura, Takushi; Yamamoto, Hiroyuki; Takahashi, Naofumi; Naruse, Taeko K; Kimura, Akinori; Matano, Tetsuro

2014-07-25

Virus-specific CD8(+) T-cell responses are crucial for the control of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) replication. Multiple studies on HIV-infected individuals and SIV-infected macaques have indicated association of several major histocompatibility complex class I (MHC-I) genotypes with lower viral loads and delayed AIDS progression. Understanding of the viral control mechanism associated with these MHC-I genotypes would contribute to the development of intervention strategy for HIV control. We have previously reported a rhesus MHC-I haplotype, 90-120-Ia, associated with lower viral loads after SIVmac239 infection. Gag206-216 and Gag241-249 epitope-specific CD8(+) T-cell responses have been shown to play a central role in the reduction of viral loads, whereas the effect of Nef-specific CD8(+) T-cell responses induced in all the 90-120-Ia(+) macaques on SIV replication remains unknown. Here, we identified three CD8(+) T-cell epitopes, Nef9-19, Nef89-97, and Nef193-203, associated with 90-120-Ia. Nef9-19 and Nef193-203 epitope-specific CD8(+) T-cell responses frequently selected for mutations resulting in viral escape from recognition by these CD8(+) T cells, indicating that these CD8(+) T cells exert strong suppressive pressure on SIV replication. Results would be useful for elucidation of the viral control mechanism associated with 90-120-Ia. Copyright © 2014 Elsevier Inc. All rights reserved.

Human urine-derived stem cells play a novel role in the treatment of STZ-induced diabetic mice.

PubMed

Zhao, Tianxue; Luo, Deng; Sun, Yun; Niu, Xin; Wang, Yang; Wang, Chen; Jia, Weiping

2018-04-19

Human urine-derived stem cells (hUSCs) are a potential stem cell source for cell therapy. However, the effect of hUSCs on glucose metabolism regulation in type 1 diabetes was not clear. Therefore, the aim of the study was to evaluate whether hUSCs have protective effect on streptozotocin (STZ)-induced diabetes. hUSCs were extracted and cultivated with a special culture medium. Flow cytometry analysis was applied to detect cell surface markers. BALB/c male nude mice were either injected with high-dose STZ (HD-STZ) or multiple low-dose STZ (MLD-STZ). Serum and pancreatic insulin were measured, islet morphology and its vascularization were investigated. hUSCs highly expressed CD29, CD73, CD90 and CD146, and could differentiate into, at least, bone and fat in vitro. Transplantation of hUSCs into HD-STZ treated mice prolonged the median survival time and improved their blood glucose, and into those with MLD-STZ improved the glucose tolerance, islet morphology and increased the serum and pancreas insulin content. Furthermore, CD31 expression increased significantly in islets of BALB/c nude mice treated with hUSCs compared to those of un-transplanted MLD-STZ mice. hUSCs could improve the median survival time and glucose homeostasis in STZ-treated mice through promoting islet vascular regeneration and pancreatic beta-cell survival.

40 CFR 413.74 - Pretreatment standards for existing sources.

Code of Federal Regulations, 2011 CFR

2011-07-01

....5 2.7 Ni 4.1 2.6 Cr 7.0 4.0 Zn 4.2 2.6 Pb 0.6 0.4 Cd 1.2 0.7 Total metals 10.5 6.8 (d) Alternatively... days shall not exceed CN,T 74 39 Cu 176 105 Ni 160 100 Cr 273 156 Zn 164 102 Pb 23 16 Cd 47 29 Total...

40 CFR 413.74 - Pretreatment standards for existing sources.

Code of Federal Regulations, 2010 CFR

2010-07-01

....5 2.7 Ni 4.1 2.6 Cr 7.0 4.0 Zn 4.2 2.6 Pb 0.6 0.4 Cd 1.2 0.7 Total metals 10.5 6.8 (d) Alternatively... days shall not exceed CN,T 74 39 Cu 176 105 Ni 160 100 Cr 273 156 Zn 164 102 Pb 23 16 Cd 47 29 Total...

40 CFR 413.24 - Pretreatment standards for existing sources.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 1.9 1.0 Cu 4.5 2.7 Ni 4.1 2.6 Cr 7.0 4.0 Zn 4.2 2.6 Pb .6 .4 Cd 1.2 .7 Total metals 10.5 6.8 (d... exceed Ag 47 29 CN, T 74 39 Cu 176 105 Ni 160 100 Cr 273 156 Zn 164 102 Pb 23 16 Cd 47 29 Total metals...

40 CFR 413.24 - Pretreatment standards for existing sources.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 1.9 1.0 Cu 4.5 2.7 Ni 4.1 2.6 Cr 7.0 4.0 Zn 4.2 2.6 Pb .6 .4 Cd 1.2 .7 Total metals 10.5 6.8 (d... exceed Ag 47 29 CN, T 74 39 Cu 176 105 Ni 160 100 Cr 273 156 Zn 164 102 Pb 23 16 Cd 47 29 Total metals...

Multi-color CD34⁺ progenitor-focused flow cytometric assay in evaluation of myelodysplastic syndromes in patients with post cancer therapy cytopenia.

PubMed

Tang, Guilin; Jorgensen, L Jeffrey; Zhou, Yi; Hu, Ying; Kersh, Marian; Garcia-Manero, Guillermo; Medeiros, L Jeffrey; Wang, Sa A

2012-08-01

Bone marrow assessment for myelodysplastic syndrome (MDS) in a patient who develops cytopenia(s) following cancer therapy is challenging. With recent advances in multi-color flow cytometry immunophenotypic analysis, a CD34(+) progenitor-focused 7-color assay was developed and tested in this clinical setting. This assay was first performed in 73 MDS patients and 53 non-MDS patients (developmental set). A number of immunophenotypic changes were differentially observed in these two groups. Based on the sensitivity, specificity and reproducibility, a core panel of markers was selected for final assessment that included increased total CD34(+) myeloblasts; decreased stage I hematogones; altered CD45/side scatter; altered expression of CD13, CD33, CD34, CD38, CD117, and CD123; aberrant expression of lymphoid or mature myelomonocytic antigens on CD34(+) myeloblasts; and several marked alterations in maturing myelomonocytic cells. The data were translated into a simplified scoring system which was then used in 120 patients with cytopenia(s) secondary to cancer therapy over a 2-year period (validation set). With a median follow-up of 11 months, this assay demonstrated 89% sensitivity, 94% specificity, and 92% accuracy in establishing or excluding a diagnosis of MDS. Copyright © 2012 Elsevier Ltd. All rights reserved.

CD4+ memory T cells retain surface expression of CD31 independently of thymic function in patients with lymphoproliferative disorders following autologous hematopoietic stem-cell transplantation.

PubMed

Batorov, Egor V; Tikhonova, Marina A; Kryuchkova, Irina V; Sergeevicheva, Vera V; Sizikova, Svetlana A; Ushakova, Galina Y; Batorova, Dariya S; Gilevich, Andrey V; Ostanin, Alexander A; Shevela, Ekaterina Y; Chernykh, Elena R

2017-07-01

High-dose chemotherapy with autologous hematopoietic stem-cell transplantation (AHSCT) causes severe and long-lasting immunodeficiency in patients with lymphoproliferative disorders. The thymus begins to restore the T-cell repertoire approximately from the sixth month post-transplant. We assessed the dynamics of post-transplant recovery of CD4 + CD45RA + CD31 + T cells, "recent thymic emigrants" (RTEs), and a poorly described subtype of CD4 + CD45RA - CD31 + T cells in 90 patients with lymphoproliferative disorders following high-dose chemotherapy with AHSCT. Relative and absolute counts of CD4 + CD31 + naïve and memory T cells were evaluated before AHSCT, at the day of engraftment, and 6- and 12-month post-transplant. The pre-transplant count of CD4 + CD45RA + CD31 + T cells was lower than in healthy controls, and did not reach donors' values during the 12-month period. The pre-transplant number of CD4 + CD45RA - CD31 + T cells was higher than in healthy controls and was restored rapidly following AHSCT. Post-transplant mediastinal radiotherapy reduced counts of RTEs and elongated recovery period. Non-thymic tissue irradiation did not reduce this subset. The obtained data indicate that homeostatic proliferation may decrease the significance of CD31 expression on CD4 + CD45RA + T cells as a marker of RTEs, and suggest that evaluation of RTEs recovery by flow cytometry requires an accurate gating strategy to exclude CD31 + memory T cells.

CD19+CD21low B cells and CD4+CD45RA+CD31+ T cells correlate with first diagnosis of chronic graft-versus-host disease.

PubMed

Greinix, Hildegard T; Kuzmina, Zoya; Weigl, Roman; Körmoczi, Ulrike; Rottal, Arno; Wolff, Daniel; Kralj, Mateja; Kalhs, Peter; Mitterbauer, Margit; Rabitsch, Werner; Edinger, Matthias; Holler, Ernst; Pickl, Winfried F

2015-02-01

Chronic graft-versus-host disease (cGVHD) is a serious and frequent complication of allogeneic hematopoietic stem cell transplantation (HCT). Currently, no biomarkers for prediction and diagnosis of cGVHD are available. We performed a large prospective study focusing on noninvasive biomarkers for National Institutes of Health-defined cGVHD patients (n = 163) in comparison to time-matched HCT recipients who never experienced cGVHD (n = 64), analyzed from day 100 after HCT. In logistic regression analysis, CD19(+)CD21(low) B cells (P = .002; hazard ratio [HR], 3.31; 95% confidence interval [CI], 1.53 to 7.17) and CD4(+)CD45RA(+)CD31(+) T cells (P < .001; HR, 3.88; 95% CI, 1.88 to 7.99) assessed on day 100 after HCT were significantly associated with subsequent development of cGVHD, independent of clinical parameters. A significant association with diagnosis of cGVHD was only observed for CD19(+)CD21(low) B cells (P = .008; HR, 3.00; 95% CI, 1.33 to 6.75) and CD4(+)CD45RA(+)CD31(+) T cells (P = .017; HR, 2.80; 95% CI, 1.19 to 6.55). CD19(+)CD21(low) B cells were found to have the highest discriminatory value with an area under the receiver operating curve of .77 (95% CI, .64 to .90). Our results demonstrate that CD19(+)CD21(low) B cells and CD4(+)CD45RA(+)CD31(+) T cells are significantly elevated in patients with newly diagnosed cGVHD. Copyright © 2015 American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.

Extracellular ATP is Differentially Metabolized on Papillary Thyroid Carcinoma Cells Surface in Comparison to Normal Cells.

PubMed

Bertoni, Ana Paula Santin; de Campos, Rafael Paschoal; Tsao, Marisa; Braganhol, Elizandra; Furlanetto, Tania Weber; Wink, Márcia Rosângela

2018-02-17

The incidence of differentiated thyroid cancer has been increasing. Nevertheless, its molecular mechanisms are not well understood. In recent years, extracellular nucleotides and nucleosides have emerged as important modulators of tumor microenvironment. Extracellular ATP is mainly hydrolyzed by NTPDase1/CD39 and NTPDase2/CD39L1, generating AMP, which is hydrolyzed by ecto-5'-nucleotidase (CD73) to adenosine, a possible promoter of tumor growth and metastasis. There are no studies evaluating the expression and functionality of these ectonucleotidases on normal or tumor-derived thyroid cells. Thus, we investigated the ability of thyroid cancer cells to hydrolyze extracellular ATP generating adenosine, and the expression of ecto-enzymes, as compared to normal cells. We found that normal thyroid derived cells presented a higher ability to hydrolyze ATP and higher mRNA levels for ENTDP1-2, when compared to papillary thyroid carcinoma (PTC) derived cells, which had a higher ability to hydrolyze AMP and expressed CD73 mRNA and protein at higher levels. In addition, adenosine induced an increase in proliferation and migration in PTC derived cells, whose effect was blocked by APCP, a non-hydrolysable ADP analogue, which is an inhibitor of CD73. Taken together, these results showed that thyroid follicular cells have a functional purinergic signaling. The higher expression of CD73 in PTC derived cells might favor the accumulation of extracellular adenosine in the tumor microenvironment, which could promote tumor progression. Therefore, as already shown for other tumors, the purinergic signaling should be considered a potential target for thyroid cancer management and treatment.

Ex Vivo Restimulation of Human PBMC Expands a CD3+CD4−CD8− γδ + T Cell Population That Can Confound the Evaluation of CD4 and CD8 T Cell Responses to Vaccination

PubMed Central

Sedgmen, B. J.; Papalia, L.; Wang, L.; Dyson, A. R.; McCallum, H. A.; Simson, C. M.; Pearse, M. J.; Maraskovsky, E.; Hung, D.; Eomois, P. P.; Hartel, G.; Barnden, M. J.; Rockman, S. P.

2013-01-01

The measurement of vaccine-induced humoral and CD4+ and CD8+ cellular immune responses represents an important correlate of vaccine efficacy. Accurate and reliable assays evaluating such responses are therefore critical during the clinical development phase of vaccines. T cells play a pivotal role both in coordinating the adaptive and innate immune responses and as effectors. During the assessment of cell-mediated immunity (CMI) in subjects participating in a large-scale influenza vaccine trial, we identified the expansion of an IFN-γ-producing CD3+CD4−CD8− γδ + T cell population in the peripheral blood of 90/610 (15%) healthy subjects. The appearance of CD3+CD4−CD8− γδ + T cells in the blood of subjects was transient and found to be independent of the study cohort, vaccine group, subject gender and ethnicity, and ex vivo restimulation conditions. Although the function of this population and relevance to vaccination are unclear, their inclusion in the total vaccine-specific T-cell response has the potential to confound data interpretation. It is thus recommended that when evaluating the induction of IFN-γ-producing CD4+ and CD8+ immune responses following vaccination, the CD3+CD4−CD8− γδ + T cells are either excluded or separately enumerated from the overall frequency determination. PMID:24066003

Generation and characterisation of human umbilical cord derived mesenchymal stem cells by explant method.

PubMed

Yusoff, Z; Maqbool, M; George, E; Hassan, R; Ramasamy, R

2016-06-01

Mesenchymal stem cells (MSCs) derived from human umbilical cord (UC) have been considered as an important tool for treating various malignancies, tissue repair and organ regeneration. Umbilical cord-derived mesenchymal stem cells (UC-MSCs) are better alternative to MSCs that derived from bone marrow (BM-MSCs) as they are regarded as medical waste with little ethical concern for research and easily culture-expanded. In this present study, the foetal distal end of human UC was utilised to generate MSC by explant method. Upon in vitro culture, adherent cells with fibroblastic morphology were generated with rapid growth kinetics. Under the respective inductive conditions, these cells were capable of differentiating into adipocytes and osteocytes; express an array of standard MSC's surface markers CD29, CD73, CD90, CD106 and MHC-class I. Further assessment of immunosuppression activity revealed that MSCs generated from UC had profoundly inhibited the proliferation of mitogen-activated T lymphocytes in a dosedependent manner. The current laboratory findings have reinforced the application of explant method to generate UCMSCs thus, exploring an ideal platform to fulfil the increasing demand of MSCs for research and potential clinical use.

Bispecific single-chain diabody-immunoliposomes targeting endoglin (CD105) and fibroblast activation protein (FAP) simultaneously.

PubMed

Rabenhold, Markus; Steiniger, Frank; Fahr, Alfred; Kontermann, Roland E; Rüger, Ronny

2015-03-10

Liposomes are well-established drug delivery systems with cancer chemotherapy as main focus. To increase the cellular drug delivery, liposomes can be endowed with ligands, e.g. recombinant antibody fragments, which ensure specific cell interaction. Multispecific immunoliposomes can be prepared to improve the liposome to cell interaction by targeting multiple different targets at the same time, for instance by coupling two or more different ligands to the liposomal surface, resulting in a synergistic or additive increase in binding. An alternative approach is the use of bispecific ligands to address at least two different targets. For this purpose we cloned a single-chain diabody fragment (scDb`), a bispecific molecule targeting two antigens, endoglin (CD105) and fibroblast activation protein (FAP), expressed on cells of the tumor microenvironment. As model cell system, a human fibrosarcoma cell line was used expressing endoglin and FAP simultaneously. Monospecific immunoliposomes directed either against endoglin or FAP were compared in vitro for cell binding and cytotoxic activity with bispecific dual-targeted scFv`-IL (bispecific scFv`FAP/CD105-IL) and bispecific single-chain diabody`-IL (scDb`CD105/FAP-IL) targeting endoglin and FAP simultaneously. In the underlying study, bispecific scFv`FAP/CD105-IL interacted stronger with cells expressing FAP and endoglin (both targets simultaneously) compared to the monospecific immunoliposomes. Furthermore, bispecific scDb`-immunoliposomes increased the cell interaction massively and showed enhanced cytotoxicity against target cells using doxorubicin-loaded immunoliposomes. The use of recombinant bispecific ligands as scDb`-molecules facilitates the generation of bispecific immunoliposomes by using the established post-insertion technique, enabling an extension of the ligand specificity spectrum via genetic modification. Copyright © 2015 Elsevier B.V. All rights reserved.

Ex Vivo Expansion of CD34+CD90+CD49f+ Hematopoietic Stem and Progenitor Cells from Non‐Enriched Umbilical Cord Blood with Azole Compounds

PubMed Central

Bari, Sudipto; Zhong, Qixing; Fan, Xiubo; Poon, Zhiyong; Lim, Alvin Soon Tiong; Lim, Tse Hui; Dighe, Niraja; Li, Shang; Chiu, Gigi Ngar Chee; Chai, Christina Li Lin

2018-01-01

Abstract Umbilical cord blood (UCB) transplants in adults have slower hematopoietic recovery compared to bone marrow (BM) or peripheral blood (PB) stem cells mainly due to low number of total nucleated cells and hematopoietic stem and progenitor cells (HSPC). As such in this study, we aimed to perform ex vivo expansion of UCB HSPC from non‐enriched mononucleated cells (MNC) using novel azole‐based small molecules. Freshly‐thawed UCB–MNC were cultured in expansion medium supplemented with small molecules and basal cytokine cocktail. The effects of the expansion protocol were measured based on in vitro and in vivo assays. The proprietary library of >50 small molecules were developed using structure‐activity‐relationship studies of SB203580, a known p38‐MAPK inhibitor. A particular analog, C7, resulted in 1,554.1 ± 27.8‐fold increase of absolute viable CD45+CD34+CD38–CD45RA– progenitors which was at least 3.7‐fold higher than control cultures (p < .001). In depth phenotypic analysis revealed >600‐fold expansion of CD34+/CD90+/CD49f+ rare HSPCs coupled with significant (p < .01) increase of functional colonies from C7 treated cells. Transplantation of C7 expanded UCB grafts to immunodeficient mice resulted in significantly (p < .001) higher engraftment of human CD45+ and CD45+CD34+ cells in the PB and BM by day 21 compared to non‐expanded and cytokine expanded grafts. The C7 expanded grafts maintained long‐term human multilineage chimerism in the BM of primary recipients with sustained human CD45 cell engraftment in secondary recipients. In conclusion, a small molecule, C7, could allow for clinical development of expanded UCB grafts without pre‐culture stem cell enrichment that maintains in vitro and in vivo functionality. Stem Cells Translational Medicine 2018;7:376–393 PMID:29392885

ARS-Interacting Multi-Functional Protein 1 Induces Proliferation of Human Bone Marrow-Derived Mesenchymal Stem Cells by Accumulation of β-Catenin via Fibroblast Growth Factor Receptor 2-Mediated Activation of Akt

PubMed Central

Kim, Seo Yoon; Son, Woo Sung; Park, Min Chul; Kim, Chul Min; Cha, Byung Hyun; Yoon, Kang Jun; Lee, Soo-Hong

2013-01-01

ARS-Interacting Multi-functional Protein 1 (AIMP1) is a cytokine that is involved in the regulation of angiogenesis, immune activation, and fibroblast proliferation. In this study, fibroblast growth factor receptor 2 (FGFR2) was isolated as a binding partner of AIMP peptide (amino acids 6–46) in affinity purification using human bone marrow-derived mesenchymal stem cells (BMMSCs). AIMP1 peptide induced the proliferation of adult BMMSCs by activating Akt, inhibiting glycogen synthase kinase-3β, and thereby increasing the level of β-catenin. In addition, AIMP1 peptide induced the translocation of β-catenin to the nucleus and increased the transcription of c-myc and cyclin D1 by activating the β-catenin/T-cell factor (TCF) complex. By contrast, transfection of dominant negative TCF abolished the effect of AIMP1. The inhibition of Akt, using LY294002, abolished the accumulation and nuclear translocation of β-catenin induced by AIMP1, leading to a decrease in c-myc and cyclin D1 expression, which decreased the proliferation of BMMSCs. An intraperitoneal injection of AIMP1 peptide into C57/BL6 mice increased the colony formation of fibroblast-like cells. Fluorescence activated cell sorting analysis showed that the colony-forming cells were CD29+/CD44+/CD90+/CD105+/CD34−/CD45−, which is characteristic of MSCs. In addition, the fibroblast-like cells differentiated into adipocytes, chondrocytes, and osteocytes. Taken together, these data suggest that AIMP1 peptide promotes the proliferation of BMMSCs by activating the β-catenin/TCF complex via FGFR2-mediated activation of Akt, which leads to an increase in MSCs in peripheral blood. PMID:23672191

Autologous Adipose-Derived Tissue Matrix Part I: Biologic Characteristics.

PubMed

Schendel, Stephen A

2017-10-01

Autologous collagen is an ideal soft tissue filler and may serve as a matrix for stem cell implantation and growth. Procurement of autologous collagen has been limited, though, secondary to a sufficient source. Liposuction is a widely performed and could be a source of autologous collagen. The amount of collagen and its composition in liposuctioned fat remains unknown. The purpose of this research was to characterize an adipose-derived tissue-based product created using ultrasonic cavitation and cryo-grinding. This study evaluated the cellular and protein composition of the final product. Fat was obtained from individuals undergoing routine liposuction and was processed by a 2 step process to obtain only the connective tissue. The tissue was then evaluated by scanning electronic microscope, Western blot analysis, and flow cytometry. Liposuctioned fat was obtained from 10 individuals with an average of 298 mL per subject. After processing an average of 1 mL of collagen matrix was obtained from each 100 mL of fat. Significant viable cell markers were present in descending order for adipocytes > CD90+ > CD105+ > CD45+ > CD19+ > CD144+ > CD34+. Western blot analysis showed collagen type II, III, IV, and other proteins. Scanning electronic microscope study showed a regular pattern of cross-linked, helical collagen. Additionally, vital staing demonstrated that the cells were still viable after processing. Collagen and cells can be easily obtained from liposuctioned fat by ultrasonic separation without alteration of the overall cellular composition of the tissue. Implantation results in new collagen and cellular growth. Collagen matrix with viable cells for autologous use can be obtained from liposuctioned fat and may provide long term results. 5. © 2017 The American Society for Aesthetic Plastic Surgery, Inc. Reprints and permission: journals.permissions@oup.com

ARS-interacting multi-functional protein 1 induces proliferation of human bone marrow-derived mesenchymal stem cells by accumulation of β-catenin via fibroblast growth factor receptor 2-mediated activation of Akt.

PubMed

Kim, Seo Yoon; Son, Woo Sung; Park, Min Chul; Kim, Chul Min; Cha, Byung Hyun; Yoon, Kang Jun; Lee, Soo-Hong; Park, Sang Gyu

2013-10-01

ARS-Interacting Multi-functional Protein 1 (AIMP1) is a cytokine that is involved in the regulation of angiogenesis, immune activation, and fibroblast proliferation. In this study, fibroblast growth factor receptor 2 (FGFR2) was isolated as a binding partner of AIMP peptide (amino acids 6-46) in affinity purification using human bone marrow-derived mesenchymal stem cells (BMMSCs). AIMP1 peptide induced the proliferation of adult BMMSCs by activating Akt, inhibiting glycogen synthase kinase-3β, and thereby increasing the level of β-catenin. In addition, AIMP1 peptide induced the translocation of β-catenin to the nucleus and increased the transcription of c-myc and cyclin D1 by activating the β-catenin/T-cell factor (TCF) complex. By contrast, transfection of dominant negative TCF abolished the effect of AIMP1. The inhibition of Akt, using LY294002, abolished the accumulation and nuclear translocation of β-catenin induced by AIMP1, leading to a decrease in c-myc and cyclin D1 expression, which decreased the proliferation of BMMSCs. An intraperitoneal injection of AIMP1 peptide into C57/BL6 mice increased the colony formation of fibroblast-like cells. Fluorescence activated cell sorting analysis showed that the colony-forming cells were CD29(+)/CD44(+)/CD90(+)/CD105(+)/CD34(-)/CD45(-), which is characteristic of MSCs. In addition, the fibroblast-like cells differentiated into adipocytes, chondrocytes, and osteocytes. Taken together, these data suggest that AIMP1 peptide promotes the proliferation of BMMSCs by activating the β-catenin/TCF complex via FGFR2-mediated activation of Akt, which leads to an increase in MSCs in peripheral blood.

Motor Neuron Transdifferentiation of Neural Stem Cell from Adipose-Derived Stem Cell Characterized by Differential Gene Expression.

PubMed

Darvishi, Marzieh; Tiraihi, Taki; Mesbah-Namin, Seyed A; Delshad, AliReza; Taheri, Taher

2017-03-01

Adipose-derived stem cells (ADSC) are adult stem cells which can be induced into motor neuron-like cells (MNLC) with a preinduction-induction protocol. The purpose of this study is to generate MNLC from neural stem cells (NSC) derived from ADSC. The latter were isolated from the perinephric regions of Sprague-Dawley rats, transdifferentiated into neurospheres (NS) using B27, EGF, and bFGF. After generating NSC from the NS, they induced into MNLC by treating them with Shh and RA, then with GDNF, CNTF, BDNF, and NT-3. The ADSC lineage was evaluated by its mesodermal differentiation and was characterized by immunostaining with CD90, CD105, CD49d, CD106, CD31, CD45, and stemness genes (Oct4, Nanog, and Sox2). The NS and the NSC were evaluated by immunostaining with nestin, NF68, and Neurod1, while the MNLC were evaluated by ISLET1, Olig2, and HB9 genes. The efficiency of MNLC generation was more than 95 ± 1.4 % (mean ± SEM). The in vitro generated myotubes were innervated by the MNLC. The induced ADSC adopted multipolar motor neuron morphology, and they expressed ISLET1, Olig2, and HB9. We conclude that ADSC can be induced into motor neuron phenotype with high efficiency, associated with differential expression of the motor neuron gene. The release of MNLC synaptic vesicles was demonstrated by FM1-43, and they were immunostained with synaptophysin. This activity was correlated with the intracellular calcium ion shift and membrane depolarization upon stimulation as was demonstrated by the calcium indicator and the voltage-sensitive dye, respectively.

Update on the pathogenesis of Scleroderma: focus on circulating progenitor cells.

PubMed

Brunasso, Alexandra Maria Giovanna; Massone, Cesare

2016-01-01

In systemic sclerosis (SSc), the development of fibrosis seems to be a consequence of the initial ischemic process related to an endothelial injury. The initial trigger event in SSc is still unknown, but circulating progenitor cells (CPCs) might play a key role. Such cells have the ability to traffic into injury sites, exhibiting inflammatory features of macrophages, tissue remodeling properties of fibroblasts, and vasculogenesis functions of endothelial cells. The different subsets of CPCs described thus far in SSc arise from a pool of circulating monocyte precursors (CD14 (+) cells) and probably correspond to a different degree of differentiation of a single cell of origin. Several subsets of CPCs have been described in patients with SSc, all have a monocytic origin but may or may not express CD14, and all of these cells have the ability to give origin to endothelial cells, or collagen (Col)-producing cells, or both. We were able to identify six subsets of CPCs: pluripotent stem cells (CD14 (+), CD45 (+), and CD34 (+)), monocyte-derived multipotential cells (MOMCs) or monocyte-derived mesenchymal progenitors (CD14 (+), CD45 (+), CD34 (+), Col I (+), CD11b (+), CD68 (+), CD105 (+), and VEGFR1 (+)), early endothelial progenitor cells (EPCs) or monocytic pro-angiogenic hematopoietic cells or circulating hematopoietic cells (CD14 (+), CD45 (+), CD34 (low/-), VEGFR2 (+/-), CXCR4 (+), c-kit (+), and DC117 (+)), late EPCs (CD14 (-), CD133 (+), VEGFR2 (+), CD144 (+) [VE-cadherin (+)], and CD146 (+)), fibroblast-like cells (FLCs)/circulating Col-producing monocytes (CD14 (+), CD45 (+), CD34 (+/-), and Col I (+)), and fibrocytes (CD14 (-), CD45 (+), CD34 (+), Col I (+), and CXCR4 (+)). It has been demonstrated that circulating CD14 (+) monocytes with an activated phenotype are increased in patients with SSc when compared with normal subjects. CD14 (+), CD34 (+), and Col I (+) spindle-shaped cells have been found in increased numbers in lungs of SSc patients with interstitial lung disease. Elevated blood amounts of early EPCs have been found in patients with SSc by different groups of researchers and such levels correlate directly with the interstitial lung involvement. The prevalence of hematopoietic markers expressed by CPCs that migrate from blood into injury sites in SSc differs and changes according to the degree of differentiation. CXCR4 is the most commonly expressed marker, followed by CD34 and CD45 at an end stage of differentiation. Such difference also indicates a continuous process of cell differentiation that might relate to the SSc clinical phenotype (degree of fibrosis and vascular involvement). A deeper understanding of the role of each subtype of CPCs in the development of the disease will help us to better classify patients in order to offer them targeted approaches in the future.

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.

Effects of exogenous salinity (NaCl) gradient on Cd release in acidified contaminated brown soil

NASA Astrophysics Data System (ADS)

Zhang, Lina; Rong, Yong; Mao, Li; Gao, Zhiyuan; Liu, Xiaoyu; Dong, Zhicheng

2018-02-01

Taking acidified Cd contaminated brown soil in Yantai as the research object, based on different exogenous salinity (NaCl) gradient (0%, 0.3%, 0.6%, 0.9%, 1.5%, 2% and 5%), indoor simulation experiments of Cd release were carried out after field investigation. Results showed that there was a significantly positive relation (r>0.90) between Cd release concentration/amount/ratio and exogenous salt (NaCl). Besides, the more exogenous salt (NaCl) was added; maximum release concentration/amount of Cd appeared the earlier. It was found that exogenous salt (NaCl) addition could obviously promote Cd release from acidified Cd contaminated brown soil. It was believed that this could be mainly due to the cation exchange between Cd2+ and Na+, together with the dissociation and/or complexation between Cl- and Cd2+. In addition, available adsorption sites reduction by exchange base in soil causing Cd changed from solid state to soil solution was also a probable reason.

CD147 Required for Corneal Endothelial Lactate Transport

PubMed Central

Li, Shimin; Nguyen, Tracy T.; Bonanno, Joseph A.

2014-01-01

Purpose. CD147/basigin is a chaperone for lactate:H+ cotransporters (monocarboxylate transporters) MCT1 and MCT4. We tested the hypothesis that MCT1 and -4 in corneal endothelium contribute to lactate efflux from stroma to anterior chamber and that silencing CD147 expression would cause corneal edema. Methods. CD147 was silenced via small interfering ribonucleic acid (siRNA) transfection of rabbit corneas ex vivo and anterior chamber lenti-small hairpin RNA (shRNA) pseudovirus in vivo. CD147 and MCT expression was examined by Western blot, RT-PCR, and immunofluorescence. Functional effects were examined by measuring lactate-induced cell acidification, corneal lactate efflux, [lactate], central cornea thickness (CCT), and Azopt (a carbonic anhydrase inhibitor) sensitivity. Results. In ex vivo corneas, 100 nM CD147 siRNA reduced CD147, MCT1, and MCT4 expression by 85%, 79%, and 73%, respectively, while MCT2 expression was unaffected. CD147 siRNA decreased lactate efflux from 3.9 ± 0.81 to 1.5 ± 0.37 nmol/min, increased corneal [lactate] from 19.28 ± 7.15 to 56.73 ± 8.97 nmol/mg, acidified endothelial cells (pHi = 6.83 ± 0.07 vs. 7.19 ± 0.09 in control), and slowed basolateral lactate-induced acidification from 0.0034 ± 0.0005 to 0.0012 ± 0.0005 pH/s, whereas apical acidification was unchanged. In vivo, CD147 shRNA increased CCT by 28.1 ± 0.9 μm at 28 days; Azopt increased CCT to 24.4 ± 3.12 vs. 12.0 ± 0.48 μm in control, and corneal [lactate] was 47.63 ± 6.29 nmol/mg in shCD147 corneas and 17.82 ± 4.93 nmol/mg in paired controls. Conclusions. CD147 is required for the expression of MCT1 and MCT4 in the corneal endothelium. Silencing CD147 slows lactate efflux, resulting in stromal lactate accumulation and corneal edema, consistent with lactate efflux as a significant component of the corneal endothelial pump. PMID:24970254

CD147 required for corneal endothelial lactate transport.

PubMed

Li, Shimin; Nguyen, Tracy T; Bonanno, Joseph A

2014-06-26

CD147/basigin is a chaperone for lactate:H(+) cotransporters (monocarboxylate transporters) MCT1 and MCT4. We tested the hypothesis that MCT1 and -4 in corneal endothelium contribute to lactate efflux from stroma to anterior chamber and that silencing CD147 expression would cause corneal edema. CD147 was silenced via small interfering ribonucleic acid (siRNA) transfection of rabbit corneas ex vivo and anterior chamber lenti-small hairpin RNA (shRNA) pseudovirus in vivo. CD147 and MCT expression was examined by Western blot, RT-PCR, and immunofluorescence. Functional effects were examined by measuring lactate-induced cell acidification, corneal lactate efflux, [lactate], central cornea thickness (CCT), and Azopt (a carbonic anhydrase inhibitor) sensitivity. In ex vivo corneas, 100 nM CD147 siRNA reduced CD147, MCT1, and MCT4 expression by 85%, 79%, and 73%, respectively, while MCT2 expression was unaffected. CD147 siRNA decreased lactate efflux from 3.9 ± 0.81 to 1.5 ± 0.37 nmol/min, increased corneal [lactate] from 19.28 ± 7.15 to 56.73 ± 8.97 nmol/mg, acidified endothelial cells (pHi = 6.83 ± 0.07 vs. 7.19 ± 0.09 in control), and slowed basolateral lactate-induced acidification from 0.0034 ± 0.0005 to 0.0012 ± 0.0005 pH/s, whereas apical acidification was unchanged. In vivo, CD147 shRNA increased CCT by 28.1 ± 0.9 μm at 28 days; Azopt increased CCT to 24.4 ± 3.12 vs. 12.0 ± 0.48 μm in control, and corneal [lactate] was 47.63 ± 6.29 nmol/mg in shCD147 corneas and 17.82 ± 4.93 nmol/mg in paired controls. CD147 is required for the expression of MCT1 and MCT4 in the corneal endothelium. Silencing CD147 slows lactate efflux, resulting in stromal lactate accumulation and corneal edema, consistent with lactate efflux as a significant component of the corneal endothelial pump. Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.

Characterization of high-resistivity CdTe and Cd0.9Zn0.1Te crystals grown by Bridgman method for radiation detector applications

NASA Astrophysics Data System (ADS)

Mandal, Krishna C.; Krishna, Ramesh M.; Pak, Rahmi O.; Mannan, Mohammad A.

2014-09-01

CdTe and Cd0.9Zn0.1Te (CZT) crystals have been studied extensively for various applications including x- and γ-ray imaging and high energy radiation detectors. The crystals were grown from zone refined ultra-pure precursor materials using a vertical Bridgman furnace. The growth process has been monitored, controlled, and optimized by a computer simulation and modeling program developed in our laboratory. The grown crystals were thoroughly characterized after cutting wafers from the ingots and processed by chemo-mechanical polishing (CMP). The infrared (IR) transmission images of the post-treated CdTe and CZT crystals showed average Te inclusion size of ~10 μm for CdTe and ~8 μm for CZT crystal. The etch pit density was ≤ 5×104 cm-2 for CdTe and ≤ 3×104 cm-2 for CZT. Various planar and Frisch collar detectors were fabricated and evaluated. From the current-voltage measurements, the electrical resistivity was estimated to be ~ 1.5×1010 Ω-cm for CdTe and 2-5×1011 Ω-cm for CZT. The Hecht analysis of electron and hole mobility-lifetime products (μτe and μτh) showed μτe = 2×10-3 cm2/V (μτh = 8×10-5 cm2/V) and 3-6×10-3 cm2/V (μτh = 4- 6×10-5 cm2/V) for CdTe and CZT, respectively. Detectors in single pixel, Frisch collar, and coplanar grid geometries were fabricated. Detectors in Frisch grid and guard-ring configuration were found to exhibit energy resolution of 1.4% and 2.6 %, respectively, for 662 keV gamma rays. Assessments of the detector performance have been carried out also using 241Am (60 keV) showing energy resolution of 4.2% FWHM.

Articular cartilage-derived cells hold a strong osteogenic differentiation potential in comparison to mesenchymal stem cells in vitro

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

Salamon, Achim, E-mail: achim.salamon@med.uni-rostock.de; Jonitz-Heincke, Anika, E-mail: anika.jonitz@med.uni-rostock.de; Adam, Stefanie, E-mail: stefanie.adam@med.uni-rostock.de

Cartilaginous matrix-degenerative diseases like osteoarthritis (OA) are characterized by gradual cartilage erosion, and also by increased presence of cells with mesenchymal stem cell (MSC) character within the affected tissues. Moreover, primary chondrocytes long since are known to de-differentiate in vitro and to be chondrogenically re-differentiable. Since both findings appear to conflict with each other, we quantitatively assessed the mesenchymal differentiation potential of OA patient cartilage-derived cells (CDC) towards the osteogenic and adipogenic lineage in vitro and compared it to that of MSC isolated from adipose tissue (adMSC) of healthy donors. We analyzed expression of MSC markers CD29, CD44, CD105, andmore » CD166, and, following osteogenic and adipogenic induction in vitro, quantified their expression of osteogenic and adipogenic differentiation markers. Furthermore, CDC phenotype and proliferation were monitored. We found that CDC exhibit an MSC CD marker expression pattern similar to adMSC and a similar increase in proliferation rate during osteogenic differentiation. In contrast, the marked reduction of proliferation observed during adipogenic differentiation of adMSC was absent in CDC. Quantification of differentiation markers revealed a strong osteogenic differentiation potential for CDC, however almost no capacity for adipogenic differentiation. Since in the pathogenesis of OA, cartilage degeneration coincides with high bone turnover rates, the high osteogenic differentiation potential of OA patient-derived CDC may affect clinical therapeutic regimens aiming at autologous cartilage regeneration in these patients. - Highlights: • We analyze the mesenchymal differentiation capacity of cartilage-derived cells (CDC). • CDC express mesenchymal stem cell (MSC) markers CD29, CD44, CD105, and CD166. • CDC and MSC proliferation is reduced in adipogenesis and increased in osteogenesis. • Adipogenic differentiation is virtually absent in CDC, but strong in MSC. • Osteogenic differentiation is significantly stronger for CDC than for MSC.« less

Effects of bacteria on CdS thin films used in technological devices

NASA Astrophysics Data System (ADS)

Alpdoğan, S.; Adıgüzel, A. O.; Sahan, B.; Tunçer, M.; Metin Gubur, H.

2017-04-01

Cadmium sulfide (CdS) thin films were fabricated on glass substrates by the chemical bath deposition method at 70 {}^\\circ \\text{C} considering deposition times ranging from 2 h to 5 h. The optical band gaps of CdS thin films were found to be in the 2.42-2.37 eV range. CdS thin films had uniform spherical nano-size grains which had polycrystalline, hexagonal and cubic phases. The films had a characteristic electrical resistivity of the order of {{10}5} Ω \\text{cm} and n-type conductivity at room condition. CdS thin films were incubated in cultures of B.domonas aeruginosa and Staphylococcus aureus, which exist abundantly in the environment, and form biofilms. SEM images showed that S. aureus and K. pneumonia were detected significantly on the film surfaces with a few of P. aeruginosa and B. subtilis cells attached. CdS thin film surface exhibits relatively good resistance to the colonization of P. aeruginosa and B. subtilis. Optical results showed that the band gap of CdS thin films which interacted with the bacteria is 2.42 \\text{eV} . The crystal structure and electrical properties of CdS thin films were not affected by bacterial adhesion. The antimicrobial effect of CdS nanoparticles was different for different bacterial strains.

Application and comparison of high-speed countercurrent chromatography and high performance liquid chromatography in preparative enantioseparation of α-substitution mandelic acids

PubMed Central

Tong, Shengqiang; Zhang, Hu; Shen, Mangmang; Ito, Yoichiro; Yan, Jizhong

2014-01-01

Preparative enantioseparations of α-cyclopentylmandelic acid and α-methylmandelic acid by high-speed countercurrent chromatography (HSCCC) and high performance liquid chromatography (HPLC) were compared using hydroxypropy-β-cyclodextrin (HP-β-CD) and sulfobutyl ether-β-cyclodextrin (SBE-β-CD) as the chiral mobile phase additives. In preparative HPLC the enantioseparation was achieved on the ODS C18 reverse phase column with the mobile phase composed of a mixture of acetonitrile and 0.10 mol L−1 phosphate buffer at pH 2.68 containing 20 mmol L−1 HP-β-CD for α-cyclopentylmandelic acid and 20 mmol L−1 SBE-β-CD for α-methylmandelic acid. The maximum sample size for α-cyclopentylmandelic acid and α-methylmandelic acid was only about 10 mg and 5 mg, respectively. In preparative HSCCC the enantioseparations of these two racemates were performed with the two-phase solvent system composed of n-hexane-methyl tert.-butyl ether-0.1 molL−1 phosphate buffer solution at pH 2.67 containing 0.1 mol L−1 HP-β-CD for α-cyclopentylmandelic acid (8.5:1.5:10, v/v/v) and 0.1 mol L−1 SBE-β-CD for α-methylmandelic acid (3:7:10, v/v/v). Under the optimum separation conditions, total 250 mg of racemic α-cyclopentylmandelic acid could be completely enantioseparated by HSCCC with HP-β-CD as a chiral mobile phase additive in a single run, yielding 105-110 mg of enantiomers with 95-98% purity and 85-90% recovery. But, no complete enantioseparation of α-methylmandelic acid was achieved by preparative HSCCC with either of the chiral selectors due to their limited enantioselectivity. In this paper preparative enantioseparation by HSCCC and HPLC was compared from various aspects. PMID:25983356

Application and comparison of high-speed countercurrent chromatography and high performance liquid chromatography in preparative enantioseparation of α-substitution mandelic acids.

PubMed

Tong, Shengqiang; Zhang, Hu; Shen, Mangmang; Ito, Yoichiro; Yan, Jizhong

2015-04-01

Preparative enantioseparations of α-cyclopentylmandelic acid and α-methylmandelic acid by high-speed countercurrent chromatography (HSCCC) and high performance liquid chromatography (HPLC) were compared using hydroxypropy-β-cyclodextrin (HP-β-CD) and sulfobutyl ether-β-cyclodextrin (SBE-β-CD) as the chiral mobile phase additives. In preparative HPLC the enantioseparation was achieved on the ODS C 18 reverse phase column with the mobile phase composed of a mixture of acetonitrile and 0.10 mol L -1 phosphate buffer at pH 2.68 containing 20 mmol L -1 HP-β-CD for α-cyclopentylmandelic acid and 20 mmol L -1 SBE-β-CD for α-methylmandelic acid. The maximum sample size for α-cyclopentylmandelic acid and α-methylmandelic acid was only about 10 mg and 5 mg, respectively. In preparative HSCCC the enantioseparations of these two racemates were performed with the two-phase solvent system composed of n -hexane-methyl tert. -butyl ether-0.1 molL -1 phosphate buffer solution at pH 2.67 containing 0.1 mol L -1 HP-β-CD for α-cyclopentylmandelic acid (8.5:1.5:10, v/v/v) and 0.1 mol L -1 SBE-β-CD for α-methylmandelic acid (3:7:10, v/v/v). Under the optimum separation conditions, total 250 mg of racemic α-cyclopentylmandelic acid could be completely enantioseparated by HSCCC with HP-β-CD as a chiral mobile phase additive in a single run, yielding 105-110 mg of enantiomers with 95-98% purity and 85-90% recovery. But, no complete enantioseparation of α-methylmandelic acid was achieved by preparative HSCCC with either of the chiral selectors due to their limited enantioselectivity. In this paper preparative enantioseparation by HSCCC and HPLC was compared from various aspects.

Toxic metals in cigarettes and human health risk assessment associated with inhalation exposure.

PubMed

Benson, Nsikak U; Anake, Winifred U; Adedapo, Adebusayo E; Fred-Ahmadu, Omowunmi H; Ayejuyo, Olusegun O

2017-11-08

This study evaluated the concentrations of cadmium (Cd), copper (Cu), iron (Fe), manganese (Mn), lead (Pb), and zinc (Zn) in 10 branded cigarettes commonly consumed in Nigeria. Chemical sequential extraction method and pseudo-total metal digestion procedure were used for extraction of metals from filler tobacco and filter samples. Samples were analyzed using flame atomic absorption spectrometry (FAAS). The filler tobacco of cigarettes had Cd, Cu, Fe, Mn, Pb, and Zn concentrations in the ranges of 5.90-7.94, 18.26-34.94, 192.61-3494.05, 44.67-297.69, 17.21-74.78, and 47.02-167.31 μg/cigarette, respectively. The minimum and maximum concentrations in the filter samples were 8.67-12.34 μg/g of Cd, 1.77-36.48 μg/g of Cu, 1.83-15.27 μg/g of Fe, 3.82-7.44 μg/g of Mn, 4.09-13.78 μg/g of Pb, and 30.07-46.70 μg/g of Zn. The results of this study showed that the concentrations of heavy metals in the filler tobacco samples were consistently higher than those obtained for the cigarette filters except for Cd. Toxic metals were largely found in the most labile chemical fractions. Moderate to very high risks are found associated with potential exposure to Cd and Pb. The carcinogenic risks posed by Cd and Pb ranged between 1.87E-02 and 2.52E-02, 1.05E-03 and 4.76E-03, respectively, while the non-carcinogenic risk estimates for Cd and Pb were greater than 1.0 (HI > 1). Toxic metals in cigarette may have significant carcinogenic and non-carcinogenic health effects associated with inhalation exposure. Continuous monitoring and regulations of the ingredients of imported and locally produced tobacco products are advocated.

Ethanol Tolerance Affects Endogenous Adenosine Signaling in Mouse Hippocampus

PubMed Central

Zhang, Dali; Xiong, Wei; Jackson, Michael F.

2016-01-01

Ethanol has many pharmacological effects, including increases in endogenous adenosine levels and adenosine receptor activity in brain. Ethanol consumption is associated with both positive and negative health outcomes, but tolerance to the behavioral effects of ethanol can lead to increased consumption, which increases the risk of negative health outcomes. The present study was performed to test whether a 7-day treatment with ethanol is linked to reduced adenosine signaling and whether this is a consequence of reduced ecto-5′-nucleotidase activity. Wild-type (CD73+/+) and ecto-5′-nucleotidase-deficient (CD73−/−) mice were treated with ethanol (2 g/kg) or saline for 7 days. In CD73+/+ mice, repeated ethanol treatment reduced the hypothermic and ataxic effects of acute ethanol, indicating the development of tolerance to the acute effects of ethanol. In CD73+/+ mice, this 7-day ethanol treatment led to increased hippocampal synaptic activity and reduced adenosine A1 receptor activity under both basal and low Mg2+ conditions. These effects of ethanol tolerance were associated with an 18% decrease in activity of ecto-5′-nucleotidase activity in hippocampal cell membranes. In contrast, ethanol treatment was not associated with changes in synaptic activity or adenosine signaling in hippocampus from CD73−/− mice. These data indicate that ethanol treatment is associated with a reduction in adenosine signaling through adenosine A1 receptors in hippocampus, mediated, at least in part, via reduced ecto-5′-nucleotidase activity. PMID:27189965

Ethanol Tolerance Affects Endogenous Adenosine Signaling in Mouse Hippocampus.

PubMed

Zhang, Dali; Xiong, Wei; Jackson, Michael F; Parkinson, Fiona E

2016-07-01

Ethanol has many pharmacological effects, including increases in endogenous adenosine levels and adenosine receptor activity in brain. Ethanol consumption is associated with both positive and negative health outcomes, but tolerance to the behavioral effects of ethanol can lead to increased consumption, which increases the risk of negative health outcomes. The present study was performed to test whether a 7-day treatment with ethanol is linked to reduced adenosine signaling and whether this is a consequence of reduced ecto-5'-nucleotidase activity. Wild-type (CD73(+/+)) and ecto-5'-nucleotidase-deficient (CD73(-/-)) mice were treated with ethanol (2 g/kg) or saline for 7 days. In CD73(+/+) mice, repeated ethanol treatment reduced the hypothermic and ataxic effects of acute ethanol, indicating the development of tolerance to the acute effects of ethanol. In CD73(+/+) mice, this 7-day ethanol treatment led to increased hippocampal synaptic activity and reduced adenosine A1 receptor activity under both basal and low Mg(2+) conditions. These effects of ethanol tolerance were associated with an 18% decrease in activity of ecto-5'-nucleotidase activity in hippocampal cell membranes. In contrast, ethanol treatment was not associated with changes in synaptic activity or adenosine signaling in hippocampus from CD73(-/-) mice. These data indicate that ethanol treatment is associated with a reduction in adenosine signaling through adenosine A1 receptors in hippocampus, mediated, at least in part, via reduced ecto-5'-nucleotidase activity. Copyright © 2016 The Author(s).

Evaluation of synovium-derived mesenchymal stem cells and 3D printed nanocomposite scaffolds for tissue engineering

NASA Astrophysics Data System (ADS)

Pan, Jian-Feng; Li, Shuo; Guo, Chang-An; Xu, Du-Liang; Zhang, Feng; Yan, Zuo-Qin; Mo, Xiu-Mei

2015-08-01

Stem cells and scaffolds play a very important role in tissue engineering. Here, we isolated synovium-derived mesenchymal stem cells (SMSCs) from synovial membrane tissue and characterized stem-cell properties. Gelatin nanoparticles (NP) were prepared using a two-step desolvation method and then pre-mixed into different host matrix (silk fibroin (SF), gelatin (Gel), or SF-Gel mixture) to generate various 3D printed nanocomposite scaffolds (NP/SF, NP/SF-Gel, NP/Gel-1, and NP/Gel-2). The microstructure was examined by scanning electron microscopy. Biocompatibility assessment was performed through CCK-8 assay by coculturing with SMSCs at 1, 3, 7 and 14 days. According to the results, SMSCs are similar to other MSCs in their surface epitope expression, which are negative for CD45 and positive for CD44, CD90, and CD105. After incubation in lineage-specific medium, SMSCs could differentiate into chondrocytes, osteocytes and adipocytes. 3D printed nanocomposite scaffolds exhibited a good biocompatibility in the process of coculturing with SMSCs and had no negative effect on cell behavior. The study provides a strategy to obtain SMSCs and fabricate 3D printed nanocomposite scaffolds, the combination of which could be used for practical applications in tissue engineering.

Immunotherapy of BALB/c mice bearing Ehrlich ascites tumor with vitamin D-binding protein-derived macrophage activating factor.

PubMed

Yamamoto, N; Naraparaju, V R

1997-06-01

Vitamin D3-binding protein (DBP; human DBP is known as Gc protein) is the precursor of macrophage activating factor (MAF). Treatment of mouse DBP with immobilized beta-galactosidase or treatment of human Gc protein with immobilized beta-galactosidase and sialidase generated a remarkably potent MAF, termed DBPMAF or GcMAF, respectively. The domain of Gc protein responsible for macrophage activation was cloned and enzymatically converted to the cloned MAF, designated CdMAF. In Ehrlich ascites tumor-bearing mice, tumor-specific serum alpha-N-acetylgalactosaminidase (NaGalase) activity increased linearly with time as the transplanted tumor cells grew in the peritoneal cavity. Therapeutic effects of DBPMAF, GcMAF, and CdMAF on mice bearing Ehrlich ascites tumor were assessed by survival time, the total tumor cell count in the peritoneal cavity, and serum NaGalase activity. Mice that received a single administration of DBPMAF or GcMAF (100 pg/mouse) on the same day after transplantation of tumor (1 x 10(5) cells) showed a mean survival time of 35 +/- 4 days, whereas tumor-bearing controls had a mean survival time of 16 +/- 2 days. When mice received the second DBPMAF or GcMAF administration at day 4, they survived more than 50 days. Mice that received two DBPMAF administrations, at days 4 and 8 after transplantation of 1 x 10(5) tumor cells, survived up to 32 +/- 4 days. At day 4 posttransplantation, the total tumor cell count in the peritoneal cavity was approximately 5 x 10(5) cells. Mice that received two DBPMAF administrations, at days 0 and 4 after transplantation of 5 x 10(5) tumor cells, also survived up to 32 +/- 4 days, while control mice that received the 5 x 10(5) ascites tumor cells only survived for 14 +/- 2 days. Four DBPMAF, GcMAF, or CdMAF administrations to mice transplanted with 5 x 10(5) Ehrlich ascites tumor cells with 4-day intervals showed an extended survival of at least 90 days and an insignificantly low serum NaGalase level between days 30 and 90.

IRGM Variants and Susceptibility to Inflammatory Bowel Disease in the German Population

PubMed Central

Bues, Stephanie; Stallhofer, Johannes; Fries, Christoph; Olszak, Torsten; Tsekeri, Eleni; Wetzke, Martin; Beigel, Florian; Steib, Christian; Friedrich, Matthias; Göke, Burkhard; Diegelmann, Julia; Czamara, Darina; Brand, Stephan

2013-01-01

Background & Aims Genome-wide association studies identified the autophagy gene IRGM to be strongly associated with Crohn's disease (CD) but its impact in ulcerative colitis (UC), its phenotypic effects and potential epistatic interactions with other IBD susceptibility genes are less clear which we therefore analyzed in this study. Methodology/Principal Findings Genomic DNA from 2060 individuals including 817 CD patients, 283 UC patients, and 961 healthy, unrelated controls (all of Caucasian origin) was analyzed for six IRGM single nucleotide polymorphisms (SNPs) (rs13371189, rs10065172 = p.Leu105Leu, rs4958847, rs1000113, rs11747270, rs931058). In all patients, a detailed genotype-phenotype analysis and testing for epistasis with the three major CD susceptibility genes NOD2, IL23R and ATG16L1 were performed. Our analysis revealed an association of the IRGM SNPs rs13371189 (p = 0.02, OR 1.31 [95% CI 1.05–1.65]), rs10065172 = p.Leu105Leu (p = 0.016, OR 1.33 [95% CI 1.06–1.66]) and rs1000113 (p = 0.047, OR 1.27 [95% CI 1.01–1.61]) with CD susceptibility. There was linkage disequilibrium between these three IRGM SNPs. In UC, several IRGM haplotypes were weakly associated with UC susceptibility (p<0.05). Genotype-phenotype analysis revealed no significant associations with a specific IBD phenotype or ileal CD involvement. There was evidence for weak gene-gene-interaction between several SNPs of the autophagy genes IRGM and ATG16L1 (p<0.05), which, however, did not remain significant after Bonferroni correction. Conclusions/Significance Our results confirm IRGM as susceptibility gene for CD in the German population, supporting a role for the autophagy genes IRGM and ATG16L1 in the pathogenesis of CD. PMID:23365659

Evidence of two distinct functionally specialized fibroblast lineages in breast stroma.

PubMed

Morsing, Mikkel; Klitgaard, Marie Christine; Jafari, Abbas; Villadsen, René; Kassem, Moustapha; Petersen, Ole William; Rønnov-Jessen, Lone

2016-11-03

The terminal duct lobular unit (TDLU) is the most dynamic structure in the human breast and the putative site of origin of human breast cancer. Although stromal cells contribute to a specialized microenvironment in many organs, this component remains largely understudied in the human breast. We here demonstrate the impact on epithelium of two lineages of breast stromal fibroblasts, one of which accumulates in the TDLU while the other resides outside the TDLU in the interlobular stroma. The two lineages are prospectively isolated by fluorescence activated cell sorting (FACS) based on different expression levels of CD105 and CD26. The characteristics of the two fibroblast lineages are assessed by immunocytochemical staining and gene expression analysis. The differentiation capacity of the two fibroblast populations is determined by exposure to specific differentiating conditions followed by analysis of adipogenic and osteogenic differentiation. To test whether the two fibroblast lineages are functionally imprinted by their site of origin, single cell sorted CD271 low /MUC1 high normal breast luminal epithelial cells are plated on fibroblast feeders for the observation of morphological development. Epithelial structure formation and polarization is shown by immunofluorescence and digitalized quantification of immunoperoxidase-stained cultures. Lobular fibroblasts are CD105 high /CD26 low while interlobular fibroblasts are CD105 low /CD26 high . Once isolated the two lineages remain phenotypically stable and functionally distinct in culture. Lobular fibroblasts have properties in common with bone marrow derived mesenchymal stem cells and they specifically convey growth and branching morphogenesis of epithelial progenitors. Two distinct functionally specialized fibroblast lineages exist in the normal human breast, of which the lobular fibroblasts have properties in common with mesenchymal stem cells and support epithelial growth and morphogenesis. We propose that lobular fibroblasts constitute a specialized microenvironment for human breast luminal epithelial progenitors, i.e. the putative precursors of breast cancer.

Acetate supplementation modulates brain adenosine metabolizing enzymes and adenosine A₂A receptor levels in rats subjected to neuroinflammation.

PubMed

Smith, Mark D; Bhatt, Dhaval P; Geiger, Jonathan D; Rosenberger, Thad A

2014-06-04

Acetate supplementation reduces neuroglia activation and pro-inflammatory cytokine expression in rat models of neuroinflammation and Lyme neuroborreliosis. Because single-dose glyceryl triacetate (GTA) treatment increases brain phosphocreatine and reduces brain AMP levels, we postulate that GTA modulates adenosine metabolizing enzymes and receptors, which may be a possible mechanism to reduce neuroinflammation. To test this hypothesis, we quantified the ability of GTA to alter brain levels of ecto-5'-nucleotidase (CD73), adenosine kinase (AK), and adenosine A2A receptor using western blot analysis and CD73 activity by measuring the rate of AMP hydrolysis. Neuroinflammation was induced by continuous bacterial lipopolysaccharide (LPS) infusion in the fourth ventricle of the brain for 14 and 28 days. Three treatment strategies were employed, one and two where rats received prophylactic GTA through oral gavage with LPS infusion for 14 or 28 days. In the third treatment regimen, an interventional strategy was used where rats were subjected to 28 days of neuroinflammation, and GTA treatment was started on day 14 following the start of the LPS infusion. We found that rats subjected to neuroinflammation for 28 days had a 28% reduction in CD73 levels and a 43% increase in AK levels that was reversed with prophylactic acetate supplementation. CD73 activity in these rats was increased by 46% with the 28-day GTA treatment compared to the water-treated rats. Rats subjected to neuroinflammation for 14 days showed a 50% increase in levels of the adenosine A2A receptor, which was prevented with prophylactic acetate supplementation. Interventional GTA therapy, beginning on day 14 following the induction of neuroinflammation, resulted in a 67% increase in CD73 levels and a 155% increase in adenosine A2A receptor levels. These results support the hypothesis that acetate supplementation can modulate brain CD73, AK and adenosine A2A receptor levels, and possibly influence purinergic signaling.

Expression of recombinant CD59 with an N-terminal peptide epitope facilitates analysis of residues contributing to its complement-inhibitory function.

PubMed

Zhou, Q; Zhao, J; Hüsler, T; Sims, P J

1996-10-01

CD59 is a plasma membrane-anchored glycoprotein that serves to protect human cells from lysis by the C5b-9 complex of complement. The immunodominant epitopes of CD59 are known to be sensitive to disruption of native tertiary structure, complicating immunological measurement of expressed mutant constructs for structure function analysis. In order to quantify cell-surface expression of wild-type and mutant forms of this complement inhibitor, independent of CD59 antigen, an 11-residue peptide (TAG) recognized by monoclonal antibody (mAb) 9E10 was inserted before the N-terminal codon (L1) of mature CD59, in a pcDNA3 expression plasmid. SV-T2 cells were transfected with this plasmid, yielding cell lines expressing 0 to > 10(5) CD59/cell. The TAG-CD59 fusion protein was confirmed to be GPI-anchored, N-glycosylated and showed identical complement-inhibitory function to wild-type CD59, lacking the TAG peptide sequence. Using this construct, the contribution of each of four surface-localized aromatic residues (4Y, 47F, 61Y, and 62Y) to CD59's complement-inhibitory function was examined. These assays revealed normal surface expression with complete loss of complement-inhibitory function in the 4Y --> S, 47F --> G and 61Y --> S mutants. By contrast, 62Y --> S mutants retained approximately 40% of function of wild-type CD59. These studies confirmed the utility of the TAG-CD59 construct for quantifying CD59 surface expression and activity, and implicate surface aromatic residues 4Y, 47F, 61Y and 62Y as essential to maintenance of CD59's normal complement-regulatory function.

40 CFR 413.14 - Pretreatment standards for existing sources.

Code of Federal Regulations, 2011 CFR

2011-07-01

... exceed CN, T 1.9 1.0 Cu 4.5 2.7 Ni 4.1 2.6 Cr 7.0 4.0 Zn 4.2 2.6 Pb .6 .4 Cd 1.2 .7 Total metals 10.5 6.8... monitoring days shall not exceed CN, T 74 39 Cu 176 105 Ni 160 100 Cr 273 156 Zn 164 102 Pb 23 16 Cd 47 29...

40 CFR 413.14 - Pretreatment standards for existing sources.

Code of Federal Regulations, 2010 CFR

2010-07-01

... exceed CN, T 1.9 1.0 Cu 4.5 2.7 Ni 4.1 2.6 Cr 7.0 4.0 Zn 4.2 2.6 Pb .6 .4 Cd 1.2 .7 Total metals 10.5 6.8... monitoring days shall not exceed CN, T 74 39 Cu 176 105 Ni 160 100 Cr 273 156 Zn 164 102 Pb 23 16 Cd 47 29...

Iron differentially modulates the CD4-lck and CD8-lck complexes in resting peripheral blood T-lymphocytes.

PubMed

Arosa, F A; de Sousa, M

1995-03-01

Clinical and experimental studies performed in situations of iron overload have demonstrated that iron impairs several T-cell functions. We have examined the effect of iron in the form of ferric citrate on the CD4-lck and CD8-lck complexes in view of the key role played by the tyrosine kinase p56lck in regulating T-cell functions. Ferric citrate was seen to differentially modulate the CD4-lck and CD8-lck complexes in resting peripheral blood T-lymphocytes (PBLs) cultured in the presence of this metal salt for periods of 20 to 24 hr. Thus, whereas ferric citrate invariably induced a marked decrease in the in vitro activity of the CD4-associated lck by three- to fourfold at 100 microM (P < 3 x 10(-5)), it did not affect significantly the in vitro activity of the CD8-associated lck, although modest decreases were observed in some experiments. Immunoprecipitation and subsequent lck-immunoblotting revealed that the marked decrease in CD4-lck activity induced by 100 microM of ferric citrate was due to a decrease in the amount of p56lck on CD4 immunoprecipitates. Furthermore, flow cytometry analysis showed a decrease in the surface expression of the CD4 molecule in iron-treated PBLs, as judged by a decrease in the mean fluorescence intensity (MFI), that was accompanied by a decrease in the percentage of CD4+ T-lymphocytes. In marked contrast, whereas the surface expression of the CD8 molecule was slightly decreased, the percentage of CD8+ T-lymphocytes remained constant. This differential effect of ferric citrate on the CD4+ and CD8+ T-cell subsets led to a marked decrease in the CD4/CD8 ratios in iron-treated PBLs after the 20- to 24-hr period (P < 0.001). The present results indicate that iron in the form of ferric citrate can modulate key molecules involved in the process of T-cell activation and therefore influence T-cell-mediated functions.

Econometric models for predicting confusion crop ratios

NASA Technical Reports Server (NTRS)

Umberger, D. E.; Proctor, M. H.; Clark, J. E.; Eisgruber, L. M.; Braschler, C. B. (Principal Investigator)

1979-01-01

Results for both the United States and Canada show that econometric models can provide estimates of confusion crop ratios that are more accurate than historical ratios. Whether these models can support the LACIE 90/90 accuracy criterion is uncertain. In the United States, experimenting with additional model formulations could provide improved methods models in some CRD's, particularly in winter wheat. Improved models may also be possible for the Canadian CD's. The more aggressive province/state models outperformed individual CD/CRD models. This result was expected partly because acreage statistics are based on sampling procedures, and the sampling precision declines from the province/state to the CD/CRD level. Declining sampling precision and the need to substitute province/state data for the CD/CRD data introduced measurement error into the CD/CRD models.

[Cellular immunophenotypes in 97 adults with acute leukemia].

PubMed

Piedras, J; López-Karpovitch, X; Cárdenas, M R

1997-01-01

To analyze hematopoietic cell surface antigen reactivity in acute leukemia (AL) by flow cytometry and identify acute mixed-lineage leukemias (AMLL) employing the most widely accepted criteria. Ninety seven patients with de novo AL were studied. Cell surface antigens were investigated with monoclonal antibodies directed to: B lymphoid (CD10, CD19, CD20, CD21, CD22); T lymphoid (CD2, CD3, CD5, CD7); and myeloid (CD13, CD14, CD15, CD33, CD41) cell lineages. Maturation cell-associated antigens (CD34, HLA-DR and TdT) were also studied. Twelve patients unclassified by cytomorphology could be classified by immunophenotype. Using cytomorphologic, cytochemical and immunophenotypic data, 54 cases corresponded to acute lymphoblastic leukemia (ALL) and 43 were acute myeloblastic leukemia (AML). In All there were 63% B lineage, 15% T, 7% T/B, 6% undifferentiated and 9% mixed-lineage (coexpression of two or more myeloid-associated antigens). In AML, myeloid immunophenotype was observed in 86% undifferentiated in 2%, and mixed-lineage in 12% (coexpression of two or more lymphoid-associated antigens). In addition, 26% of ALL cases and 12% of AML cases expressed a single myeloid and lymphoid antigen respectively. The most common aberrant antigens in ALL and AML were CD13 and CD7 respectively. The highest frequency of CD34 antigen expression (90%) was detected in patients with AMLL. Flow cytometric immunophenotypic analysis allowed to: a) establish diagnosis in cytomorphologically unclassified cases; b) identify AMLL with a frequency similar to that reported in other series; and c) confirm the heterogeneity of AL.

Peripheral Blood CD38 Bright CD8+ Effector Memory T Cells Predict Acute Graft-versus-Host Disease.

PubMed

Khandelwal, Pooja; Lane, Adam; Chaturvedi, Vijaya; Owsley, Erika; Davies, Stella M; Marmer, Daniel; Filipovich, Alexandra H; Jordan, Michael B; Marsh, Rebecca A

2015-07-01

Acute graft-versus-host disease (aGVHD) is mediated by allogeneic T cell responses. We hypothesized that increases of peripheral blood-activated CD8+ effector memory T (TEM) cells would be observed after hematopoietic stem cell transplantation (HSCT) before onset of aGVHD symptoms. Blood was collected twice weekly after HSCT for 7 weeks in 49 consecutive pediatric and adult HSCT recipients. Samples were incubated with fluorochrome-conjugated antibodies against CD45, CD3, CD8, CD38, CD45RA, and CCR7 and analyzed using flow cytometry. TEM cells were defined as CD3+ CD8+ CCR7- CD45RA(-) lymphocytes. CD38 expression was used as a marker of T cell activation. Patients were followed for 100 days for development of aGVHD. Twenty-three patients developed grade 1 to 4 aGVHD at a median of 37 days (range, 15 to 79 days) after HCST. Absolute CD38 bright CD8+ TEM of > 35 cells/μL predicted aGVHD at a median of 8 days (range, 1 to 34) before aGVHD onset with a sensitivity of 82.6% and specificity of 91.6%. The cumulative incidence of aGVHD was 90% in patients with absolute CD38 bright CD8+ TEM >35 cells/μL and 15% in patients without (P < .0001). Quantification of CD38 bright CD8+ TEM cells may predict aGVHD in children and young adult HSCT recipients. Copyright © 2015 American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.

Interaction of Synuclein and Inflammation in Dopaminergic Neurodegeneration

DTIC Science & Technology

2014-06-01

are less responsive to the blocking effects of anti-CD36 antibodies. The CD36 inhibitor, ursolic acid , at concentrations of 10-5 and 10-6, reduced...N9 chemotaxis across native synuclein by almost 50%. Equally interesting is the fact that ursolic acid had a small effect on CD36-deficient N9 cell...chemotaxis across synuclein which suggests that ursolic acid may also inhibit other scavenger receptors such as SR1 and RAGE. Ursolic acid was

CD30, a marker to detect the high-risk kidney transplant recipients.

PubMed

Spiridon, Camelia; Nikaein, Afzal; Lerman, Mark; Hunt, Judson; Dickerman, Richard; Mack, Michael

2008-01-01

Sensitization of potential renal transplant recipients may impact the selection of donors and the outcome of transplant. Another element of the potential kidney transplant recipient immune system that provides useful information regarding the transplant outcome is the immunologic CD30 molecule. This study shows a significant correlation between the pre-transplant high level of soluble CD30 and increased incidence of post-transplant infection. Only 7/34 (20.6%) of the patients who had a low level of sCD30 (< 90 U/mL) developed infection as compared with the 25/58 (43.1%) of the patients who had a high level (> 90 U/mL) of sCD30 (p < 0.04). Higher level of sCD30 pre-transplant was also correlated with the increased level of serum creatinine (p < 0.05) and pre-transplant malignancy (p < 0.04). A significant higher level of sCD30 was also noted among females (74%), as compared with males (50%) with p < 0.03. In addition, significant effect of 3-6 human leukocyte antigen (HLA) mismatches on rejection was seen. These results show that higher pre-transplant immunologic reactivity measured by sCD30 level was associated with post-transplant outcome. The high level of sCD30 among females may indicate an active immunologic status, perhaps because of previous pregnancies.

Exciton and intracenter radiative recombination in ZnMnTe and CdMnTe quantum wells with optically active manganese ions

NASA Astrophysics Data System (ADS)

Agekyan, V. F.; Akai, I.; Vasil'Ev, N. N.; Karasawa, T.; Karczewski, G.; Serov, A. Yu.; Filosofov, N. G.

2007-06-01

The emission spectra of Zn1-x Mn x Te/Zn0.6Mg0.4Te and Cd1-x Mn x Te/Cd0.5Mg0.5Te quantum-well structures with different manganese concentrations and quantum-well widths are studied at excitation power densities ranging from 105 to 107 W cm-2. Under strong optical pumping, intracenter luminescence of Mn2+ ions degrades as a result of the interaction of excited managanese ions with high-density excitons. This process is accompanied by a strong broadening of the emission band of quantum-well excitons due to the exciton-exciton interaction and saturation of the exciton ground state. Under pumping at a power density of 105 W cm-2, stimulated emission of quantum-well excitons arises in CdTe/Cd0.5Mg0.5Te. The luminescence kinetics of the quantum-well and barrier excitons is investigated with a high temporal resolution. The effect of the quantum-well width and the managanese concentration on the kinetics and band shape of the Mn2+ intracenter luminescence characterized by the contribution of the manganese interface ions is determined.

Alterations in the adenosine metabolism and CD39/CD73 adenosinergic machinery cause loss of Treg cell function and autoimmunity in ADA-deficient SCID

PubMed Central

Sauer, Aisha V.; Brigida, Immacolata; Carriglio, Nicola; Jofra Hernandez, Raisa; Scaramuzza, Samantha; Clavenna, Daniela; Sanvito, Francesca; Poliani, Pietro L.; Gagliani, Nicola; Carlucci, Filippo; Tabucchi, Antonella; Roncarolo, Maria Grazia; Traggiai, Elisabetta; Villa, Anna

2012-01-01

Adenosine acts as anti-inflammatory mediator on the immune system and has been described in regulatory T cell (Treg)–mediated suppression. In the absence of adenosine deaminase (ADA), adenosine and other purine metabolites accumulate, leading to severe immunodeficiency with recurrent infections (ADA-SCID). Particularly ADA-deficient patients with late-onset forms and after enzyme replacement therapy (PEG-ADA) are known to manifest immune dysregulation. Herein we provide evidence that alterations in the purine metabolism interfere with Treg function, thereby contributing to autoimmune manifestations in ADA deficiency. Tregs isolated from PEG-ADA–treated patients are reduced in number and show decreased suppressive activity, whereas they are corrected after gene therapy. Untreated murine ADA−/− Tregs show alterations in the plasma membrane CD39/CD73 ectonucleotidase machinery and limited suppressive activity via extracellular adenosine. PEG-ADA–treated mice developed multiple autoantibodies and hypothyroidism in contrast to mice treated with bone marrow transplantation or gene therapy. Tregs isolated from PEG-ADA–treated mice lacked suppressive activity, suggesting that this treatment interferes with Treg functionality. The alterations in the CD39/CD73 adenosinergic machinery and loss of function in ADA-deficient Tregs provide new insights into a predisposition to autoimmunity and the underlying mechanisms causing defective peripheral tolerance in ADA-SCID. Trials were registered at www.clinicaltrials.gov as NCT00598481/NCT00599781. PMID:22184407

Predicting plant uptake of cadmium: validated with long-term contaminated soils.

PubMed

Lamb, Dane T; Kader, Mohammed; Ming, Hui; Wang, Liang; Abbasi, Sedigheh; Megharaj, Mallavarapu; Naidu, Ravi

2016-10-01

Cadmium accumulates in plant tissues at low soil loadings and is a concern for human health. Yet at higher levels it is also of concern for ecological receptors. We determined Cd partitioning constants for 41 soils to examine the role of soil properties controlling Cd partitioning and plant uptake. From a series of sorption and dose response studies, transfer functions were developed for predicting Cd uptake in Cucumis sativa L. (cucumber). The parameter log K f was predicted with soil pH ca , logCEC and log OC. Transfer of soil pore-water Cd 2+ to shoots was described with a power function (R 2  = 0.73). The dataset was validated with 13 long-term contaminated soils (plus 2 control soils) ranging in Cd concentration from 0.2 to 300 mg kg -1 . The series of equations predicting Cd shoot from pore-water Cd 2+ were able to predict the measured data in the independent dataset (root mean square error = 2.2). The good relationship indicated that Cd uptake to cucumber shoots could be predicted with Cd pore and Cd 2+ without other pore-water parameters such as pH or Ca 2+ . The approach may be adapted to a range of plant species.

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.

CIRCULATING MICROPARTICLES IN PATIENTS WITH ANTIPHOSPHOLIPID ANTIBODIES: CHARACTERIZATION AND ASSOCIATIONS

PubMed Central

Chaturvedi, Shruti; Cockrell, Erin; Espinola, Ricardo; Hsi, Linda; Fulton, Stacey; Khan, Mohammad; Li, Liang; Fonseca, Fabio; Kundu, Suman; McCrae, Keith R.

2014-01-01

The antiphospholipid syndrome is characterized by venous or arterial thrombosis and/or recurrent fetal loss in the presence of circulating antiphospholipid antibodies. These antibodies cause activation of endothelial and other cell types leading to the release of microparticles with procoagulant and pro-inflammatory properties. The aims of this study were to characterize the levels of endothelial cell, monocyte, platelet derived, and tissue factor-bearing microparticles in patients with antiphospholipid antibodies, to determine the association of circulating microparticles with anticardiolipin and anti-β2-glycoprotein antibodies, and to define the cellular origin of microparticles that express tissue factor. Microparticle content within citrated blood from 47 patients with antiphospholipid antibodies and 144 healthy controls was analyzed within 2 hours of venipuncture. Levels of Annexin-V, CD105 and CD144 (endothelial derived), CD41 (platelet derived) and tissue factor positive microparticles were significantly higher in patients than controls. Though levels of CD14 (monocyte-derived) microparticles in patient plasma were not significantly increased, increased levels of CD14 and tissue factor positive microparticles were observed in patients. Levels of microparticles that stained for CD105 and CD144 showed a positive correlation with IgG (R = 0.60, p=0.006) and IgM anti-beta2-glycoprotein I antibodies (R=0.58, p=0.006). The elevation of endothelial and platelet derived microparticles in patients with APS and their correlation with anti-β2-glycoprotein I antibodies suggests a chronic state of vascular cell activation in these individuals and an important role for β2-glycoprotein I in development of the pro-thrombotic state associated with antiphospholipid antibodies. PMID:25467081

Intracarotid injection of fluorescence activated cell-sorted CD49d-positive neural stem cells improves targeted cell delivery and behavior after stroke in a mouse stroke model.

PubMed

Guzman, Raphael; De Los Angeles, Alejandro; Cheshier, Samuel; Choi, Raymond; Hoang, Stanley; Liauw, Jason; Schaar, Bruce; Steinberg, Gary

2008-04-01

Intravascular delivery of neural stem cells (NSCs) after stroke has been limited by the low efficiency of transendothelial migration. Vascular cell adhesion molecule-1 is an endothelial adhesion molecule known to be upregulated early after stroke and is responsible for the firm adhesion of inflammatory cells expressing the surface integrin, CD49d. We hypothesize that enriching for NSCs that express CD49d and injecting them into the carotid artery would improve targeted cell delivery to the injured brain. Mouse NSCs were analyzed for the expression of CD49d by fluorescence activated cell sorting. A CD49d-enriched (CD49d(+)) (>95%) and -depleted (CD49d(-); <5%) NSC population was obtained by cell sorting. C57/Bl6 mice underwent left-sided hypoxia-ischemia surgery and were assigned to receive 3 x 10(5) CD49d(+), CD49d(-) NSCs, or vehicle injection into the left common carotid artery 48 hours after stroke. Behavioral recovery was measured using a rotarod for 2 weeks after cell injection. Fluorescence activated cell sorting analysis revealed 25% CD49d(+) NSCs. In a static adhesion assay, NSCs adhered to vascular cell adhesion molecule-1 in a dose-dependent manner. Significantly more NSCs were found in the cortex, the hippocampus, and the subventricular zone in the ischemic hemisphere in animals receiving CD49d(+) NSCs as compared with CD49d(-) NSCs (P<0.05). Animals treated with CD49d(+) cells showed a significantly better behavioral recovery as compared with CD49d(-) and vehicle-treated animals. We show that enrichment of NSCs by fluorescence activated cell sorting for the surface integrin, CD49d, and intracarotid delivery promotes cell homing to the area of stroke in mice and improves behavioral recovery.

[Association of CD(+)4 T lymphocyte count and gingival crevicular fluid prostaglandin E2 with periodontal parameters in HIV-positive periodontitis patients].

PubMed

Jia, Hongcheng; Wang, Xuan; Hua, Wenhao; Li, Xiaoguang; Hou, Wen; Fu, Qian

2014-02-01

To investigate the correlation of CD(+)4 T lymphocyte count and prostaglandin E2 (PGE2) in gingival crevicular fluid (GCF) with periodontal status in HIV-positive patients with periodontitis. Twenty subjects were selected according to inclusion criteria. The plasmatic CD(+)4 T lymphocytes were counted. All the individuals were divided into three groups, group A (CD(+)4 T lymphocyte count < 200 cell/mm(3)), group B (200 cell/mm(3) ≤ CD(+)4 T lymphocyte count ≤ 500 cell/mm(3)) and group C (CD(+)4 T lymphocyte count > 500 cell/mm(3)). Periodontal indexes, including plaque index(PLI), bleeding index(BI), attachment level(AL) and probing depth(PD) were recorded.GCF samples were taken from 120 index teeth by means of sterile paper strips.GCF PGE2 levels were determined by radioimmunoassays. Mann-Whitney was used to compare the periodontal indexes and PGE2 levels among the three groups. Partial correlations and Spearman correlations were applied to analyze the correlation of CD(+)4 T lymphocytes count and PGE2 in gingival crevicular fluid with periodontal status. BI value, PGE2 concentration and total PGE2 were 3.00(2.00), 90.75(30.60) µg/L, 447.58 (243.08) pg in group B, which were higher than those in group A[2.00(1.25), 79.75(30.50) µg/L and 339.52 (200.97) pg respectively] and group C[2.00(1.00), 73.38 (14.83) µg/L and 299.18 (108.33) pg respectively] (P < 0.0167). But the differences of PD and AL among the three groups were not significantly different(P > 0.0167). The correlations were observed between CD(+)4 T lymphocyte count and BI for the subpopulations with CD(+)4 T lymphocyte count <200 cells/mm(3) (r = 0.657, P < 0.05) and between 200-500 cells/mm(3) (r = -0.369, P < 0.05). PGE2 concentration was negatively correlated with BI, PD and AL (P < 0.05), and total PGE2 was positively correlated with PD and AL(P < 0.05). There was an association between the periodontal status and CD(+)4 T lymphocyte count in HIV(+) patients.GCF PGE2 level was related to periodontal parameters including BI, PD and AL.

Adaptive Immunity against Leishmania Nucleoside Hydrolase Maps Its C-Terminal Domain as the Target of the CD4+ T Cell–Driven Protective Response

PubMed Central

Nico, Dirlei; Claser, Carla; Borja-Cabrera, Gulnara P.; Travassos, Luiz R.; Palatnik, Marcos; da Silva Soares, Irene; Rodrigues, Mauricio Martins; Palatnik-de-Sousa, Clarisa B.

2010-01-01

Nucleoside hydrolases (NHs) show homology among parasite protozoa, fungi and bacteria. They are vital protagonists in the establishment of early infection and, therefore, are excellent candidates for the pathogen recognition by adaptive immune responses. Immune protection against NHs would prevent disease at the early infection of several pathogens. We have identified the domain of the NH of L. donovani (NH36) responsible for its immunogenicity and protective efficacy against murine visceral leishmaniasis (VL). Using recombinant generated peptides covering the whole NH36 sequence and saponin we demonstrate that protection against L. chagasi is related to its C-terminal domain (amino-acids 199–314) and is mediated mainly by a CD4+ T cell driven response with a lower contribution of CD8+ T cells. Immunization with this peptide exceeds in 36.73±12.33% the protective response induced by the cognate NH36 protein. Increases in IgM, IgG2a, IgG1 and IgG2b antibodies, CD4+ T cell proportions, IFN-γ secretion, ratios of IFN-γ/IL-10 producing CD4+ and CD8+ T cells and percents of antibody binding inhibition by synthetic predicted epitopes were detected in F3 vaccinated mice. The increases in DTH and in ratios of TNFα/IL-10 CD4+ producing cells were however the strong correlates of protection which was confirmed by in vivo depletion with monoclonal antibodies, algorithm predicted CD4 and CD8 epitopes and a pronounced decrease in parasite load (90.5–88.23%; p = 0.011) that was long-lasting. No decrease in parasite load was detected after vaccination with the N-domain of NH36, in spite of the induction of IFN-γ/IL-10 expression by CD4+ T cells after challenge. Both peptides reduced the size of footpad lesions, but only the C-domain reduced the parasite load of mice challenged with L. amazonensis. The identification of the target of the immune response to NH36 represents a basis for the rationale development of a bivalent vaccine against leishmaniasis and for multivalent vaccines against NHs-dependent pathogens. PMID:21085470

Lead and cadmium interactions in Cynodon nlemfuensis and sandy soil subjected to treated wastewater application under greenhouse conditions

NASA Astrophysics Data System (ADS)

Madyiwa, Simon; Chimbari, Moses John; Schutte, Frederik

Pb and Cd are known to influence each other’s uptake by some plants when the two metals exist in the soil in significant amounts. This influence may be beneficial if it reduces uptake of metal by plants but may be detrimental if it increases uptake of the metal. This study was carried out to investigate the interaction of Pb and Cd in sandy soils and Cynodon nlemfluensis (star grass). Star grass was grown under greenhouse conditions in 33 fertilized pots containing sandy soils. Three weeks after planting the grass the pots were randomly assigned to the following treatments replicated three times; (a) application of three varying concentrations of Pb or Cd in addition to effluent and sludge, (b) application of three varying concentrations of combined Pb and Cd in addition to effluent and sludge, (c) application of water and (d) application of only effluent and sludge. Analysis of grass samples was done 45 and 90 days after addition of Pb and Cd to pots and that of the soil was done 90 days after addition of Pb and Cd to pots. The log normal mean level (in mg/kg) of Pb detected in the soil was 1.75 and that of Cd was 0.057 in mixed treatments while for single treatments the levels were 1.67 for Pb and 0.03 for Cd. The presence of Cd in the soil had no effect on the bio-available level of Pb but Pb significantly ( p < 0.05) increased the bio-available concentration of Cd. The log normal mean levels of Pb in grass re-growth from mixed treatment was 1.68 and that of Cd was 0.57 while the values for single treatments were 1.47 for Pb and 0.31 for Cd. There was no significant change in the level of uptake of Pb between single treatments and mixed treatments. However, Pb significantly increased uptake of Cd in mixed treatments compared to single treatments ( p < 0.05). The results of this study indicate that co-presence of Pb and Cd may have the detrimental effect of increasing uptake of Cd in star grass.

A truncated form of CD9-partner 1 (CD9P-1), GS-168AT2, potently inhibits in vivo tumour-induced angiogenesis and tumour growth

PubMed Central

Colin, S; Guilmain, W; Creoff, E; Schneider, C; Steverlynck, C; Bongaerts, M; Legrand, E; Vannier, J P; Muraine, M; Vasse, M; Al-Mahmood, S

2011-01-01

Background: Tetraspanins are transmembrane proteins known to contribute to angiogenesis. CD9 partner-1 (CD9P-1/EWI-F), a glycosylated type 1 transmembrane immunoglobulin, is a member of the tetraspanin web, but its role in angiogenesis remains to be elucidated. Methods: We measured the expression of CD9P-1 under angiogenic and angiostatic conditions, and the influence of its knockdown onto capillary structures formation by human endothelial cells (hECs). A truncated form of CDP-1, GS-168AT2, was produced and challenged vs hEC proliferation, migration and capillaries' formation. Its association with CD9P-1, CD9, CD81 and CD151 and the expressions of these later at hEC surface were analysed. Finally, its effects onto in vivo tumour-induced angiogenesis and tumour growth were investigated. Results: Vascular endothelial growth factor (VEGF)-induced capillary tube-like formation was inhibited by tumour necrosis factor α and was associated with a rise in CD9P-1 mRNA expression (P<0.05); accordingly, knockdown of CD9P-1 inhibited VEGF-dependent in vitro angiogenesis. GS-168AT2 dose-dependently inhibited in vitro angiogenesis, hEC migration and proliferation (P<0.05). Co-precipitation experiments suggest that GS-168AT2 corresponds to the sequence by which CD9P-1 physiologically associates with CD81. GS-168AT2 induced the depletion of CD151, CD9 and CD9P-1 from hEC surface, correlating with GS-168AT2 degradation. Finally, in vivo injections of GS-168AT2 inhibited tumour-associated angiogenesis by 53.4±9.5% (P=0.03), and reduced tumour growth of Calu 6 tumour xenografts by 73.9±16.4% (P=0.007) without bodyweight loss. Conclusion: The truncated form of CD9P-1, GS-168AT2, potently inhibits angiogenesis and cell migration by at least the downregulation of CD151 and CD9, which provides the first evidences for the central role of CD9P-1 in tumour-associated angiogenesis and tumour growth. PMID:21863033

Growth and Cadmium Phytoextraction by Swiss Chard, Maize, Rice, Noccaea caerulescens, and Alyssum murale in Ph Adjusted Biosolids Amended Soils.

PubMed

Broadhurst, C Leigh; Chaney, Rufus L; Davis, Allen P; Cox, Albert; Kumar, Kuldip; Reeves, Roger D; Green, Carrie E

2015-01-01

Past applications of biosolids to soils at some locations added higher Cd levels than presently permitted. Cadmium phytoextraction would alleviate current land use constraints. Unamended farm soil, and biosolids amended farm and mine soils were obtained from a Fulton Co., IL biosolids management facility. Soils contained 0.16, 22.8, 45.3 mg Cd kg(-1) and 43.1, 482, 812 mg Zn kg(-1) respectively with initial pH 6.0, 6.1, 6.4. In greenhouse studies, Swiss chard (Beta vulgaris var. cicla), a Cd-accumulator maize (inbred B37 Zea mays) and a southern France Cd-hyperaccumulator genotype of Noccaea caerulescens were tested for Cd accumulation and phytoextraction. Soil pH was adjusted from ∼5.5-7.0. Additionally 100 rice (Oryza sativa) genotypes and the Ni-hyperaccumulator Alyssum murale were screened for potential phytoextraction use. Chard suffered phytotoxicity at low pH and accumulated up to 90 mg Cd kg(-1) on the biosolids amended mine soil. The maize inbred accumulated up to 45 mg Cd kg(-1) with only mild phytotoxicity symptoms during early growth at pH>6.0. N. caerulescens did not exhibit phytotoxicity symptoms at any pH, and accumulated up to 235 mg Cd kg(-1) in 3 months. Reharvested N. caerulescens accumulated up to 900 mg Cd kg(-1) after 10 months. Neither Alyssum nor 90% of rice genotypes survived acceptably. Both N. caerulescens and B37 maize show promise for Cd phytoextraction in IL and require field evaluation; both plants could be utilized for nearly continuous Cd removal. Other maize inbreds may offer higher Cd phytoextraction at lower pH, and mono-cross hybrids higher shoot biomass yields. Further, maize grown only for biomass Cd maximum removal could be double-cropped.

BCR-ABL1 molecular remission after 90Y-epratuzumab tetraxetan radioimmunotherapy in CD22+ Ph+ B-ALL: proof of principle.

PubMed

Chevallier, Patrice; Bodet-Milin, Caroline; Robillard, Nelly; Eugene, Thomas; Menard, Audrey; Le Houerou, Claire; Guillaume, Thierry; Delaunay, Jacques; Escoffre-Barbe, Martine; Wegener, William A; Goldenberg, David M; Kraeber-Bodéré, Francoise

2013-12-01

Although targeted therapies are used increasingly in hematologic malignancies, we are unaware of any prior studies of radioimmunotherapy (RAIT) in B-acute lymphoblastic leukemia (ALL), even though this radiosensitive tumor expresses CD22, potentially a good target for this approach. Here, we report a patient with Philadelphia chromosome-positive B-ALL in third relapse who received RAIT with (90) yttrium ((90) Y)-labeled anti-CD22 epratuzumab tetraxetan. Seven weeks after initiating therapy, the patient achieved a BCR-ABL1 molecular remission documented by RT-qPCR, which is now continuing at 6 months while awaiting an allogeneic hematopoietic stem cell transplant. (90) Y-Epratuzumab tetraxetan may be a promising therapeutic option for CD22(+) B-ALL patients. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Overtraining and immune system: a prospective longitudinal study in endurance athletes.

PubMed

Gabriel, H H; Urhausen, A; Valet, G; Heidelbach, U; Kindermann, W

1998-07-01

A prospective longitudinal study investigated for 19 +/- 3) months whether immunophenotypes of peripheral leukocytes were altered in periods of severe training. Leukocyte membrane antigens (CD3, CD4, CD8, CD14, CD16, CD19, CD45, CD45RO, and CD56) of endurance athletes were immunophenotyped (dual-color flow cytometry) and list mode data analyzed by a self-learning classification system in a state of an overtraining syndrome (OT; N = 15) and several occasions without symptoms of staleness (NS; N = 70). Neither at physical rest nor after a short-term highly intensive cycle ergometer exercise session at 110% of the individual anaerobic threshold did cell counts of neutrophils, T, B, and natural killer cells differ between OT and NS. Eosinophils were lower during OT, activated T cells (CD3+HLA/DR+) showed slight increases (NS: 5.5 +/- 2.7; OT 7.3 +/- 2.4% CD3+ of cells; means +/- SD; P < 0.01) during OT without reaching pathological ranges. The cell-surface expression of CD45RO (P < 0.001) on T cells, but not cell concentrations of CD45RO+ T cells, were higher during OT. OT could be classified with high specificities (92%) and sensitivities (93%). It is concluded that OT does not lead to clinically relevant alterations of immunophenotypes in peripheral blood and especially that an immunosuppressive effect cannot be detected. Immunophenotyping may provide help with the diagnosis of OT in future, but the diagnostic approach presented here requires improvements before use in sports medicine practice is enabled.

Clonal precursor of bone, cartilage, and hematopoietic niche stromal cells

PubMed Central

Chan, Charles K. F.; Lindau, Paul; Jiang, Wen; Chen, James Y.; Zhang, Lillian F.; Chen, Ching-Cheng; Seita, Jun; Sahoo, Debashis; Kim, Jae-Beom; Lee, Andrew; Park, Sujin; Nag, Divya; Gong, Yongquan; Kulkarni, Subhash; Luppen, Cynthia A.; Theologis, Alexander A.; Wan, Derrick C.; DeBoer, Anthony; Seo, Eun Young; Vincent-Tompkins, Justin D.; Loh, Kyle; Walmsley, Graham G.; Kraft, Daniel L.; Wu, Joseph C.; Longaker, Michael T.; Weissman, Irving L.

2013-01-01

Organs are composites of tissue types with diverse developmental origins, and they rely on distinct stem and progenitor cells to meet physiological demands for cellular production and homeostasis. How diverse stem cell activity is coordinated within organs is not well understood. Here we describe a lineage-restricted, self-renewing common skeletal progenitor (bone, cartilage, stromal progenitor; BCSP) isolated from limb bones and bone marrow tissue of fetal, neonatal, and adult mice. The BCSP clonally produces chondrocytes (cartilage-forming) and osteogenic (bone-forming) cells and at least three subsets of stromal cells that exhibit differential expression of cell surface markers, including CD105 (or endoglin), Thy1 [or CD90 (cluster of differentiation 90)], and 6C3 [ENPEP glutamyl aminopeptidase (aminopeptidase A)]. These three stromal subsets exhibit differential capacities to support hematopoietic (blood-forming) stem and progenitor cells. Although the 6C3-expressing subset demonstrates functional stem cell niche activity by maintaining primitive hematopoietic stem cell (HSC) renewal in vitro, the other stromal populations promote HSC differentiation to more committed lines of hematopoiesis, such as the B-cell lineage. Gene expression analysis and microscopic studies further reveal a microenvironment in which CD105-, Thy1-, and 6C3-expressing marrow stroma collaborate to provide cytokine signaling to HSCs and more committed hematopoietic progenitors. As a result, within the context of bone as a blood-forming organ, the BCSP plays a critical role in supporting hematopoiesis through its generation of diverse osteogenic and hematopoietic-promoting stroma, including HSC supportive 6C3(+) niche cells. PMID:23858471

CD34 Antigen and the MPL Receptor Expression Defines a Novel Class of Human Cord Blood-Derived Primitive Hematopoietic Stem Cells.

PubMed

Matsuoka, Yoshikazu; Takahashi, Masaya; Sumide, Keisuke; Kawamura, Hiroshi; Nakatsuka, Ryusuke; Fujioka, Tatsuya; Sonoda, Yoshiaki

2017-06-09

In the murine hematopoietic stem cell (HSC) compartment, thrombopoietin (THPO)/MPL (THPO receptor) signaling plays an important role in the maintenance of adult quiescent HSCs. However, the role of THPO/MPL signaling in the human primitive HSC compartment has not yet been elucidated. We have identified very primitive human cord blood (CB)-derived CD34- severe combined immunodeficiency (SCID)-repopulating cells (SRCs) using the intra-bone marrow injection method. In this study, we investigated the roles of the MPL expression in the human primitive HSC compartment. The SRC activities of the highly purified CB-derived 18Lin-CD34+/-MPL+/- cells were analyzed using NOG mice. In the primary recipient mice, nearly all mice that received CD34+/-MPL+/- cells were repopulated with human CD45+ cells. Nearly all of these mice that received CD34+MPL+/- and CD34-MPL- cells showed a secondary repopulation. Interestingly, the secondary recipient mice that received CD34+/-MPL- cells showed a distinct tertiary repopulation. These results clearly indicate that the CD34+/- SRCs not expressing MPL sustain a long-term (LT) (>1 year) human cell repopulation in NOG mice. Moreover, CD34- SRCs generate CD34+CD38-CD90+ SRCs in vitro and in vivo. These findings provide a new concept that CD34-MPL- SRCs reside at the apex of the human HSC hierarchy.

Double electron capture of {sup 106}Cd in the TGV-2 experiment

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

Rukhadze, N. I.; Egorov, V. G.; Kovalik, A.

2015-10-28

A new experimental run of searching for EC/EC decay of {sup 106}Cd was performed at the Modane underground laboratory (4800 m w.e.) using the TGV-2 spectrometer and ∼23.2 g {sup 106}Cd with enrichment of 99.57%. The limit on 2νEC/EC decay of {sup 106}Cd - T{sub 1/2}(2νEC/EC) > 3.1×10{sup 20} y, at 90% C.L was obtained from the preliminary calculation of experimental data accumulated for 7018 h of measurement. The limits on the resonance neutrino-less double electron capture decay of {sup 106}Cd were obtained from the measurement of ∼23.2 g of {sup 106}Cd with the low-background HPGe spectrometer OBELIX lasted 395more » h - T{sub 1/2}(KL, 2741 keV) > 0.9×10{sup 20} y and T{sub 1/2}(KK, 2718 keV) > 1.4×10{sup 20} y at 90% C.L.« less

Update on the pathogenesis of Scleroderma: focus on circulating progenitor cells

PubMed Central

Brunasso, Alexandra Maria Giovanna; Massone, Cesare

2016-01-01

In systemic sclerosis (SSc), the development of fibrosis seems to be a consequence of the initial ischemic process related to an endothelial injury. The initial trigger event in SSc is still unknown, but circulating progenitor cells (CPCs) might play a key role. Such cells have the ability to traffic into injury sites, exhibiting inflammatory features of macrophages, tissue remodeling properties of fibroblasts, and vasculogenesis functions of endothelial cells. The different subsets of CPCs described thus far in SSc arise from a pool of circulating monocyte precursors (CD14 + cells) and probably correspond to a different degree of differentiation of a single cell of origin. Several subsets of CPCs have been described in patients with SSc, all have a monocytic origin but may or may not express CD14, and all of these cells have the ability to give origin to endothelial cells, or collagen (Col)-producing cells, or both. We were able to identify six subsets of CPCs: pluripotent stem cells (CD14 +, CD45 +, and CD34 +), monocyte-derived multipotential cells (MOMCs) or monocyte-derived mesenchymal progenitors (CD14 +, CD45 +, CD34 +, Col I +, CD11b +, CD68 +, CD105 +, and VEGFR1 +), early endothelial progenitor cells (EPCs) or monocytic pro-angiogenic hematopoietic cells or circulating hematopoietic cells (CD14 +, CD45 +, CD34 low/−, VEGFR2 +/−, CXCR4 +, c-kit +, and DC117 +), late EPCs (CD14 −, CD133 +, VEGFR2 +, CD144 + [VE-cadherin +], and CD146 +), fibroblast-like cells (FLCs)/circulating Col-producing monocytes (CD14 +, CD45 +, CD34 +/−, and Col I +), and fibrocytes (CD14 −, CD45 +, CD34 +, Col I +, and CXCR4 +). It has been demonstrated that circulating CD14 + monocytes with an activated phenotype are increased in patients with SSc when compared with normal subjects. CD14 +, CD34 +, and Col I + spindle-shaped cells have been found in increased numbers in lungs of SSc patients with interstitial lung disease. Elevated blood amounts of early EPCs have been found in patients with SSc by different groups of researchers and such levels correlate directly with the interstitial lung involvement. The prevalence of hematopoietic markers expressed by CPCs that migrate from blood into injury sites in SSc differs and changes according to the degree of differentiation. CXCR4 is the most commonly expressed marker, followed by CD34 and CD45 at an end stage of differentiation. Such difference also indicates a continuous process of cell differentiation that might relate to the SSc clinical phenotype (degree of fibrosis and vascular involvement). A deeper understanding of the role of each subtype of CPCs in the development of the disease will help us to better classify patients in order to offer them targeted approaches in the future. PMID:27158466

Characterization of stem and progenitor cells in the dental pulp of erupted and unerupted murine molars

PubMed Central

Balic, Anamaria; Aguila, H. Leonardo; Caimano, Melissa J.; Francone, Victor P.; Mina, Mina

2010-01-01

In the past few years there have been significant advances in the identification of putative stem cells also referred to as “mesenchymal stem cells” (MSC) in dental tissues including the dental pulp. It is thought that MSC in dental pulp share certain similarities with MSC isolated from other tissues. However, cells in dental pulp are still poorly characterized. This study focused on the characterization of progenitor and stem cells in dental pulps of erupted and unerupted mice molars. Our study showed that dental pulps from unerupted molars contain a significant number of cells expressing CD90+/CD45-, CD117+/CD45-, Sca-1+/CD45- and little if any CD45+ cells. Our in vitro functional studies showed that dental pulp cells from unerupted molars displayed extensive osteo-dentinogenic potential but were unable to differentiate into chondrocytes and adipocytes. Dental pulp from erupted molars displayed a reduced number of cells, contained higher percentage of CD45+ and lower percentage of cells expressing CD90+/CD45-, CD117+/CD45- as compared to unerupted molars. In vitro functional assays demonstrated the ability of a small fraction of cells to differentiate into odontoblasts, osteoblasts, adipocytes and chondrocytes. There was a significant reduction in the osteo-dentinogenic potential of the pulp cells derived from erupted molars compared to unerupted molars. Furthermore, the adipogenic and chondrogenic differentiation of pulp cells from erupted molars was dependent on a long induction period and infrequent. Based on these findings we propose that the dental pulp of the erupted molars contain a small population of multipotent cells, whereas the dental pulp of the unerupted molars does not contain multipotent cells but is enriched in osteo-dentinogenic progenitors engaged in the formation of coronal and radicular odontoblasts. PMID:20193787

The Significance of SDF-1α-CXCR4 Axis in in vivo Angiogenic Ability of Human Periodontal Ligament Stem Cells.

PubMed

Bae, Yoon-Kyung; Kim, Gee-Hye; Lee, Jae Cheoun; Seo, Byoung-Moo; Joo, Kyeung-Min; Lee, Gene; Nam, Hyun

2017-06-30

Periodontal ligament stem cells (PDLSCs) are multipotent stem cells derived from periodontium and have mesenchymal stem cell (MSC)-like characteristics. Recently, the perivascular region was recognized as the developmental origin of MSCs, which suggests the in vivo angiogenic potential of PDLSCs. In this study, we investigated whether PDLSCs could be a potential source of perivascular cells, which could contribute to in vivo angiogenesis. PDLSCs exhibited typical MSC-like characteristics such as the expression pattern of surface markers (CD29, CD44, CD73, and CD105) and differentiation potentials (osteogenic and adipogenic differentiation). Moreover, PDLSCs expressed perivascular cell markers such as NG2, αsmooth muscle actin, platelet-derived growth factor receptor β, and CD146. We conducted an in vivo Matrigel plug assay to confirm the in vivo angiogenic potential of PDLSCs. We could not observe significant vessel-like structures with PDLSCs alone or human umbilical vein endothelial cells (HU-VECs) alone at day 7 after injection. However, when PDLSCs and HUVECs were co-injected, there were vessel-like structures containing red blood cells in the lumens, which suggested that anastomosis occurred between newly formed vessels and host circulatory system. To block the SDF-1α and CXCR4 axis between PDLSCs and HUVECs, AMD3100, a CXCR4 antagonist, was added into the Matrigel plug. After day 3 and day 7 after injection, there were no significant vessel-like structures. In conclusion, we demonstrated the peri-vascular characteristics of PDLSCs and their contribution to in vivo angiogenesis, which might imply potential application of PDLSCs into the neovascularization of tissue engineering and vascular diseases.

Mesenchymal stem cell and regenerative medicine: regeneration versus immunomodulatory challenges

PubMed Central

Law, Sujata; Chaudhuri, Samaresh

2013-01-01

Mesenchymal Stem cells (MSC) are now presented with the opportunities of multifunctional therapeutic approaches. Several reports are in support of their self-renewal, capacity for multipotent differentiation, and immunomodulatory properties. They are unique to contribute to the regeneration of mesenchymal tissues such as bone, cartilage, muscle, ligament, tendon, and adipose. In addition to promising trials in regenerative medicine, such as in the treatment of major bone defects and myocardial infarction, MSC has shown a therapeutic effect other than direct hematopoiesis support in hematopoietic reconstruction. MSCs are identified by the expression of many molecules including CD105 (SH2) and CD73(SH3/4) and are negative for the hematopoietic markers CD34, CD45, and CD14. Manufacturing of MSC for clinical trials is also an important aspect as their differentiation, homing and Immunomodulatory properties may differ. Their suppressive effects on immune cells, including T cells, B cells, NK cells and DC cells, suggest MSCs as a novel therapy for GVHD and other autoimmune disorders. Since the cells by themselves are non-immunogenic, tissue matching between MSC donor and recipient is not essential and, MSC may be the first cell type able to be used as an “off-the-shelf” therapeutic product. Following a successful transplantation, the migration of MSC to the site of injury refers to the involvement of chemokines and chemokine receptors of respective specificity. It has been demonstrated that cultured MSCs have the ability to engraft into healthy as well as injured tissue and can differentiate into several cell types in vivo, which facilitates MSC to be an ideal tool for regenerative therapy in different disease types. However, some observations have raised questions about the limitations for proper use of MSC considering some critical factors that warn regular clinical use. PMID:23671814

Differentiation of isolated human umbilical cord mesenchymal stem cells into neural stem cells

PubMed Central

Chen, Song; Zhang, Wei; Wang, Ji-Ming; Duan, Hong-Tao; Kong, Jia-Hui; Wang, Yue-Xin; Dong, Meng; Bi, Xue; Song, Jian

2016-01-01

AIM To investigate whether umbilical cord human mesenchymal stem cell (UC-MSC) was able to differentiate into neural stem cell and neuron in vitro. METHODS The umbilical cords were obtained from pregnant women with their written consent and the approval of the Clinic Ethnics Committee. UC-MSC were isolated by adherent culture in the medium contains 20% fetal bovine serum (FBS), then they were maintained in the medium contain 10% FBS and induced to neural cells in neural differentiation medium. We investigated whether UC-MSC was able to differentiate into neural stem cell and neuron in vitro by using flow cytometry, reverse transcriptase-polymerase chain reaction (RT-PCR) and immunofluorescence (IF) analyzes. RESULTS A substantial number of UC-MSC was harvested using the tissue explants adherent method at about 2wk. Flow cytometric study revealed that these cells expressed common markers of MSCs, such as CD105 (SH2), CD73 (SH3) and CD90. After induction of differentiation of neural stem cells, the cells began to form clusters; RT-PCR and IF showed that the neuron specific enolase (NSE) and neurogenic differentiation 1-positive cells reached 87.3%±14.7% and 72.6%±11.8%, respectively. Cells showed neuronal cell differentiation after induced, including neuron-like protrusions, plump cell body, obviously and stronger refraction. RT-PCR and IF analysis showed that microtubule-associated protein 2 (MAP2) and nuclear factor-M-positive cells reached 43.1%±10.3% and 69.4%±19.5%, respectively. CONCLUSION Human umbilical cord derived MSCs can be cultured and proliferated in vitro and differentiate into neural stem cells, which may be a valuable source for cell therapy of neurodegenerative eye diseases. PMID:26949608

Comparison of Osteogenic and Chondrogenic Differentiation Ability of Buccal Fat Pad Derived Mesenchymal Stem Cells and Gingival Derived Cells.

PubMed

Ghaderi, Hamid; Razmkhah, Mahboobeh; Kiany, Farin; Chenari, Nooshafarin; Haghshenas, Mohammad Reza; Ghaderi, Abbas

2018-06-01

One major goal of tissue engineering and regenerative medicine is to find an appropriate source of mesenchymal stem cells (MSCs) with higher differentiation ability. In this experimental study, the osteogenic and chondrogenic differentiation ability of buccal fat pad derived MSCs (BFP-MSCs) with gingival derived cells (GDCs) were compared. BFP-MSCs and GDCs were cultured enzymatically and expanded. The expanded cells were analyzed for membrane-associated markers, using flow cytometry. Then the ability of these cells to differentiate into osteocyte and chondrocyte was assessed morphologically and by mRNA expression of collagen I (COLL), BGLA and bone morphogenetic protein 2 (BMP2) using qRT-PCR. Flow cytometry analysis showed that both BFP-MSCs and GDCs expressed the characteristic stem cell markers such as CD73, CD44, and CD90, whereas they did not express hematopoietic markers. Mineralized calcium deposition was observed apparently in BFP-MSCs cultured in osteogenic medium but GDCs showed fewer mineralized nodules. The mRNA expression levels of BGLA and BMP2 showed 7×105 and 733-fold more mRNA expression in BFP-MSCs treated with differentiation media compared to the control group. In chondrogenic differentiation, BFP-MSCs transformed from a spindle to a cuboidal shape while GDCs showed only a slight transformation. In addition, mRNA expression of COLL showed 282-fold higher expression in BFP-MSCs in comparison to the control group. Such significant difference in mRNA expression of BGLA, BMP2, and COLL was not observed in GDCs compared to their corresponding controls. Based on the present results, BFP yields a greater proportion of stem cells compared to gingiva. Therefore, this tissue can be introduced as an easily available source for the treatment of periodontal defects and other maxillofacial injuries.

A novel 3D mesenchymal stem cell model of the multiple myeloma bone marrow niche: biologic and clinical applications

PubMed Central

Jakubikova, Jana; Cholujova, Danka; Hideshima, Teru; Gronesova, Paulina; Soltysova, Andrea; Harada, Takeshi; Joo, Jungnam; Kong, Sun-Young; Szalat, Raphael E.; Richardson, Paul G.; Munshi, Nikhil C.; Dorfman, David M.; Anderson, Kenneth C.

2016-01-01

Specific niches within the tumor bone marrow (BM) microenvironment afford a sanctuary for multiple myeloma (MM) clones due to stromal cell-tumor cell interactions, which confer survival advantage and drug resistance. Defining the sequelae of tumor cell interactions within the MM niches on an individualized basis may provide the rationale for personalized therapies. To mimic the MM niche, we here describe a new 3D co-culture ex-vivo model in which primary MM patient BM cells are co-cultured with mesenchymal stem cells (MSC) in a hydrogel 3D system. In the 3D model, MSC with conserved phenotype (CD73+CD90+CD105+) formed compact clusters with active fibrous connections, and retained lineage differentiation capacity. Extracellular matrix molecules, integrins, and niche related molecules including N-cadherin and CXCL12 are expressed in 3D MSC model. Furthermore, activation of osteogenesis (MMP13, SPP1, ADAMTS4, and MGP genes) and osteoblastogenic differentiation was confirmed in 3D MSC model. Co-culture of patient-derived BM mononuclear cells with either autologous or allogeneic MSC in 3D model increased proliferation of MM cells, CXCR4 expression, and SP cells. We carried out immune profiling to show that distribution of immune cell subsets was similar in 3D and 2D MSC model systems. Importantly, resistance to novel agents (IMiDs, bortezomib, carfilzomib) and conventional agents (doxorubicin, dexamethasone, melphalan) was observed in 3D MSC system, reflective of clinical resistance. This 3D MSC model may therefore allow for studies of MM pathogenesis and drug resistance within the BM niche. Importantly, ongoing prospective trials are evaluating its utility to inform personalized targeted and immune therapy in MM. PMID:27764795

In vivo targeting and imaging of tumor vasculature with radiolabeled, antibody-conjugated nanographene.

PubMed

Hong, Hao; Yang, Kai; Zhang, Yin; Engle, Jonathan W; Feng, Liangzhu; Yang, Yunan; Nayak, Tapas R; Goel, Shreya; Bean, Jero; Theuer, Charles P; Barnhart, Todd E; Liu, Zhuang; Cai, Weibo

2012-03-27

Herein we demonstrate that nanographene can be specifically directed to the tumor neovasculature in vivo through targeting of CD105 (i.e., endoglin), a vascular marker for tumor angiogenesis. The covalently functionalized nanographene oxide (GO) exhibited excellent stability and target specificity. Pharmacokinetics and tumor targeting efficacy of the GO conjugates were investigated with serial noninvasive positron emission tomography imaging and biodistribution studies, which were validated by in vitro, in vivo, and ex vivo experiments. The incorporation of an active targeting ligand (TRC105, a monoclonal antibody that binds to CD105) led to significantly improved tumor uptake of functionalized GO, which was specific for the neovasculature with little extravasation, warranting future investigation of these GO conjugates for cancer-targeted drug delivery and/or photothermal therapy to enhance therapeutic efficacy. Since poor extravasation is a major hurdle for nanomaterial-based tumor targeting in vivo, this study also establishes CD105 as a promising vascular target for future cancer nanomedicine. © 2012 American Chemical Society

Elevated pretransplantation soluble CD30 is associated with decreased early allograft function after human lung transplantation.

PubMed

Shah, Ashish S; Leffell, M Sue; Lucas, Donna; Zachary, Andrea A

2009-02-01

Early allograft function after lung transplantation is variable. Clinical criteria have limited predictive value for early graft function. Recipient immunologic state before LTx may affect early lung function. We investigated the association between pretransplantation soluble CD30 (sCD30), a marker of Th2-type T-cell activation, and early clinical parameters of allograft function. Between September 2002 and January 2007, a total of 80 transplantations were performed at Johns Hopkins Hospital. Of the patients, 43 had a pretransplantation sCD30 level determined. Pre- and postoperative patient variables were collected, and patients were stratified into two groups: sCD30 <20 (low sCD30) and >20 (high sCD30). High sCD30 (n = 26) and low sCD30 (n = 17) groups were similar in age, gender, and ischemia time. In the high sCD30 group, a higher percentage of patients had pulmonary fibrosis and a lower percentage had emphysema. Oxygenation at 48 hours was significantly worse in the high sCD30 group as compared with the low sCD30 (p = 0.003). Moreover, prolonged intubation and 90-day mortality were greater in the high sCD30 group. This represents the first report of the use of sCD30 as a marker for early allograft function in human lung transplanation. Increased pretransplantation recipient sCD30 appears to be associated with decreased early post-transplantation gas exchange, prolonged intubation, and early mortality.

Extension of activation cross-section data of deuteron induced nuclear reactions on cadmium up to 50 MeV

NASA Astrophysics Data System (ADS)

Hermanne, A.; Tárkányi, F.; Takács, S.; Ditrói, F.

2016-10-01

The excitation functions for 109,110g,111m+g,113m,114m,115mIn, 107,109,115m,115gCd and 105g,106m,110g,111Ag are presented for stacked foil irradiations on natCd targets in the 49-33 MeV deuteron energy domain. Reduced uncertainty is obtained by determining incident particle flux and energy scale relative to re-measured monitor reactions natAl(d,x)22,24Na. The results were compared to our earlier studies on natCd and on enriched 112Cd targets. The merit of the values predicted by the TALYS 1.6 code (resulting from a weighted combination of reaction cross-section data on all stable Cd isotopes as available in the on-line libraries TENDL-2014 and TENDL-2015) is discussed. Influence on optimal production routes for several radionuclides with practical applications (111In, 114mIn, 115Cd, 109,107Cd….) is reviewed.

Evaluation of soluble CD30 as an immunologic marker in heart transplant recipients.

PubMed

Spiridon, C; Hunt, J; Mack, M; Rosenthal, J; Anderson, A; Eichhorn, E; Magee, M; Dewey, T; Currier, M; Nikaein, A

2006-12-01

CD30 is an immunologic molecule that belongs to the TNF-R superfamily. CD30 serves as a T-cell signal transducing molecule that is expressed by a subset of activated T lymphocytes, CD45RO+ memory T cells. Augmentation of soluble CD30 during kidney transplant rejection has been reported. Our study sought to determine whether the level of sCD30 prior to heart transplant could categorize patients into high versus low immunologic risk for a poor outcome. A significant correlation was observed between high levels of soluble CD30 and a reduced incidence of infection. None of the 35 patients with high pretransplant levels of sCD30 level (>90 U/mL) developed infections posttransplantation. However, 9 of 65 patients who had low levels of sCD30 (<90 U/mL) developed infections posttransplantation (P < .02). No remarkable differences were noted among the other clinical parameters. The results also showed that the high-definition flow-bead (HDB) assay detected both weak and strong class I and class II HLA antibodies, some of which (weak class II HLA Abs) were undetectable by the anti-human globulin cytotoxicity method. In addition, more antibody specificities were detected by HDB. In conclusion, we have observed that high levels of sCD30 prior to heart transplant may be associated with greater immunologic ability and therefore produce a protective effect on the development of infection post heart transplant. We have also shown that the HDB assay is superior to the visual cytotoxicity method to detect HLA antibodies, especially those to class II HLA antigens.

Significant differences in B-cell subpopulations characterize patients with chronic graft-versus-host disease-associated dysgammaglobulinemia.

PubMed

Kuzmina, Zoya; Greinix, Hildegard T; Weigl, Roman; Körmöczi, Ulrike; Rottal, Arno; Frantal, Sophie; Eder, Sandra; Pickl, Winfried F

2011-02-17

Manifestations of chronic graft-versus-host disease (cGVHD) can resemble those seen in immunodeficiency states and autoimmune disorders. Reports by us and others suggest an involvement of B cells in the pathogenesis of cGVHD. We investigated B-lymphocyte subpopulations in cGVHD cohorts defined by serum immunoglobulin G (IgG) levels to characterize novel biomarkers for impairment of humoral immunity after allogeneic hematopoietic stem cell transplantation. Seventy-six patients were enrolled a median of 46 months after hematopoietic stem cell transplantation. The hypogammaglobulinemia group had significantly diminished CD19(+) B cells (165 vs 454 vs 417 × 10⁶L) with elevated CD19(+)CD21(low) immature (16.5%, 7.7%, and 9.1%) and CD19(+)CD21(int-high)CD38(high)IgM(high) transitional (10.5% vs 4.2% vs 6.3%) B-cell proportions compared with the normogammaglobulinemia and hypergammaglobulinemia groups. CD19(+)CD10(-)CD27(-)CD21(high) naive B cells were highly elevated in all patients with cGVHD. CD19(+)CD27(+)IgD(+) non-class-switched (4 vs 12 vs 11 × 10⁶/L) and class-switched (7 vs 35 vs 42 × 10⁶/L) memory B cells were significantly lower in the hypogammaglobulinemia group compared with the others. Besides significantly higher B-cell activation factor/B-cell ratios, significantly more cGVHD patients with hypergammaglobulinemia had autoantibodies compared with the hypogammaglobulinemia subgroup (68% vs 24%, P = .024). In conclusion, B-cell subpopulations can serve as novel cellular biomarkers for immunodeficiency and autoimmunity indicating different pathogenetic mechanisms of cGVHD and encouraging future prospective longitudinal studies.

Increased peripheral blood CD4+ T cell responses to deamidated but not to native gliadin in children with coeliac disease.

PubMed

Lammi, A; Arikoski, P; Vaarala, O; Kinnunen, T; Ilonen, J

2012-05-01

T cell recognition of gliadin from dietary gluten is essential for the pathogenesis of coeliac disease (CD). The aim of the present study was to analyse whether gliadin-specific T cells are detectable in the circulation of children with newly diagnosed coeliac disease by using a sensitive carboxfluorescein diacetate succinimidyl ester (CFSE) dilution method. Peripheral blood CD4(+) T cell responses were analysed in 20 children at diagnosis of CD and compared to those in 64 healthy control children carrying the CD-associated human leucocyte antigen (HLA)-DQ2 or -DQ8 alleles. Deamidated gliadin (gTG)-specific T cells were detectable in the peripheral blood of more than half the children with CD (11 of 20, 55%) compared to 15 of 64 (23.4%) of the control children (P = 0.008). Proliferative responses to gTG were also significantly stronger in children with CD than in controls (P = 0.01). In contrast, T cells specific to native gliadin were detectable at comparable frequencies in children with CD (two of 19, 10.5%) and controls (13 of 64, 20.3%). gTG-specific T cells had a memory phenotype more often than those specific to native gliadin in children with CD (P = 0.02), whereas controls had similar percentages of memory cells in both stimulations. Finally, gTG-specific CD4(+) T cells had a higher expression of the gut-homing molecule β7 integrin than those specific to the control antigen tetanus toxoid. Collectively, our current results demonstrate that the frequency of circulating memory CD4(+) T cells specific to gTG but not native gliadin is increased in children with newly diagnosed CD. © 2012 The Authors;Clinical and Experimental Immunology © 2012 British Society for Immunology.

Human CD34+ cells engineered to express membrane-bound tumor necrosis factor-related apoptosis-inducing ligand target both tumor cells and tumor vasculature.

PubMed

Lavazza, Cristiana; Carlo-Stella, Carmelo; Giacomini, Arianna; Cleris, Loredana; Righi, Marco; Sia, Daniela; Di Nicola, Massimo; Magni, Michele; Longoni, Paolo; Milanesi, Marco; Francolini, Maura; Gloghini, Annunziata; Carbone, Antonino; Formelli, Franca; Gianni, Alessandro M

2010-03-18

Adenovirus-transduced CD34+ cells expressing membrane-bound tumor necrosis factor-related apoptosis-inducing ligand (CD34-TRAIL+ cells) exert potent antitumor activity. To further investigate the mechanism(s) of action of CD34-TRAIL+ cells, we analyzed their homing properties as well as antitumor and antivascular effects using a subcutaneous myeloma model in immunodeficient mice. After intravenous injection, transduced cells homed in the tumor peaking at 48 hours when 188 plus or minus 25 CD45+ cells per 10(5) tumor cells were detected. Inhibition experiments showed that tumor homing of CD34-TRAIL+ cells was largely mediated by vascular cell adhesion molecule-1 and stromal cell-derived factor-1. Both CD34-TRAIL+ cells and soluble (s)TRAIL significantly reduced tumor volume by 40% and 29%, respectively. Computer-aided analysis of TdT-mediated dUTP nick end-labeling-stained tumor sections demonstrated significantly greater effectiveness for CD34-TRAIL+ cells in increasing tumor cell apoptosis and necrosis over sTRAIL. Proteome array analysis indicated that CD34-TRAIL+ cells and sTRAIL activate similar apoptotic machinery. In vivo staining of tumor vasculature with sulfosuccinimidyl-6-(biotinamido) hexanoate-biotin revealed that CD34-TRAIL+ cells but not sTRAIL significantly damaged tumor vasculature, as shown by TdT-mediated dUTP nick end-labeling+ endothelial cells, appearance of hemorrhagic areas, and marked reduction of endothelial area. These results demonstrate that tumor homing of CD34-TRAIL+ cells induces early vascular disruption, resulting in hemorrhagic necrosis and tumor destruction.

Protective Cytomegalovirus (CMV)-Specific T-Cell Immunity Is Frequent in Kidney Transplant Patients without Serum Anti-CMV Antibodies

PubMed Central

Litjens, Nicolle H. R.; Huang, Ling; Dedeoglu, Burç; Meijers, Ruud W. J.; Kwekkeboom, Jaap; Betjes, Michiel G. H.

2017-01-01

The absence of anti-cytomegalovirus (CMV) immunoglobulin G (IgG) is used to classify pretransplant patients as naïve for CMV infection (CMVneg patients). This study assessed whether pretransplant CMV-specific T-cell immunity exists in CMVneg patients and whether it protects against CMV infection after kidney transplantation. The results show that CMV-specific CD137+IFNγ+CD4+ and CD137+IFNγ+CD8+ memory T cells were present in 46 and 39% of CMVneg patients (n = 28) although at much lower frequencies compared to CMVpos patients (median 0.01 versus 0.58% for CD4+ and 0.05 versus 0.64% for CD8+ T cells) with a less differentiated CD28-expressing phenotype. In line with these data, CMV-specific proliferative CD4+ and CD8+ T cells were observed in CMVneg patients, which significantly correlated with the frequency of CMV-specific T cells. CMV-specific IgG antibody-secreting cells (ASC) could be detected at low frequency in 36% of CMVneg patients (1 versus 45 ASC/105 cells in CMVpos patients). CMVneg patients with pretransplant CMV-specific CD137+IFNγ+CD4+ T cells had a lower risk to develop CMV viremia after transplantation with a CMVpos donor kidney (relative risk: 0.43, P = 0.03). In conclusion, a solitary CMV-specific T-cell response without detectable anti-CMV antibodies is frequent and clinically relevant as it is associated with protection to CMV infection following transplantation with a kidney from a CMVpos donor. PMID:28955345

Prognostic role of CD133 expression in colorectal cancer: a meta-analysis.

PubMed

Wang, Ke; Xu, Jianjun; Zhang, Junshu; Huang, Jian

2012-12-05

CD133 has been identified as a putative cancer stem cell marker in colorectal cancer (CRC). However, the clinical and prognostic significance of CD133 in CRC remains controversial. Publications were identified which assessed the clinical or prognostic significance of CD133 in CRC up to October 2012. A meta-analysis was performed to clarify the association between CD133 expression and clinical outcomes. A total of 12 studies met the inclusion criteria, and comprised 3652 cases. Analysis of these data showed that CD133 was not significantly associated with the depth of CRC invasion (odds ratio [OR] = 1.44, 95% confidence interval [CI]: 0.77-2.68, Z = 1.15, P = 0.252) or tumor differentiation (OR = 0.63, 95% CI: 0.28-1.46, Z = -1.06, P = 0.286). Also, there was no statistically significant association of CD133 with lymph node metastasis (OR = 1.16, 95% CI: 0.87-1.54, Z = 1.05, P = 0.315) or lymphatic invasion (OR = 1.08, 95% CI: 0.81-1.43, Z = 0.53, P = 0.594). However, in identified studies, overexpression of CD133 was highly correlated with reduced overall survival (relative risk [RR] = 2.14, 95% CI: 1.45-3.17, Z = 3.81, P = 0.0001). CD133 may play an important role in the progression of CRC, and overexpression of CD133 is closely related with poorer patient survival. If these findings are confirmed by well-designed prospective studies, CD133 may be a useful maker for clinical applications.

As, Cd, Cr, Ni and Pb pressurized liquid extraction with acetic acid from marine sediment and soil samples

NASA Astrophysics Data System (ADS)

Moreda-Piñeiro, Jorge; Alonso-Rodríguez, Elia; López-Mahía, Purificación; Muniategui-Lorenzo, Soledad; Prada-Rodríguez, Darío; Moreda-Piñeiro, Antonio; Bermejo-Barrera, Adela; Bermejo-Barrera, Pilar

2006-12-01

Rapid leaching procedures by Pressurized Liquid Extraction (PLE) have been developed for As, Cd, Cr, Ni and Pb leaching from environmental matrices (marine sediment and soil samples). The Pressurized Liquid Extraction is completed after 16 min. The released elements by acetic acid Pressurized Liquid Extraction have been evaluated by inductively coupled plasma-optical emission spectrometry. The optimum multi-element leaching conditions when using 5.0 ml stainless steel extraction cells, were: acetic acid concentration 8.0 M, extraction temperature 100 °C, pressure 1500 psi, static time 5 min, flush solvent 60%, two extraction steps and 0.50 g of diatomaceous earth as dispersing agent (diatomaceous earth mass/sample mass ratio of 2). Results have showed that high acetic acid concentrations and high extraction temperatures increase the metal leaching efficiency. Limits of detection (between 0.12 and 0.5 μg g - 1 ) and repeatability of the over-all procedure (around 6.0%) were assessed. Finally, accuracy was studied by analyzing PACS-2 (marine sediment), GBW-07409 (soil), IRANT-12-1-07 (cambisol soil) and IRANT-12-1-08 (luvisol soil) certified reference materials (CRMs). These certified reference materials offer certified concentrations ranges between 2.9 and 26.2 μg g - 1 for As, from 0.068 to 2.85 μg g - 1 for Cd, between 26.4 and 90.7 μg g - 1 for Cr, from 9.3 to 40.0 μg g - 1 for Ni and between 16.3 and 183.0 μg g - 1 for Pb. Recoveries after analysis were between 95.7 and 105.1% for As, 96.2% for Cd, 95.2 and 100.6% for Cr, 95.7 and 103% for Ni and 94.2 and 105.5% for Pb.

Dynamics of three organic acids (malic, acetic and succinic acid) in sunflower exposed to cadmium and lead.

PubMed

Niu, Zhixin; Li, Xiaodong; Sun, Lina; Sun, Tieheng

2013-01-01

Sunflower (Helianthus annuus L.) has been considered as a good candidate for bioaccumulation of heavy metals. In the present study, sunflower was used to enrich the cadmium and lead in sand culture during 90 days. Biomass, Cd and Pb uptake, three organic acids and pH in cultures were investigated. Results showed that the existence of Cd and Pb showed different interactions on the organic acids exudation. In single Cd treatments, malic and acetic acids in Cd10 showed an incremental tendency with time. In the mixed treatments of Cd and Pb, malic acids increased when 10 and 40 mg x L(-1) Cd were added into Pb50, but acetic acids in Pb50 were inhibited by Cd addition. The Cd10 supplied in Pb10 stimulated the secretion of malic and succinic acids. Moreover, the Cd or Pb uptake in sunflower showed various correlations with pH and some organic acids, which might be due to the fact that the Cd and Pb interfere with the organic acids secretion in rhizosphere of sunflower, and the changes of organic acids altered the form and bioavailability of Cd and Pb in cultures conversely.

CD4 Lymphocyte Enumeration and Hemoglobin Assessment Aid for Priority Decisions: A Multisite Evaluation of the BD FACSPresto™ System

PubMed Central

Thakar, Madhuri; Angira, Francis; Pattanapanyasat, Kovit; Wu, Alan H.B.; O’Gorman, Maurice; Zeng, Hui; Qu, Chenxue; Mahajan, Bharati; Sukapirom, Kasama; Chen, Danying; Hao, Yu; Gong, Yan; Indig, Monika De Arruda; Graminske, Sharon; Orta, Diana; d’Empaire, Nicole; Lu, Beverly; Omana-Zapata, Imelda; Zeh, Clement

2017-01-01

Background: The BD FACSPresto™ system uses capillary and venous blood to measure CD4 absolute counts (CD4), %CD4 in lymphocytes, and hemoglobin (Hb) in approximately 25 minutes. CD4 cell count is used with portable CD4 counters in resource-limited settings to manage HIV/AIDS patients. A method comparison was performed using capillary and venous samples from seven clinical laboratories in five countries. The BD FACSPresto system was assessed for variability between laboratory, instrument/operators, cartridge lots and within-run at four sites. Methods: Samples were collected under approved voluntary consent. EDTA-anticoagulated venous samples were tested for CD4 and %CD4 T cells using the gold-standard BD FACSCalibur™ system, and for Hb, using the Sysmex® KX-21N™ analyzer. Venous and capillary samples were tested on the BD FACSPresto system. Matched data was analyzed for bias (Deming linear regression and Bland-Altman methods), and for concordance around the clinical decision point. The coefficient of variation was estimated per site, instrument/operator, cartridge-lot and between-runs. Results: For method comparison, 93% of the 720 samples were from HIV-positive and 7% from HIV-negative or normal subjects. CD4 and %CD4 T cells venous and capillary results gave slopes within 0.96–1.05 and R2 ≥0.96; Hb slopes were ≥1.00 and R2 ≥0.89. Variability across sites/operators gave %CV <5.8% for CD4 counts, <1.9% for %CD4 and <3.2% for Hb. The total %CV was <7.7% across instrument/cartridge lot. Conclusion: The BD FACSPresto system provides accurate, reliable, precise CD4/%CD4/Hb results compared to gold-standard methods, irrespective of venous or capillary blood sampling. The data showed good agreement between the BD FACSPresto, BD FACSCalibur and Sysmex systems. PMID:29290885

Nanosized UCMSC-derived extracellular vesicles but not conditioned medium exclusively inhibit the inflammatory response of stimulated T cells: implications for nanomedicine.

PubMed

Monguió-Tortajada, Marta; Roura, Santiago; Gálvez-Montón, Carolina; Pujal, Josep Maria; Aran, Gemma; Sanjurjo, Lucía; Franquesa, Marcel la; Sarrias, Maria-Rosa; Bayes-Genis, Antoni; Borràs, Francesc E

2017-01-01

Undesired immune responses have drastically hampered outcomes after allogeneic organ transplantation and cell therapy, and also lead to inflammatory diseases and autoimmunity. Umbilical cord mesenchymal stem cells (UCMSCs) have powerful regenerative and immunomodulatory potential, and their secreted extracellular vesicles (EVs) are envisaged as a promising natural source of nanoparticles to increase outcomes in organ transplantation and control inflammatory diseases. However, poor EV preparations containing highly-abundant soluble proteins may mask genuine vesicular-associated functions and provide misleading data. Here, we used Size-Exclusion Chromatography (SEC) to successfully isolate EVs from UCMSCs-conditioned medium. These vesicles were defined as positive for CD9, CD63, CD73 and CD90, and their size and morphology characterized by NTA and cryo-EM. Their immunomodulatory potential was determined in polyclonal T cell proliferation assays, analysis of cytokine profiles and in the skewing of monocyte polarization. In sharp contrast to the non-EV containing fractions, to the complete conditioned medium and to ultracentrifuged pellet, SEC-purified EVs from UCMSCs inhibited T cell proliferation, resembling the effect of parental UCMSCs. Moreover, while SEC-EVs did not induce cytokine response, the non-EV fractions, conditioned medium and ultracentrifuged pellet promoted the secretion of pro-inflammatory cytokines by polyclonally stimulated T cells and supported Th17 polarization. In contrast, EVs did not induce monocyte polarization, but the non-EV fraction induced CD163 and CD206 expression and TNF-α production in monocytes. These findings increase the growing evidence confirming that EVs are an active component of MSC's paracrine immunosuppressive function and affirm their potential for therapeutics in nanomedicine. In addition, our results highlight the importance of well-purified and defined preparations of MSC-derived EVs to achieve the immunosuppressive effect.

Controlling congenital and paediatric chagas disease through a community health approach with active surveillance and promotion of paediatric awareness.

PubMed

Soriano-Arandes, Antoni; Basile, Luca; Ouaarab, Hakima; Clavería, Isabel; Gómez i Prat, Jordi; Cabezos, Juan; Ciruela, Pilar; Albajar-Viñas, Pedro; Jané, Mireia

2014-11-21

Chagas disease (CD) is endemic in countries of continental Latin America. Congenital transmission is a major concern worldwide. In 2010, the Public Health Agency of Catalonia (ASPCAT) launched a screening protocol for Trypanosoma cruzi infection in pregnant women and their newborns. In 2012, ASPCAT detected appropriate follow-up of pregnant women but incomplete information about their offspring. The PROSICS community health team carried out active surveillance and community health action in target populations. These activities included active case searches, group awareness workshops and visualization campaigns as well as investigation of all lost children born from pregnant women with CD and their families. Overall, 42/179 (23.5%) cases were included in the study: 35/42 (83.3%) children were born in Hospitalet de Llobregat (Catalonia, Spain); 4/42 (16.7%) were born in Latin America; two were miscarried and one was stillborn. The mean age of pregnant women was 31.3 years (SD 5.52; range: 21-44): 90.5% were Bolivian, of whom 74% were diagnosed with CD during pregnancy. Of the 35 newborns, 31 were recovered by community health action; 12/31 were correctly controlled at Hospitalet de Llobregat and 19/31 were controlled at a primary health centre. Of these 19 (73.7%) cases, 14 were not tested for CD by family paediatricians and were recovered by the PROSICS community health team. Finally, two (6.9%) of the 29 newborns tested with serology were positive. It is essential to implement active surveillance, education and information activities at paediatric primary care and community levels to avoid the loss of CD-infected mothers and their newborns. Training sessions addressed to paediatricians and other involved health professionals would consolidate surveillance and care reference circuits, improving the control of congenital CD.

Cadmium telluride leaching behavior: Discussion of Zeng et al. (2015).

PubMed

Sinha, Parikhit

2015-11-01

Zeng et al. (2015) evaluate the leaching behavior and surface chemistry of II-VI semiconductor materials, CdTe and CdSe, in response to pH and O2. Under agitation in acidic and aerobic conditions, the authors found approximately 3.6%-6.4% (w/w) solubility of Cd content in CdTe in the Toxicity Characteristic Leaching Procedure (TCLP), Waste Extraction Test (WET), and dissolution test, with lower solubility (0.56-0.58%) under agitation in acidic and anoxic conditions. This range is comparable with prior long-term transformation and dissolution testing and bio-elution testing of CdTe (2.3%-4.1% w/w solubility of Cd content in CdTe). The implications for potential leaching behavior of CdTe-containing devices require further data. Since CdTe PV modules contain approximately 0.05% Cd content by mass, the starting Cd content in the evaluation of CdTe-containing devices would be lower by three orders of magnitude than the starting Cd content in the authors' study, and leaching potential would be further limited by the monolithic glass-adhesive laminate-glass structure of the device that encapsulates the semiconductor material. Experimental evaluation of leaching potential of CdTe PV modules crushed by landfill compactor has been conducted, with results of TCLP and WET tests on the crushed material below regulatory limits for Cd. CdTe PV recycling technology has been in commercial operation since 2005 with high yields for semiconductor (95%) and glass (90%) recovery. Copyright © 2015 Elsevier Ltd. All rights reserved.

21 CFR 73.3120 - 16,17-Dimethoxydinaphtho [1,2,3-cd:3′,2′,1′-lm] perylene-5,10-dione.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 1 2013-04-01 2013-04-01 false 16,17-Dimethoxydinaphtho [1,2,3-cd:3â²,2â²,1â²-lm] perylene-5,10-dione. 73.3120 Section 73.3120 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL LISTING OF COLOR ADDITIVES EXEMPT FROM CERTIFICATION Medical Devices...

21 CFR 73.3120 - 16,17-Dimethoxydinaphtho [1,2,3-cd:3′,2′,1′-lm] perylene-5,10-dione.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 1 2011-04-01 2011-04-01 false 16,17-Dimethoxydinaphtho [1,2,3-cd:3â²,2â²,1â²-lm] perylene-5,10-dione. 73.3120 Section 73.3120 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL LISTING OF COLOR ADDITIVES EXEMPT FROM CERTIFICATION Medical Devices...

21 CFR 73.3120 - 16,17-Dimethoxydinaphtho [1,2,3-cd:3′,2′,1′-lm] perylene-5,10-dione.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 1 2014-04-01 2014-04-01 false 16,17-Dimethoxydinaphtho [1,2,3-cd:3â²,2â²,1â²-lm] perylene-5,10-dione. 73.3120 Section 73.3120 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL LISTING OF COLOR ADDITIVES EXEMPT FROM CERTIFICATION Medical Devices...

21 CFR 73.3120 - 16,17-Dimethoxydinaphtho [1,2,3-cd:3′,2′,1′-lm] perylene-5,10-dione.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 1 2012-04-01 2012-04-01 false 16,17-Dimethoxydinaphtho [1,2,3-cd:3â²,2â²,1â²-lm] perylene-5,10-dione. 73.3120 Section 73.3120 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL LISTING OF COLOR ADDITIVES EXEMPT FROM CERTIFICATION Medical Devices...

21 CFR 73.3120 - 16,17-Dimethoxydinaphtho [1,2,3-cd:3′,2′,1′-lm] perylene-5,10-dione.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 1 2010-04-01 2010-04-01 false 16,17-Dimethoxydinaphtho [1,2,3-cd:3â²,2â²,1â²-lm] perylene-5,10-dione. 73.3120 Section 73.3120 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL LISTING OF COLOR ADDITIVES EXEMPT FROM CERTIFICATION Medical Devices...

Antitumor effect of combined NAMPT and CD73 inhibition in an ovarian cancer model

PubMed Central

Magnone, Mirko; Zamporlini, Federica; Emionite, Laura; Sturla, Laura; Bianchi, Giovanna; Vigliarolo, Tiziana; Nahimana, Aimable; Nencioni, Alessio; Raffaelli, Nadia; Bruzzone, Santina

2016-01-01

Nicotinamide phosphoribosyltransferase (NAMPT) is a crucial enzyme in the biosynthesis of intracellular NAD+. NAMPT inhibitors have potent anticancer activity in several preclinical models by depleting NAD+ and ATP levels. Recently, we demonstrated that CD73 enables the utilization of extracellular NAD+/nicotinamide mononucleotide (NMN) by converting them to Nicotinamide riboside (NR), which can cross the plasmamembrane and fuel intracellular NAD+ biosynthesis in human cells. These processes are herein confirmed to also occur in a human ovarian carcinoma cell line (OVCAR-3), by means of CD73 or NRK1 specific silencing. Next, we investigated the anti-tumor activity of the simultaneous inhibition of NAMPT (with FK866) and CD73 (with α, β-methylene adenosine 5′-diphosphate, APCP), in an in vivo human ovarian carcinoma model. Interestingly, the combined therapy was found to significantly decrease intratumor NAD+, NMN and ATP levels, compared with single treatments. In addition, the concentration of these nucleotides in ascitic exudates was more remarkably reduced in animals treated with both FK866 and APCP compared with single treatments. Importantly, tumors treated with FK866 in combination with APCP contained a statistically significant lower proportion of Ki67 positive proliferating cells and a higher percentage of necrotic area. Finally, a slight but significant increase in animal survival in response to the combined therapy, compared to the single agents, could be demonstrated. Our results indicate that the pharmacological inhibition of CD73 enzymatic activity could be considered as a means to potentiate the anti-cancer effects of NAMPT inhibitors. PMID:26658104

Antitumor effect of combined NAMPT and CD73 inhibition in an ovarian cancer model.

PubMed

Sociali, Giovanna; Raffaghello, Lizzia; Magnone, Mirko; Zamporlini, Federica; Emionite, Laura; Sturla, Laura; Bianchi, Giovanna; Vigliarolo, Tiziana; Nahimana, Aimable; Nencioni, Alessio; Raffaelli, Nadia; Bruzzone, Santina

2016-01-19

Nicotinamide phosphoribosyltransferase (NAMPT) is a crucial enzyme in the biosynthesis of intracellular NAD+. NAMPT inhibitors have potent anticancer activity in several preclinical models by depleting NAD+ and ATP levels. Recently, we demonstrated that CD73 enables the utilization of extracellular NAD+/nicotinamide mononucleotide (NMN) by converting them to Nicotinamide riboside (NR), which can cross the plasmamembrane and fuel intracellular NAD+ biosynthesis in human cells. These processes are herein confirmed to also occur in a human ovarian carcinoma cell line (OVCAR-3), by means of CD73 or NRK1 specific silencing. Next, we investigated the anti-tumor activity of the simultaneous inhibition of NAMPT (with FK866) and CD73 (with α, β-methylene adenosine 5'-diphosphate, APCP), in an in vivo human ovarian carcinoma model. Interestingly, the combined therapy was found to significantly decrease intratumor NAD+, NMN and ATP levels, compared with single treatments. In addition, the concentration of these nucleotides in ascitic exudates was more remarkably reduced in animals treated with both FK866 and APCP compared with single treatments. Importantly, tumors treated with FK866 in combination with APCP contained a statistically significant lower proportion of Ki67 positive proliferating cells and a higher percentage of necrotic area. Finally, a slight but significant increase in animal survival in response to the combined therapy, compared to the single agents, could be demonstrated. Our results indicate that the pharmacological inhibition of CD73 enzymatic activity could be considered as a means to potentiate the anti-cancer effects of NAMPT inhibitors.

Low numbers of CD8+ T lymphocytes in hereditary haemochromatosis are explained by a decrease of the most mature CD8+ effector memory T cells.

PubMed

Macedo, M F; Porto, G; Costa, M; Vieira, C P; Rocha, B; Cruz, E

2010-03-01

Low CD8(+) T lymphocyte numbers have long been described in hereditary haemochromatosis (HH). Recently, two conserved haplotypes localized near the microsatellite D6S105 at the major histocompatibility complex (MHC) class I region were described predicting the clinical expression of HH and the CD8(+) T lymphocyte numbers. The A-A-T haplotype was associated with a severe clinical expression of HH and low CD8(+) T lymphocyte numbers, while the G-G-G haplotype was associated with a milder clinical expression of HH and high CD8(+) T lymphocyte numbers. As CD8(+) T lymphocytes are a very heterogeneous population, in this study we analysed the CD8(+) subpopulations of naive, central memory (T(CM)) and effector memory (T(EM)), and further subsets of CD8(+) T(EM) cells in 47 HH patients and 68 controls. In addition, association studies were conducted between the conserved haplotypes and the CD8(+) T cell subpopulations in HH. Variations of the numbers of naive and central memory cells with age were similar between HH patients and controls. For T(EM) cells and the T(EM) CD27(-)CD28(-) subset no effect of age was observed in HH [R(2) = 0.001, not significant (n.s.) and R(2) = 0.01, n.s., respectively] contrasting with the increasing of these subpopulations with age in controls (R(2) = 0.09, P = 0.017 and R(2) = 0.22, P = 0.0005, respectively). Interestingly, patients homozygous for the A-A-T haplotype have lower numbers of CD8(+) T(EM) cells due especially to lower numbers of T(EM) CD27(-)CD28(-) (0.206 +/- 0.119 and 0.066 +/- 0.067 x 10(6) cells/ml, respectively) than patients carrying the G-G-G haplotype (0.358 +/- 0.195 and 0.246 +/- 0.202 x 10(6) cells/ml, respectively). This may suggest an inability of HH patients to differentiate the CD8(+) T cells into the most mature phenotype.

Low numbers of CD8+ T lymphocytes in hereditary haemochromatosis are explained by a decrease of the most mature CD8+ effector memory T cells

PubMed Central

Macedo, M F; Porto, G; Costa, M; Vieira, C P; Rocha, B; Cruz, E

2010-01-01

Low CD8+ T lymphocyte numbers have long been described in hereditary haemochromatosis (HH). Recently, two conserved haplotypes localized near the microsatellite D6S105 at the major histocompatibility complex (MHC) class I region were described predicting the clinical expression of HH and the CD8+ T lymphocyte numbers. The A-A-T haplotype was associated with a severe clinical expression of HH and low CD8+ T lymphocyte numbers, while the G-G-G haplotype was associated with a milder clinical expression of HH and high CD8+ T lymphocyte numbers. As CD8+ T lymphocytes are a very heterogeneous population, in this study we analysed the CD8+ subpopulations of naive, central memory (TCM) and effector memory (TEM), and further subsets of CD8+ TEM cells in 47 HH patients and 68 controls. In addition, association studies were conducted between the conserved haplotypes and the CD8+ T cell subpopulations in HH. Variations of the numbers of naive and central memory cells with age were similar between HH patients and controls. For TEM cells and the TEM CD27−CD28− subset no effect of age was observed in HH [R2 = 0·001, not significant (n.s.) and R2 = 0·01, n.s., respectively] contrasting with the increasing of these subpopulations with age in controls (R2 = 0·09, P = 0·017 and R2 = 0·22, P = 0·0005, respectively). Interestingly, patients homozygous for the A-A-T haplotype have lower numbers of CD8+ TEM cells due especially to lower numbers of TEM CD27−CD28− (0·206 ± 0·119 and 0·066 ± 0·067 × 106 cells/ml, respectively) than patients carrying the G-G-G haplotype (0·358 ± 0·195 and 0·246 ± 0·202 × 106 cells/ml, respectively). This may suggest an inability of HH patients to differentiate the CD8+ T cells into the most mature phenotype. PMID:20015273

Changing mortality risk associated with CD4 cell response to antiretroviral therapy in South Africa

PubMed Central

Lawn, Stephen D.; Little, Francesca; Bekker, Linda-Gail; Kaplan, Richard; Campbel, Elizabeth; Orrell, Catherine; Wood, Robin

2013-01-01

Objective To determine the relationship between mortality risk and the CD4 cell response to antiretroviral therapy (ART). Design Observational community-based ART cohort in South Africa. Methods CD4 cell counts were measured 4 monthly, and deaths were prospectively ascertained. Cumulative person-time accrued within a range of updated CD4 cell count strata (CD4 cell-strata) was calculated and used to derive CD4 cell-stratified mortality rates. Results Patients (2423) (median baseline CD4 cell count of 105 cells/ml) were observed for up to 5 years of ART. One hundred and ninety-seven patients died during 3155 person years of observation. In multivariate analysis, mortality rate ratios associated with 0–49, 50–99, 100–199, 200–299, 300– 399, 400–499 and at least 500 cells/ml updated CD4 cell-strata were 11.6, 4.9, 2.6, 1.7, 1.5, 1.4 and 1.0, respectively. Analysis of CD4 cell count recovery permitted calculations of person-time accrued within these CD4 cell strata. Despite rapid immune recovery, high mortality in the first year of ART was related to the large proportion of person-time accrued within CD4 cell-strata less than 200 cells/ml. Moreover, patients with baseline CD4 cell counts less than 100 cells/ml had much higher cumulative mortality estimates at 1 and 4 years (11.6 and 16.7%) compared with those of patients with baseline counts of at least 100 cells/ml (5.2 and 9.5%) largely because of greater cumulative person-time at CD4 cell counts less than 200 cells/ml. Conclusion: Updated CD4 cell counts are the variable most strongly associated with mortality risk during ART. High cumulative mortality risk is associated with person-time accrued at low CD4 cell counts. National HIV programmes in resource-limited settings should be designed to minimize the time patients spend with CD4 cell counts less than 200 cells/ml both before and during ART. PMID:19114870

High CD49f expression is associated with osteosarcoma tumor progression: a study using patient-derived primary cell cultures.

PubMed

Penfornis, Patrice; Cai, David Z; Harris, Michael R; Walker, Ryan; Licini, David; Fernandes, Joseph D A; Orr, Griffin; Koganti, Tejaswi; Hicks, Chindo; Induru, Spandana; Meyer, Mark S; Khokha, Rama; Barr, Jennifer; Pochampally, Radhika R

2014-08-01

Overall prognosis for osteosarcoma (OS) is poor despite aggressive treatment options. Limited access to primary tumors, technical challenges in processing OS tissues, and the lack of well-characterized primary cell cultures has hindered our ability to fully understand the properties of OS tumor initiation and progression. In this study, we have isolated and characterized cell cultures derived from four central high-grade human OS samples. Furthermore, we used the cell cultures to study the role of CD49f in OS progression. Recent studies have implicated CD49f in stemness and multipotency of both cancer stem cells and mesenchymal stem cells. Therefore, we investigated the role of CD49f in osteosarcomagenesis. First, single cell suspensions of tumor biopsies were subcultured and characterized for cell surface marker expression. Next, we characterized the growth and differentiation properties, sensitivity to chemotherapy drugs, and anchorage-independent growth. Xenograft assays showed that cell populations expressing CD49f(hi) /CD90(lo) cell phenotype produced an aggressive tumor. Multiple lines of evidence demonstrated that inhibiting CD49f decreased the tumor-forming ability. Furthermore, the CD49f(hi) /CD90(lo) cell population is generating more aggressive OS tumor growth and indicating this cell surface marker could be a potential candidate for the isolation of an aggressive cell type in OSs. © 2014 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

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

Synovial fluid progenitors expressing CD90+ from normal but not osteoarthritic joints undergo chondrogenic differentiation without micro-mass culture.

PubMed

Krawetz, Roman J; Wu, Yiru Elizabeth; Martin, Liam; Rattner, Jerome B; Matyas, John R; Hart, David A

2012-01-01

Mesenchymal progenitor cells (MPCs) can differentiate into osteoblasts, adipocytes, and chondrocytes, and are in part responsible for maintaining tissue integrity. Recently, a progenitor cell population has been found within the synovial fluid that shares many similarities with bone marrow MPCs. These synovial fluid MPCs (sfMPCs) share the ability to differentiate into bone and fat, with a bias for cartilage differentiation. In this study, sfMPCs were isolated from human and canine synovial fluid collected from normal individuals and those with osteoarthritis (human: clinician-diagnosed, canine: experimental) to compare the differentiation potential of CD90+ vs. CD90- sfMPCs, and to determine if CD90 (Thy-1) is a predictive marker of synovial fluid progenitors with chondrogenic capacity in vitro. sfMPCs were derived from synovial fluid from normal and OA knee joints. These cells were induced to differentiate into chondrocytes and analyzed using quantitative PCR, immunofluorescence, and electron microscopy. The CD90+ subpopulation of sfMPCs had increased chondrogenic potential compared to the CD90- population. Furthermore, sfMPCs derived from healthy joints did not require a micro-mass step for efficient chondrogenesis. Whereas sfMPCs from OA synovial fluid retain the ability to undergo chondrogenic differentiation, they require micro-mass culture conditions. Overall, this study has demonstrated an increased chondrogenic potential within the CD90+ fraction of human and canine sfMPCs and that this population of cells derived from healthy normal joints do not require a micro-mass step for efficient chondrogenesis, while sfMPCs obtained from OA knee joints do not differentiate efficiently into chondrocytes without the micro-mass procedure. These results reveal a fundamental shift in the chondrogenic ability of cells isolated from arthritic joint fluids, and we speculate that the mechanism behind this change of cell behavior is exposure to the altered milieu of the OA joint fluid, which will be examined in further studies.

CD103+ intraepithelial T cells in high-grade serous ovarian cancer are phenotypically diverse TCRαβ+ CD8αβ+ T cells that can be targeted for cancer immunotherapy

PubMed Central

Workel, Hagma H.; Tijans, Aline M.; Terwindt, Anouk L.J.; Brunekreeft, Kim L.; Plat, Annechien; Klip, Harry G.; Eggink, Florine A.; Leffers, Ninke; Helfrich, Wijnand; Samplonius, Douwe F.; Bremer, Edwin; Wisman, G. Bea A.; Daemen, Toos; Duiker, Evelien W.; Hollema, Harry; Nijman, Hans W.; de Bruyn, Marco

2016-01-01

CD103+ tumor-infiltrating lymphocytes (TIL) have been linked to specific epithelial infiltration and a prolonged survival in high-grade serous epithelial ovarian cancer (HGSC). However, whether these cells are induced as part of an ongoing anti-HGSC immune response or represent non-specifically expanded resident or mucosal lymphocytes remains largely unknown. In this study, we first confirmed that CD103+ TIL from HGSC were predominantly localized in the cancer epithelium and were strongly correlated with an improved prognosis. We further demonstrate that CD103+ TIL were almost exclusively CD3+ TCRαβ+ CD8αβ+ CD4- T cells, but heterogeneously expressed T cell memory and differentiation markers. Activation of peripheral T cells in the presence of HGSC was sufficient to trigger induction of CD103 in over 90% of all CD8+ cells in a T cell receptor (TCR)- and TGFβR1-dependent manner. Finally, CD103+ TIL isolated from primary HGSC showed signs of recent activation and dominantly co-expressed key immunotherapeutic targets PD-1 and CD27. Taken together, our data indicate CD103+ TIL in HGSC are formed as the result of an adaptive anti-tumor immune response that might be reactivated by (dual) checkpoint inhibition. PMID:27650547

Expansion and productive HIV-1 infection of Foxp3 positive CD4 T cells at pleural sites of HIV/TB co-infection

PubMed Central

Hirsch, Christina S; Baseke, Joy; Kafuluma, John Lusiba; Nserko, Mary; Mayanja-Kizza, Harriet; Toossi, Zahra

2016-01-01

Background CD4 T-cells expressing Foxp3 are expanded systemically during active tuberculosis (TB) regardless of HIV-1 co-infection. Foxp3+ CD4 T cells are targets of HIV-1 infection. However, expansion of HIV-1 infected Foxp3+ CD4 T cells at sites of HIV/TB co-infection, and whether they contribute to promotion of HIV-1 viral activity is not known. Methods Pleural fluid mononuclear cells (PFMC) from HIV/TB co-infected patients with pleural TB were characterized by immune-staining and FACS analysis for surface markers CD4, CD127, CCR5, CXCR4, HLA-DR and intracellular expression of Foxp3, HIVp24, IFN-γ and Bcl-2. Whole PFMC and bead separated CD4+CD25+CD127− T cells were assessed for HIV-1 LTR strong stop (SS) DNA by real-time PCR, which represents viral DNA post cell entry and initiation of reverse transcription. Results High numbers of HIV-1 p24 positive Foxp3+ and Foxp3+CD127− CD4 T cells were identified in PFMC from HIV/TB co-infected subjects. CD4+Foxp3+CD127− T cells displayed high expression of the cellular activation marker, HLA-DR. Further, expression of the HIV-1 co-receptors, CCR5 and CXCR4, were higher on CD4+Foxp3+T cells compared to CD4+Foxp3− T cells. Purified CD4+CD25+CD127− T cells isolated from PFMC of HIV/TB co-infected patients, were over 90% CD4+Foxp3+T cells, and exhibited higher HIV-1 SS DNA as compared to whole PFMC, and as compared to CD4+CD25+CD127− T cells from an HIV-infected subject with pleural mesothelioma. HIV-1 p24+ Foxp3+ CD4+T cells from HIV/TB patients higher in Bcl-2 expression as compared to both HIV-1 p24+ Foxp3− CD4 T cells, and Foxp3+ CD4+T cells without HIV-p24 expression. Conclusion Foxp3+ CD4 T cells in PFMC from HIV/TB co-infected subjects are predisposed to productive HIV-1 infection and have survival advantage as compared to Foxp3 negative CD4 T cells. PMID:28124031

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

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.

Genetics in arterial calcification: pieces of a puzzle and cogs in a wheel.

PubMed

Rutsch, Frank; Nitschke, Yvonne; Terkeltaub, Robert

2011-08-19

Artery calcification reflects an admixture of factors such as ectopic osteochondral differentiation with primary host pathological conditions. We review how genetic factors, as identified by human genome-wide association studies, and incomplete correlations with various mouse studies, including knockout and strain analyses, fit into "pieces of the puzzle" in intimal calcification in human atherosclerosis, and artery tunica media calcification in aging, diabetes mellitus, and chronic kidney disease. We also describe in sharp contrast how ENPP1, CD73, and ABCC6 serve as "cogs in a wheel" of arterial calcification. Specifically, each is a minor component in the function of a much larger network of factors that exert balanced effects to promote and suppress arterial calcification. For the network to normally suppress spontaneous arterial calcification, the "cogs" ENPP1, CD73, and ABCC6 must be present and in working order. Monogenic ENPP1, CD73, and ABCC6 deficiencies each drive a molecular pathophysiology of closely related but phenotypically different diseases (generalized arterial calcification of infancy (GACI), pseudoxanthoma elasticum (PXE) and arterial calcification caused by CD73 deficiency (ACDC)), in which premature onset arterial calcification is a prominent but not the sole feature.

Genetics in Arterial Calcification

PubMed Central

Rutsch, Frank; Nitschke, Yvonne; Terkeltaub, Robert

2011-01-01

Artery calcification reflects an admixture of factors such as ectopic osteochondral differentiation with primary host pathological conditions. We review how genetic factors, as identified by human genome-wide association studies, and incomplete correlations with various mouse studies, including knockout and strain analyses, fit into “pieces of the puzzle” in intimal calcification in human atherosclerosis, and artery tunica media calcification in aging, diabetes mellitus, and chronic kidney disease. We also describe in sharp contrast how ENPP1, CD73, and ABCC6 serve as “cogs in a wheel” of arterial calcification. Specifically, each is a minor component in the function of a much larger network of factors that exert balanced effects to promote and suppress arterial calcification. For the network to normally suppress spontaneous arterial calcification, the “cogs” ENPP1, CD73, and ABCC6 must be present and in working order. Monogenic ENPP1, CD73, and ABCC6 deficiencies each drive a molecular pathophysiology of closely related but phenotypically different diseases (generalized arterial calcification of infancy (GACI), pseudoxan-thoma elasticum (PXE) and arterial calcification caused by CD73 deficiency (ACDC)), in which premature onset arterial calcification is a prominent but not the sole feature. PMID:21852556

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.

The role of phytoplankton in the modulation of dissolved and oyster cadmium concentrations in Deep Bay, British Columbia, Canada.

PubMed

Cassis, David; Lekhi, Priyanka; Pearce, Christopher M; Ebell, Nadene; Orians, Kristin; Maldonado, Maria T

2011-09-15

We previously identified dissolved cadmium (Cd(diss)) as the main source of this metal in cultured Pacific oysters, Crassostrea gigas, in Deep Bay, British Columbia, Canada (Lekhi et al., 2008). Total suspended particulate Cd (Cd(part)) was not found to be a significant source of oyster Cd (Cd(oys)), with Cd(part) >20 μm negatively correlated with Cd(oys) concentration. High phytoplankton abundance in spring and summer was hypothesized to reduce Cd(oys) indirectly by drawing down Cd(diss) and increasing oyster growth. In the present study we expanded on these results by examining specifically how the phytoplankton community composition modulates both Cd(diss) and Cd(oys) concentrations in Deep Bay. Based on calculations of nutrients and Cd(diss) drawdown, phytoplankton accounted for approximately 90% of the overall summer reduction in Cd(diss) in the bay. Diatoms were the dominant phytoplankton group, being correlated negatively with Cd(oys) and positively with Cd(part). This suggests that diatom growth mediates the transfer of Cd from the dissolved to the particulate phase, resulting in lower Cd(oys). Spring blooms and sporadic harmful algal blooms may mediate a large flux of Cd(part) to the sediments. Thus, phytoplankton act as a sink, rather than a source, of Cd to oysters in Deep Bay and have a crucial role in the seasonality of Cd(oys) by reducing the concentration of Cd(diss) during the summer. Based on environmental variables, two descriptive models for annual Cd(oys) concentrations were developed using multiple linear regression. The first model (R(2)=0.870) was created to explain the maximum variability in Cd(oys) concentrations throughout the year, while the second (R(2)=0.806) was based on parameters that could be measured easily under farm conditions. Oyster age heavily affected both models, with the first model being secondarily affected by temperature and the second one being more sensitive to changes in salinity. Copyright © 2011 Elsevier B.V. All rights reserved.

CD5-expressing B-cell lymphomas/leukemias: relatively high frequency of CD5+ B-cell lymphomas with an overall poor prognosis in Nagasaki Japan.

PubMed

Kamihira, S; Hirakata, Y; Atogami, S; Sohda, H; Tsuruda, K; Yamada, Y; Tomonaga, M

1996-06-01

To characterize CD5+ B-cell neoplasms in Japan, where chronic lymphocytic leukemia (CLL) is rare and of different subtypes in comparison with Western countries, we collected 58 cases of CD5+ B-cell lymphomas/leukemias and analyzed their clinicopathologic features. According to the French-American-British (FAB) and standard histologic classification, the cases corresponded to small lymphocytic lymphoma (SLL, group I; n = 22, consisting of CLL, n = 10, CLL/PL, n = 3, and CLLmixed, n = 7); intermediate differentiated lymphoma/mantle cell lymphoma (IDL/MCL, group II, n = 18); and others with CD5-positive lymphomas (group III, n = 18). The CD5+ B-cell lymphomas showed morphologic and prognostic variability among the three groups. The clinical and immunophenotypic features were remarkably consistent in leukemic disease being seen in 73% of all cases, splenomegaly in 63%, and intense CD19, CD20, surface membrane immunogobulin M (SmIgM) or SmIgM and SmIgD, light-chain expression, and no CD10 expression. The median survival time of groups I, II, and III was 7.8, 3.3, and 0.8 years, respectively. These findings suggest that CD5 antigens may serve as valid markers for the prognosis and clinical features of B-cell lymphomas and that CD5+ B-cell lymphomas with an overall poor prognosis occurs at a relatively high frequency in Japan. This also suggests that a combination of immunophenotypic and morphologic features is of value for characterizing CD5+ B-cell neoplasms.

CD34 Antigen and the MPL Receptor Expression Defines a Novel Class of Human Cord Blood-Derived Primitive Hematopoietic Stem Cells

PubMed Central

Matsuoka, Yoshikazu; Takahashi, Masaya; Sumide, Keisuke; Kawamura, Hiroshi; Nakatsuka, Ryusuke; Fujioka, Tatsuya; Sonoda, Yoshiaki

2017-01-01

In the murine hematopoietic stem cell (HSC) compartment, thrombopoietin (THPO)/MPL (THPO receptor) signaling plays an important role in the maintenance of adult quiescent HSCs. However, the role of THPO/MPL signaling in the human primitive HSC compartment has not yet been elucidated. We have identified very primitive human cord blood (CB)-derived CD34– severe combined immunodeficiency (SCID)-repopulating cells (SRCs) using the intra-bone marrow injection method. In this study, we investigated the roles of the MPL expression in the human primitive HSC compartment. The SRC activities of the highly purified CB-derived 18Lin–CD34+/–MPL+/– cells were analyzed using NOG mice. In the primary recipient mice, nearly all mice that received CD34+/–MPL+/– cells were repopulated with human CD45+ cells. Nearly all of these mice that received CD34+MPL+/– and CD34–MPL– cells showed a secondary repopulation. Interestingly, the secondary recipient mice that received CD34+/–MPL– cells showed a distinct tertiary repopulation. These results clearly indicate that the CD34+/– SRCs not expressing MPL sustain a long-term (LT) (>1 year) human cell repopulation in NOG mice. Moreover, CD34– SRCs generate CD34+CD38–CD90+ SRCs in vitro and in vivo. These findings provide a new concept that CD34–MPL– SRCs reside at the apex of the human HSC hierarchy. PMID:27938494

Low expression of CD39 on regulatory T cells as a biomarker for resistance to methotrexate therapy in rheumatoid arthritis

PubMed Central

Peres, Raphael Sanches; Liew, Foo Y.; Talbot, Jhimmy; Carregaro, Vanessa; Oliveira, Rene D.; Almeida, Sergio L.; França, Rafael F. O.; Donate, Paula B.; Pinto, Larissa G.; Ferreira, Flavia I. S.; Costa, Diego L.; Demarque, Daniel P.; Gouvea, Dayana Rubio; Lopes, Norberto P.; Queiroz, Regina Helena C.; Silva, Joao Santana; Figueiredo, Florencio; Alves-Filho, Jose Carlos; Cunha, Thiago M.; Ferreira, Sérgio H.; Louzada-Junior, Paulo; Cunha, Fernando Q.

2015-01-01

Rheumatoid arthritis (RA) is an inflammatory autoimmune disease characterized by joint destruction and severe morbidity. Methotrexate (MTX) is the standard first-line therapy of RA. However, about 40% of RA patients are unresponsive to MTX treatment. Regulatory T cells (Tregs, CD4+CD25+FoxP3+) are thought to play an important role in attenuating RA. To investigate the role of Tregs in MTX resistance, we recruited 122 RA patients (53 responsive, R-MTX; 69 unresponsive, UR-MTX) and 33 healthy controls. Three months after MTX treatment, R-MTX but not UR-MTX showed higher frequency of peripheral blood CD39+CD4+CD25+FoxP3+ Tregs than the healthy controls. Tregs produce adenosine (ADO) through ATP degradation by sequential actions of two cell surface ectonucleotidases: CD39 and CD73. Tregs from UR-MTX expressed a lower density of CD39, produced less ADO, and had reduced suppressive activity than Tregs from R-MTX. In a prospective study, before MTX treatment, UR-MTX expressed a lower density of CD39 on Tregs than those of R-MTX or control (P < 0.01). In a murine model of arthritis, CD39 blockade reversed the antiarthritic effects of MTX treatment. Our results demonstrate that MTX unresponsiveness in RA is associated with low expression of CD39 on Tregs and the decreased suppressive activity of these cells through reduced ADO production. Our findings thus provide hitherto unrecognized mechanism of immune regulation in RA and on mode of action of MTX. Furthermore, our data suggest that low expression of CD39 on Tregs could be a noninvasive biomarker for identifying MTX-resistant RA patients. PMID:25675517

Low expression of CD39 on regulatory T cells as a biomarker for resistance to methotrexate therapy in rheumatoid arthritis.

PubMed

Peres, Raphael Sanches; Liew, Foo Y; Talbot, Jhimmy; Carregaro, Vanessa; Oliveira, Rene D; Almeida, Sergio L; França, Rafael F O; Donate, Paula B; Pinto, Larissa G; Ferreira, Flavia I S; Costa, Diego L; Demarque, Daniel P; Gouvea, Dayana Rubio; Lopes, Norberto P; Queiroz, Regina Helena C; Silva, Joao Santana; Figueiredo, Florencio; Alves-Filho, Jose Carlos; Cunha, Thiago M; Ferreira, Sérgio H; Louzada-Junior, Paulo; Cunha, Fernando Q

2015-02-24

Rheumatoid arthritis (RA) is an inflammatory autoimmune disease characterized by joint destruction and severe morbidity. Methotrexate (MTX) is the standard first-line therapy of RA. However, about 40% of RA patients are unresponsive to MTX treatment. Regulatory T cells (Tregs, CD4(+)CD25(+)FoxP3(+)) are thought to play an important role in attenuating RA. To investigate the role of Tregs in MTX resistance, we recruited 122 RA patients (53 responsive, R-MTX; 69 unresponsive, UR-MTX) and 33 healthy controls. Three months after MTX treatment, R-MTX but not UR-MTX showed higher frequency of peripheral blood CD39(+)CD4(+)CD25(+)FoxP3(+) Tregs than the healthy controls. Tregs produce adenosine (ADO) through ATP degradation by sequential actions of two cell surface ectonucleotidases: CD39 and CD73. Tregs from UR-MTX expressed a lower density of CD39, produced less ADO, and had reduced suppressive activity than Tregs from R-MTX. In a prospective study, before MTX treatment, UR-MTX expressed a lower density of CD39 on Tregs than those of R-MTX or control (P < 0.01). In a murine model of arthritis, CD39 blockade reversed the antiarthritic effects of MTX treatment. Our results demonstrate that MTX unresponsiveness in RA is associated with low expression of CD39 on Tregs and the decreased suppressive activity of these cells through reduced ADO production. Our findings thus provide hitherto unrecognized mechanism of immune regulation in RA and on mode of action of MTX. Furthermore, our data suggest that low expression of CD39 on Tregs could be a noninvasive biomarker for identifying MTX-resistant RA patients.

Polymorphisms in CD14 Gene May Modify Soluble CD14 Levels and Represent Risk Factors for Multiple Sclerosis.

PubMed

Farrokhi, Mehrdad; Moeini, Pedram; Fazilati, Mohammad; Nazem, Habibollah; Faraji, Shahla; Saadatpour, Zahra; Fadaei, Elyas; Saadatpour, Leila; Rezaei, Ali; Ansaripour, Sadra; Amani-Beni, Ali

2016-11-07

Besides the central role of the adaptive immune system, a disturbance of innate immune system is also suggested to be involved in the pathogenesis of multiple sclerosis (MS). CD14, a receptor upregulated in activated microglia, is known to be an essential mediator of inflammation in innate immune responses. Therefore, in this study we aimed to assess possible roles of CD14-159 and -260 gene polymorphisms in MS susceptibility and the effects of those polymorphisms to its protein producing capacity in Iranian population. In this case control study, CD14-159 and -260 polymorphisms were genotyped using polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) in 200 MS patients and 200 healthy controls matched in age and gender. Serum levels of soluble CD14 (sCD14) was determined by enzyme-linked immunosorbent assay (ELISA). There were significant differences in genotype distribution of CD14-159 and -260 polymorphisms between patients and controls (P = 0.01, for-both). Mean serum level of sCD14 was significantly higher in MS patients than in control subjects (3340.30 ± 612.50 ng/ml vs 2353.73 ± 539.07 ng/ml; P < 0.01). In summary, we conclude that CD14-159 and -260 polymorphisms are associated with the risk of MS in Iranian population and affects CD14 promoter activity, thereby regulating CD14 expression. Furthermore, our study provides preliminary evidence for the activation of innate immunity in the pathogenesis of MS. In addition, the findings of the present study suggest serum level of sCD14 as candidate biomarker of MS severity.

Identification of a unique hepatocellular carcinoma line, Li-7, with CD13(+) cancer stem cells hierarchy and population change upon its differentiation during culture and effects of sorafenib.

PubMed

Yamada, Takeshi; Abei, Masato; Danjoh, Inaho; Shirota, Ryoko; Yamashita, Taro; Hyodo, Ichinosuke; Nakamura, Yukio

2015-04-11

Cancer stem cell (CSC) research has highlighted the necessity of developing drugs targeting CSCs. We investigated a hepatocellular carcinoma (HCC) cell line that not only has CSC hierarchy but also shows phenotypic changes (population changes) upon differentiation of CSC during culture and can be used for screening drugs targeting CSC. Based on a hypothesis that the CSC proportion should decrease upon its differentiation into progenitors (population change), we tested HCC cell lines (HuH-7, Li-7, PLC/PRF/5, HLF, HLE) before and after 2 months culture for several markers (CD13, EpCAM, CD133, CD44, CD90, CD24, CD166). Tumorigenicity was tested using nude mice. To evaluate the CSC hierarchy, we investigated reconstructivity, proliferation, ALDH activity, spheroid formation, chemosensitivity and microarray analysis of the cell populations sorted by FACS. Only Li-7 cells showed a population change during culture: the proportion of CD13 positive cells decreased, while that of CD166 positive cells increased. The high tumorigenicity of the Li-7 was lost after the population change. CD13(+)/CD166(-) cells showed slow growth and reconstructed the bulk Li-7 populations composed of CD13(+)/CD166(-), CD13(-)/CD166(-) and CD13(-)/CD166(+) fractions, whereas CD13(-)/CD166(+) cells showed rapid growth but could not reproduce any other population. CD13(+)/CD166(-) cells showed high ALDH activity, spheroid forming ability and resistance to 5-fluorouracil. Microarray analysis demonstrated higher expression of stemness-related genes in CD166(-) than CD166(+) fraction. These results indicated a hierarchy in Li-7 cells, in which CD13(+)/CD166(-) and CD13(-)/CD166(+) cells serve as slow growing CSCs and rapid growing progenitors, respectively. Sorafenib selectively targeted the CD166(-) fraction, including CD13(+) CSCs, which exhibited higher mRNA expression for FGF3 and FGF4, candidate biomarkers for sorafenib. 5-fluorouracil followed by sorafenib inhibited the growth of bulk Li-7 cells more effectively than the reverse sequence or either alone. We identified a unique HCC line, Li-7, which not only shows heterogeneity for a CD13(+) CSC hierarchy, but also undergoes a "population change" upon CSC differentiation. Sorafenib targeted the CSC in vitro, supporting the use of this model for screening drugs targeting the CSC. This type of "heterogeneous, unstable" cell line may prove more useful in the CSC era than conventional "homogeneous, stable" cell lines.

Prevalence of Inflammatory Bowel Disease in the Canton of Vaud (Switzerland): A population-based cohort study.

PubMed

Juillerat, Pascal; Pittet, Valérie; Bulliard, Jean-Luc; Guessous, Idris; Antonino, Anca Teodora; Mottet, Christian; Felley, Christian; Vader, John-Paul; Michetti, Pierre

2008-06-01

Because of the changing epidemiology of Inflammatory Bowel Diseases (IBD), we set out to characterize the population-based prevalence of Crohn's Disease (CD) and Ulcerative Colitis (UC) in a defined population of Switzerland. Adult IBD patients were identified by a cross-matched review of histological, hospital and gastroenterologist files throughout a geographical defined population (Canton of Vaud). Demographic factors statistically significantly associated with prevalence were evaluated using a stepwise Poisson regression analysis. Results were compared to IBD prevalence rates in other population-based studies and time trends were performed, based on a systematic literature review. Age and sex-adjusted prevalence rates were 205.7 IBD (100.7 CD and 105.0 UC) cases per 10(5) inhabitants. Among 1016 IBD patients (519 CD and 497 UC), females outnumbered males in CD (p<0.001), but males were more represented in elderly UC patients (p=0.008). Thus, being a male was statistically associated with UC (Relative Risk (RR) 1.25; p=0.013), whereas being a female was associated with CD (RR 1.27; p=0.007). Living in an urban zone was associated with both CD and UC (RR 1.49; p<0.001, 1.63; p<0.001, respectively). From 1960 to 2005, increases in UC and CD prevalences of 2.4% (95%CI, 2.1%-2.8%; p<0.001) and 3.6% (95%CI, 3.1%-4.1%; p<0.001) per annum were found in industrialised countries. Extrapolating our data to all of Switzerland yields an estimate of 12,000 IBD cases for the country, or 1 in 500 inhabitants. Our study gives support to an increase in IBD prevalence in Europe.

Slow desensitization of imatinib-induced nonimmediate reactions and dynamic changes of drug-specific CD4+CD25+CD134+ lymphocytes.

PubMed

Klaewsongkram, Jettanong; Thantiworasit, Pattarawat; Sodsai, Pimpayao; Buranapraditkun, Supranee; Mongkolpathumrat, Pungjai

2016-11-01

Imatinib is a tyrosine kinase inhibitor indicated for the treatment of gastrointestinal stromal tumors (GISTs) and certain neoplastic diseases; however, nonimmediate adverse reactions are common. To describe the process of imatinib slow desensitization in patients who experienced nonimmediate reactions to imatinib and the dynamic change in drug-specific CD4 + CD25 + CD134 + T-lymphocyte percentages. Five patients diagnosed as having GISTs and with a recent history of imatinib-induced nonimmediate reactions (maculopapular exanthema with eosinophilia, exfoliative dermatitis, palmar-plantar erythrodysesthesia, and drug rash with eosinophilia and systemic symptoms) were desensitized using a slow desensitization protocol. The reintroduced imatinib dosage was stepped up every week starting from 10 mg/d and increasing to 25, 50, 75, 100, 150, 200, and 300 mg/d until the target dose of 400 mg/d was achieved. Prednisolone of up to 30 mg/d was allowed if allergic reactions recurred. The percentages of CD4 + CD25 + CD134 + T cells present after incubating peripheral blood mononuclear cells with imatinib, at baseline and after successful desensitization, were analyzed using flow cytometric analysis. By using a slow desensitization technique, all patients were able to receive 400 mg/d of imatinib, and prednisolone was gradually tapered off. The percentages of imatinib-induced CD4 + CD25 + CD134 + T cells decreased from a mean (SD) of 11.3% (6.5%) and 13.4% (7.3%) at baseline to 3.2% (0.7%) and 3.0% (1.1%) after successful desensitization, when stimulating peripheral blood mononuclear cells with 1 and 2 μM of imatinib, respectively. Slow desensitization is a helpful procedure in treating patients with imatinib-induced nonimmediate reactions other than simple maculopapular exanthema. The reduced percentages of imatinib-induced CD4 + CD25 + CD134 + T cells in these patients may be associated with immune tolerance. Copyright © 2016 American College of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

Subcellular compartmentalization of Cd and Zn in two bivalves. II. Significance of trophically available metal (TAM)

USGS Publications Warehouse

Wallace, W.G.; Luoma, S.N.

2003-01-01

This paper examines how the subcellular partitioning of Cd and Zn in the bivalves Macoma balthica and Potamocorbula amurensis may affect the trophic transfer of metal to predators. Results show that the partitioning of metals to organelles, 'enzymes' and metallothioneins (MT) comprise a subcellular compartment containing trophically available metal (TAM; i.e. metal trophically available to predators), and that because this partitioning varies with species, animal size and metal, TAM is similarly influenced. Clams from San Francisco Bay, California, were exposed for 14 d to 3.5 ??g 1-1 Cd and 20.5 ??g 1-1 Zn, including 109Cd and 65Zn as radiotracers, and were used in feeding experiments with grass shrimp Palaemon macrodatylus, or used to investigate the subcellular partitioning of metal. Grass shrimp fed Cd-contaminated P. amurensis absorbed ???60% of ingested Cd, which was in accordance with the partitioning of Cd to the bivalve's TAM compartment (i.e. Cd associated with organelles, 'enzymes' and MT); a similar relationship was found in previous studies with grass shrimp fed Cd-contaminated oligochaetes. Thus, TAM may be used as a tool to predict the trophic transfer of at least Cd. Subcellular fractionation revealed that ???34% of both the Cd and Zn accumulated by M. balthica was associated with TAM, while partitioning to TAM in P. amurensis was metal-dependent (???60% for TAM-Cd%, ???73% for TAM-Zn%). The greater TAM-Cd% of P. amurensis than M. balthica is due to preferential binding of Cd to MT and 'enzymes', while enhanced TAM-Zn% of P. amurensis results from a greater binding of Zn to organelles. TAM for most species-metal combinations was size-dependent, decreasing with increased clam size. Based on field data, it is estimated that of the 2 bivalves, P. amurensis poses the greater threat of Cd exposure to predators because of higher tissue concentrations and greater partitioning as TAM; exposure of Zn to predators would be similar between these species.

Transplanted Peripheral Blood Stem Cells Mobilized by Granulocyte Colony-Stimulating Factor Promoted Hindlimb Functional Recovery After Spinal Cord Injury in Mice.

PubMed

Takahashi, Hiroshi; Koda, Masao; Hashimoto, Masayuki; Furuya, Takeo; Sakuma, Tsuyoshi; Kato, Kei; Okawa, Akihiko; Inada, Taigo; Kamiya, Koshiro; Ota, Mitsutoshi; Maki, Satoshi; Takahashi, Kazuhisa; Yamazaki, Masashi; Mannoji, Chikato

2016-01-01

Granulocyte colony-stimulating factor (G-CSF) mobilizes peripheral blood stem cells (PBSCs) derived from bone marrow. We hypothesized that intraspinal transplantation of PBSCs mobilized by G-CSF could promote functional recovery after spinal cord injury. Spinal cords of adult nonobese diabetes/severe immunodeficiency mice were injured using an Infinite Horizon impactor (60 kdyn). One week after the injury, 3.0 µl of G-CSF-mobilized human mononuclear cells (MNCs; 0.5 × 10(5)/µl), G-CSF-mobilized human CD34-positive PBSCs (CD34; 0.5 × 10(5)/µl), or normal saline was injected to the lesion epicenter. We performed immunohistochemistry. Locomotor recovery was assessed by Basso Mouse Scale. The number of transplanted human cells decreased according to the time course. The CD31-positive area was significantly larger in the MNC and CD34 groups compared with the vehicle group. The number of serotonin-positive fibers was significantly larger in the MNC and CD34 groups than in the vehicle group. Immunohistochemistry revealed that the number of apoptotic oligodendrocytes was significantly smaller in cell-transplanted groups, and the areas of demyelination in the MNC- and CD34-transplanted mice were smaller than that in the vehicle group, indicating that cell transplantation suppressed oligodendrocyte apoptosis and demyelination. Both the MNC and CD34 groups showed significantly better hindlimb functional recovery compared with the vehicle group. There was no significant difference between the two types of transplanted cells. Intraspinal transplantation of G-CSF-mobilized MNCs or CD34-positive cells promoted angiogenesis, serotonergic fiber regeneration/sparing, and preservation of myelin, resulting in improved hindlimb function after spinal cord injury in comparison with vehicle-treated control mice. Transplantation of G-CSF-mobilized PBSCs has advantages for treatment of spinal cord injury in the ethical and immunological viewpoints, although further exploration is needed to move forward to clinical application.

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.

[Mechanism Study of the Smectite-OR-SH Compound for Reducing Cadmium Uptake by Plants in Contaminated Soils].

PubMed

Zeng, Yan-jun; Zhou, Zhi-jun; Zhao, Qiu-xiang

2015-06-01

Adsorption and desorption experiments, pot experiments and characterization test were performed to investigate the immobilization effect and mechanism of the smectite-OR-SH compound for reducing cadmium uptake by plants in contaminated soils. The results showed that the saturated adsorption capacity for the adsorption of Cd2+ on smectite raised distinctly after functionalized. The adsorption of Cd2+ on smectite-OR-SH compound was very stable and it was difficult for Cd2+ to be desorbed from it. Crop yields promoted differently in original soil, Cd 3 mg x kg(-1) soil and Cd 10 mg x kg(-1) soil after adding the smectite-OR-SH compound. And the cadmium content of the cabbage reduced 61.00%, 62.10% and 83.73% respectively compare with the control. Characterization test analysis showed that Cd was adsorbed by the compound successfully and ligand interaction occurred between Cd and the thiol group. Floc amount on the compound surface increased correspondingly. In addition to electrostatic adsorption, ion exchange and hydroxyl ligand adsorption, the reaction mechanism of smectite-OR-SH compound with Cd was mainly sulfhydryl ligand adsorption.

Disrupted Topological Patterns of Large-Scale Network in Conduct Disorder

PubMed Central

Jiang, Yali; Liu, Weixiang; Ming, Qingsen; Gao, Yidian; Ma, Ren; Zhang, Xiaocui; Situ, Weijun; Wang, Xiang; Yao, Shuqiao; Huang, Bingsheng

2016-01-01

Regional abnormalities in brain structure and function, as well as disrupted connectivity, have been found repeatedly in adolescents with conduct disorder (CD). Yet, the large-scale brain topology associated with CD is not well characterized, and little is known about the systematic neural mechanisms of CD. We employed graphic theory to investigate systematically the structural connectivity derived from cortical thickness correlation in a group of patients with CD (N = 43) and healthy controls (HCs, N = 73). Nonparametric permutation tests were applied for between-group comparisons of graphical metrics. Compared with HCs, network measures including global/local efficiency and modularity all pointed to hypo-functioning in CD, despite of preserved small-world organization in both groups. The hubs distribution is only partially overlapped with each other. These results indicate that CD is accompanied by both impaired integration and segregation patterns of brain networks, and the distribution of highly connected neural network ‘hubs’ is also distinct between groups. Such misconfiguration extends our understanding regarding how structural neural network disruptions may underlie behavioral disturbances in adolescents with CD, and potentially, implicates an aberrant cytoarchitectonic profiles in the brain of CD patients. PMID:27841320

Histological investigations on thymus of male rats prenatally exposed to bisphenol A.

PubMed

Aydemir, Işıl; Kum, Şadiye; Tuğlu, Mehmet İbrahim

2018-09-01

Bisphenol A is called as a endocrine-distrupting chemical because of the its steroid-like activity and it used in the construction of plastic containing materials. It is indicated that bisphenol A can pass the human serum, urine, follicular fluid, placenta and umblical cord as a result of the use of substances containing this agent. In this study, we aimed to investigate the effects of bisphenol A on the development of the thymus, a primary lymphoid organ which plays an important role in the specific immunity. The adult pregnant female rats were administered orally with bisphenol A (for 21 days) and postnatal thymus samples were obtained on day 21, 45 and 90 and were performed for histochemical and immunohistochemical staining for CD3, CD4, CD8 and CD79a and TUNEL assay for the apoptotic cells. Evaluation of all groups, CD3, CD4, CD8 and CD79a stainings were decreased in the experimental groups compared with control group. The apoptotic cells were determined in the all groups on day 90 as a result of the thymus involution. It is noted that there was not any histological and morphological damages in the rats prenatally exposed the bisphenol A. The effect of the bisphenol A is unknown in the future, but there is no problem in the adult rats. Copyright © 2018 Elsevier Ltd. All rights reserved.

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.

Extended flow cytometry characterization of normal bone marrow progenitor cells by simultaneous detection of aldehyde dehydrogenase and early hematopoietic antigens: implication for erythroid differentiation studies

PubMed Central

Mirabelli, Peppino; Di Noto, Rosa; Lo Pardo, Catia; Morabito, Paolo; Abate, Giovanna; Gorrese, Marisa; Raia, Maddalena; Pascariello, Caterina; Scalia, Giulia; Gemei, Marica; Mariotti, Elisabetta; Del Vecchio, Luigi

2008-01-01

Background Aldehyde dehydrogenase (ALDH) is a cytosolic enzyme highly expressed in hematopoietic precursors from cord blood and granulocyte-colony stimulating factor mobilized peripheral blood, as well as in bone marrow from patients with acute myeloblastic leukemia. As regards human normal bone marrow, detailed characterization of ALDH+ cells has been addressed by one single study (Gentry et al, 2007). The goal of our work was to provide new information about the dissection of normal bone marrow progenitor cells based upon the simultaneous detection by flow cytometry of ALDH and early hematopoietic antigens, with particular attention to the expression of ALDH on erythroid precursors. To this aim, we used three kinds of approach: i) multidimensional analytical flow cytometry, detecting ALDH and early hematopoietic antigens in normal bone marrow; ii) fluorescence activated cell sorting of distinct subpopulations of progenitor cells, followed by in vitro induction of erythroid differentiation; iii) detection of ALDH+ cellular subsets in bone marrow from pure red cell aplasia patients. Results In normal bone marrow, we identified three populations of cells, namely ALDH+CD34+, ALDH-CD34+ and ALDH+CD34- (median percentages were 0.52, 0.53 and 0.57, respectively). As compared to ALDH-CD34+ cells, ALDH+CD34+ cells expressed the phenotypic profile of primitive hematopoietic progenitor cells, with brighter expression of CD117 and CD133, accompanied by lower display of CD38 and CD45RA. Of interest, ALDH+CD34- population disclosed a straightforward erythroid commitment, on the basis of three orders of evidences. First of all, ALDH+CD34- cells showed a CD71bright, CD105+, CD45- phenotype. Secondly, induction of differentiation experiments evidenced a clear-cut expression of glycophorin A (CD235a). Finally, ALDH+CD34- precursors were not detectable in patients with pure red cell aplasia (PRCA). Conclusion Our study, comparing surface antigen expression of ALDH+/CD34+, ALDH-/CD34+ and ALDH+/CD34- progenitor cell subsets in human bone marrow, clearly indicated that ALDH+CD34- cells are mainly committed towards erythropoiesis. To the best of our knowledge this finding is new and could be useful for basic studies about normal erythropoietic differentiation as well as for enabling the employment of ALDH as a red cell marker in polychromatic flow cytometry characterization of bone marrow from patients with aplastic anemia and myelodysplasia. PMID:18510759

CD30 Expression by B and T Cells: A Frequent Finding in Angioimmunoblastic T-Cell Lymphoma and Peripheral T-Cell Lymphoma-Not Otherwise Specified.

PubMed

Onaindia, Arantza; Martínez, Nerea; Montes-Moreno, Santiago; Almaraz, Carmen; Rodríguez-Pinilla, Socorro M; Cereceda, Laura; Revert, Jose B; Ortega, César; Tardio, Antoni; González, Lucía; García, Sonia; Camacho, Francisca I; González-Vela, Carmen; Piris, Miguel A

2016-03-01

CD30 expression in peripheral T-cell lymphoma (PTCL) and angioimmunoblastic T-cell lymphoma (AITL) is currently of great interest because therapy targeting CD30 is of clinical benefit, but the clinical and therapeutic relevance of CD30 expression in these neoplasms still remains uncertain. The aim of this study was to better quantify CD30 expression in AITL and PTCL-not otherwise specified (NOS). The secondary objective was to determine whether CD30 cells exhibit a B-cell or a T-cell phenotype. Gene expression profiling was studied in a series of 37 PTCL cases demonstrating a continuous spectrum of TNFRSF8 expression. This prompted us to study CD30 immunohistochemical (IHC) expression and mRNA levels by reverse transcription polymerase chain reaction (RT-PCR) in a different series of 51 cases (43 AITLs and 8 PTCL-NOSs) in routine samples. Double stainings with PAX5/CD30, CD3/CD30, and LEF1/CD30 were performed to study the phenotype of CD30 cells. Most (90%) of the cases showed some level of CD30 expression by IHC (1% to 95%); these levels were high (>50% of tumoral cells) in 14% of cases. CD30 expression was not detected in 10% of the cases. Quantitative RT-PCR results largely confirmed these findings, demonstrating a moderately strong correlation between global CD30 IHC and mRNA levels (r=0.65, P=1.75e-7). Forty-four of the positive cases (98%) contained CD30-positive B cells (PAX5), whereas atypical CD30-positive T cells were detected in 42 cases (93%). In conclusion, our data show that most AITL and PTCL-NOS cases express CD30, exhibiting very variable levels of CD30 expression that may be measured by IHC or RT-PCR techniques.

Reference values of lymphocyte sub-populations in healthy human immunodeficiency virus-negative Iranian adults.

PubMed

Kamallou, Atefeh; Haji Abdolbaghi, Mahbobeh; Mohraz, Minoo; Rasolinejad, Mernaz; Karbasi, Ehsan; Ansaripour, Bita; Soltani, Samaneh; Rezaei, Arezou; Khalili, Neda; Amirzargar, Aliakbar

2014-12-01

Lymphocyte subsets enumeration is considered prominent in the management of primary and acquired immunodeficiency disorders. Because of local variations due to race, age, gender, and environmental conditions on lymphocyte subsets, and to improve the accuracy of interpretation of laboratory findings, reference intervals must be determined in every population. To establish a normal reference range for CD3+, CD4+, CD8+, CD19+ and CD56+ lymphocytes in a healthy Iranian adult population using flowcytometry. Blood samples were collected from 221 HIV seronegative individuals, including 112 females and 109 males, with ages ranging from 20 to 40 years old. The percentage of lymphocytes expressing either of CD3, CD4, CD8, CD19 and CD56 surface markers were determined by flowcytometry assay. Total mean percentage and absolute count of lymphocyte subsets were as follows: CD3+: 70.90 ± 7.54%, 1800.87 ± 471.09 cells/µl; CD4+: 41.04 ± 7.86%, 1039.99 ± 338.02 cells/µl; CD8+: 31.11 ± 6.60%, 783.95 ± 234.87 cells/µl; CD19+: 12.77 ± 4.56%, 328.37 ± 153.17 cells/µl; CD56+: 15.53 ± 6.34%, 388.62 ± 176.17 cells/µl, respectively. The ratio of CD4+/CD8+ lymphocytes for the studied population was 1.39 ± 0.48. Significant differences were observed between male and female subjects indicating that the average percentage of CD3+ cells (p=0.017) and CD4+ T cells (p=0.003) were higher in the female population, whereas the average percentage of CD19+ cells (p=0.02) tended to be higher among males. However, investigations on the CD56+ NK cell and CD8+ T cell sub-populations did not show any statistical differences between the two genders. In comparison with reports of other populations, we were confronted with different results. Establishing reference values of lymphocyte subsets for each population is helpful in achieving standard criteria for the prognosis of HIV infection. Therefore, normal ranges established by this survey can be used as a reference for decisions made in clinical practice.

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.

Flow cytometric detection of endothelial microparticles (EMP): effects of centrifugation and storage alter with the phenotype studied.

PubMed

van Ierssel, Sabrina H; Van Craenenbroeck, Emeline M; Conraads, Viviane M; Van Tendeloo, Viggo F; Vrints, Christiaan J; Jorens, Philippe G; Hoymans, Vicky Y

2010-04-01

Endothelial microparticles (EMP) are released into the circulation in case of endothelial disturbance, and are therefore increasingly investigated as a biomarker reflecting disease activity. Numerous pre-analytic methods have been proposed for their flow cytometric enumeration, but standardization is still lacking. In this study we evaluated the influence of centrifugation and storage conditions on EMP quantification. Platelet-poor plasma (PPP) from 10 healthy volunteers was prepared by centrifugation at 1,550 g for 20 minutes twice. A first aliquot of PPP was analyzed immediately, a second after storage at 4 degrees C for 7 hours. A third and fourth aliquot were snap-frozen and stored at -80 degrees C for 7 and 28 days. A final aliquot was further centrifuged at 10,000g for 10 minutes and analyzed immediately. EMP were defined as CD31+CD42b-, CD62E+, CD144+ or CD144+CD105+ particles, smaller than 1.0 microm. High speed centrifugation led to a significant loss of CD31+CD42b- EMP (p=0.004). A good correlation between PPP and high speed centrifuged PPP was only found for CD144+ EMP (Kendall tau b=0.611, p=0.025). Storage at 4 degrees C did not affect EMP quantification. However, freezing at -80 degrees C increased CD31+CD42b- and CD62E+ EMP counts, and lowered CD144+ EMP (p<0.05). Nevertheless, the agreement among the different storage conditions was relatively good (Kendall coefficient of concordance >0.487; p<0.05). The flow cytometric detection of EMP varies with the centrifugation protocol and the storage method used, and these changes also depend on the phenotype studied. The results of this study caution against comparing study results gathered with different EMP laboratory protocols. (c) 2010 Elsevier Ltd. All rights reserved.

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.

Trans-differentiation of the adipose tissue-derived stem cells into neuron-like cells expressing neurotrophins by selegiline.

PubMed

Abdanipour, Alireza; Tiraihi, Taki; Delshad, Alireza

2011-01-01

Adult stem cells (ASC) are undifferentiated cells found throughout the body. These cells are promising tools for cell replacement therapy in neurodegenerative disease. Adipose tissue is the most abundant and accessible source of ASC. This study was conducted to evaluate effect of selegiline on differentiation of adipose-derived stem cells (ADSC) into functional neuron-like cells (NLC), and also level of the neurotrophin expression in differentiated cells. ADSC were transdifferentiated into NLC using selegiline where CD90, CD49d, CD31, CD106 and CD45 were used as markers for ADSC identification. Lipogenic and osteogenic differentiation of ADSC were used to characterize the ADSC. ADSC were treated with selegiline at different concentrations (from 10(-6) to 10(-11) mM) and time points (3, 6, 12, 24 and 48 h). Percentage of viable cells, nestin and neurofilament 68 (NF-68) immunoreactive cells were used as markers for differentiation. The optimal dose for neurotrophin expressions in differentiating cells was evaluated using reverse transcriptase-PCR. NLC function was evaluated by loading and unloading with FM1-43 dye. ADSC were immunoreactive to CD90 (95.67 ± 2.26), CD49d (71.52 ± 6.64) and CD31 (0.6 ± 0.86), but no immunoreactivity was detected for CD106 and CD45. The results of neural differentiation showed the highest percentage of nestin and NF-68 positive cells at 10(-9) mM concentration of selegiline (exposed for 24 h). The differentiated cells expressed synapsin and neurotrophin genes except brain-derived neurotrophic factor. ADSC can be an alternative source in cell-based therapy for neurodegenerative diseases using selegiline to induce ADSC differentiation to neuronal lineage.

Cadmium Accumulation and Pathological Alterations in the Midgut Gland of Terrestrial Snail Helix pomatia L. from a Zinc Smelter Area: Role of Soil pH.

PubMed

Włostowski, Tadeusz; Kozłowski, Paweł; Łaszkiewicz-Tiszczenko, Barbara; Oleńska, Ewa

2016-04-01

The purpose of this study was to determine whether cadmium (Cd) accumulation and toxicity in the midgut gland of Helix pomatia snails living in a Cd-contaminated area were related to soil pH. Toxic responses in the midgut gland (i.e., increased vacuolization and lipid peroxidation) occurred in H. pomatia snails exhibiting the highest Cd levels in the gland (265-274 µg/g dry wt) and living on acidic soil (pH 5.3-5.5), while no toxicity was observed in snails accumulating less Cd (90 µg/g) and ranging on neutral soil (pH 7.0), despite the fact that total soil Cd was similar in the two cases. The accumulation of Cd in the gland was directly related to the water extractable Cd in soil, which in turn correlated inversely with soil pH, indicating that this factor had a significant effect on tissue Cd. It appeared further that the occurrence of Cd toxicity was associated with low levels of metallothionein in the gland of snails ranging on acidic soil.

A new technique for promoting cyclic utilization of cyclodextrins in biotransformation.

PubMed

Shen, Yanbing; Yu, Ziqi; Yang, Xu; Wang, Fang; Luo, Jianmei; Wang, Min

2017-01-01

Cyclodextrins (CDs) can improve the productivity of steroid biotransformation by enhancing substrate solubility. CDs can be recycled by grafting them with appropriate carriers. Loofah fiber is an excellent grafting material for CDs, and can be applied to the biotransformation and recycling of β-cyclodextrin (β-CD). In this work, a technique for recycling β-CD in cortisone acetate (CA) biotransformation by Arthrobacter simplex CPCC 140451 was studied. Loofah fiber-grafted β-CD (LF-β-CD) was prepared using epichlorohydrin, which is a cross-linking agent. The grafting yield of β-CD was 74.8 mg g -1 dried fibers. LF-β-CD could increase the solubility of CA and enhance biotransformation. The initial conversion rate of CA was 1.5-fold higher than that of the blank group. LF-β-CD was also used in biocatalytic reactions for eight cycles, and it maintained the conversion ratio of CA at approximately 90%. Given the above positive results, LF-β-CD can be utilized in biotechnological recycling applications. This method can also be applied to CD derivatives and hydrophobic compounds.

Sodium alginate-grafted β-cyclodextrins as a matrix for immobilized Arthrobacter simplex for cortisone acetate biotransfromation

NASA Astrophysics Data System (ADS)

Shen, Yanbing; Niu, Lulu; Yu, Ziqi; Wang, Min; Shang, Zhihua; Yang, Yan

2018-06-01

Cyclodextrins (CDs) are used to resolve the low aqueous solubility of steroids, but the high cost of CDs is still a limiting factor in biotransformation process. This study, which is based on grafting and immobilization techniques, focused on synthesizing for the first time sodium alginate (SA)-grafted β-CD (SA-β-CD) and alginate-grafted β-CD for the immobilization of Arthrobacter simplex (ASP) cells (SA-β-CD-cells) and subsequent recycling of CDs and cells. FTIR spectium and X-ray diffraction proved that β-CD was successfully grafted with SA, whereas the grafting yield of β-CD was 10.3 μmol g-1. SA-β-CD could increase the solubility of CA by 3.5-fold, whereas the transformation rate was enhanced by 10%. The conversion ratio of CA was over 92% after the SA-β-CD recycling for nine cycles. In addition, after SA-β-CD-cells were applied in biocatalytic reactions for eight cycles, the conversion ratio of CA was over 90%. These advantages suggest great potential for using both grafting and immobilized techniques in steroid transformation.

All-integrated and highly sensitive paper based device with sample treatment platform for Cd2+ immunodetection in drinking/tap waters.

PubMed

López Marzo, Adaris M; Pons, Josefina; Blake, Diane A; Merkoçi, Arben

2013-04-02

Nowadays, the development of systems, devices, or methods that integrate several process steps into one multifunctional step for clinical, environmental, or industrial purposes constitutes a challenge for many ongoing research projects. Here, we present a new integrated paper based cadmium (Cd(2+)) immunosensing system in lateral flow format, which integrates the sample treatment process with the analyte detection process. The principle of Cd(2+) detection is based on competitive reaction between the cadmium-ethylenediaminetetraacetic acid-bovine serum albumin-gold nanoparticles (Cd-EDTA-BSA-AuNP) conjugate deposited on the conjugation pad strip and the Cd-EDTA complex formed in the analysis sample for the same binding sites of the 2A81G5 monoclonal antibody (mAb), specific to Cd-EDTA but not Cd(2+) free, which is immobilized onto the test line. This platform operates without any sample pretreatment step for Cd(2+) detection thanks to an extra conjugation pad that ensures Cd(2+) complexation with EDTA and interference masking through ovalbumin (OVA). The detection and quantification limits found for the device were 0.1 and 0.4 ppb, respectively, these being the lowest limits reported up to now for metal sensors based on paper. The accuracy of the device was evaluated by addition of known quantities of Cd(2+) to different drinking water samples and subsequent Cd(2+) content analysis. Sample recoveries ranged from 95 to 105% and the coefficient of variation for the intermediate precision assay was less than 10%. In addition, the results obtained here were compared with those obtained with the well-established inductively coupled plasma emission spectroscopy (ICPES) and the analysis of certificate standard samples.

Characterization of HgCdTe Films Grown on Large-Area CdZnTe Substrates by Molecular Beam Epitaxy

NASA Astrophysics Data System (ADS)

Arkun, F. Erdem; Edwall, Dennis D.; Ellsworth, Jon; Douglas, Sheri; Zandian, Majid; Carmody, Michael

2017-09-01

Recent advances in growth of Hg1- x Cd x Te films on large-area (7 cm × 7.5 cm) CdZnTe (CZT) substrates is presented. Growth of Hg1- x Cd x Te with good uniformity on large-area wafers is achieved using a Riber 412 molecular beam epitaxy (MBE) tool designed for growth of Hg1- x Cd x Te compounds. The reactor is equipped with conventional CdTe, Te, and Hg sources for achieving uniform exposure of the wafer during growth. The composition of the Hg1- x Cd x Te compound is controlled in situ by employing a closed-loop spectral ellipsometry technique to achieve a cutoff wavelength ( λ co) of 14 μm at 78 K. We present data on the thickness and composition uniformity of films grown for large-format focal-plane array applications. The composition and thickness nonuniformity are determined to be <1% over the area of a 7 cm × 7.5 cm wafer. The films are further characterized by Fourier-transform infrared spectroscopy, optical microscopy, and Hall measurements. Additionally, defect maps show the spatial distribution of defects generated during the epitaxial growth of the Hg1- x Cd x Te films. Microdefect densities are in the low 103 cm-2 range, and void defects are below 500 cm-2. Dislocation densities less than 5 × 105 cm-2 are routinely achieved for Hg1- x Cd x Te films grown on CZT substrates. HgCdTe 4k × 4k focal-plane arrays with 15 μm pitch for astronomical wide-area infrared imagers have been produced using the recently developed MBE growth process at Teledyne Imaging Sensors.

Heavy metal tolerance and accumulation of Triarrhena sacchariflora, a large amphibious ornamental grass.

PubMed

Tian, R N; Yu, S; Wang, S G; Zhang, Y; Tang, J Y; Liu, Y L; Nie, Y H

2013-01-01

In this study, we report the tolerance and accumulation of Triarrhena sacchariflora to copper (Cu) and cadmium (Cd). The results show that T. sacchariflora had strong tolerance to Cu and Cd stress. The tolerance indexes (TI) were greater than 0.5 for all treatments. The bioconcentration factors (BCFs) to Cu and Cd were both above 1.0. The accumulation ability of roots was stronger than that of shoots, and ranges of BCF to Cu and Cd in roots were 37.89-79.08 and 83.96-300.57, respectively. However, the translocation ability to Cu and Cd was weak, with more than 86% of Cu or Cd accumulated in roots, suggesting an exclusion strategy for heavy metal tolerance. The uptake efficiency (UE) and translocation efficiency (TE) to Cu and Cd increased linearly as the Cu and Cd concentration in the substrate increased. UE was higher than TE, with a maximum of 2,118.90 μg g(-1) root dry weight (DW) (50 mg L(-1) Cu) and 1,847.51 μg g(-1) root DW (20 mg L(-1)Cd), respectively. The results indicate that T. sacchariflora is a Cu- and Cd-tolerant non-hyperaccumulator plant, suggesting that T. sacchariflora could play an important role in phytoremediation in areas contaminated with Cu and Cd.

First-principles study of atomic and electronic structures of 60° perfect and 30°/90° partial glide dislocations in CdTe

DOE PAGES

Kweon, Kyoung E.; Aberg, Daniel; Lordi, Vincenzo

2016-05-16

The atomic and electronic structures of 60° glide perfect and 30°/90° glide partial dislocations in CdTe are studied using combined semi-empirical and density functional theory calculations. The calculations predict that the dislocation cores tend to undergo significant reconstructions along the dislocation lines from the singly-periodic (SP) structures, yielding either doubly-periodic (DP) ordering by forming a dimer or quadruply-periodic (QP) ordering by alternating a dimer and a missing dimer. Charge modulation along the dislocation line, accompanied by the QP reconstruction for the Cd-/Te-core 60° perfect and 30° partials or the DP reconstruction for the Cd-core 90° partial, results in semiconducting character,more » as opposed to the metallic character of the SP dislocation cores. Dislocation-induced defect states for the 60° Cd-/Te-core are located relatively close to the band edges, whereas the defect states lie in the middle of the band gap for the 30° Cd-/Te-core partial dislocations. In addition to the intracore charge modulation within each QP core, the possibility of intercore charge transfer between two different dislocation cores when they are paired together in the same system is discussed. As a result, the analysis of the electronic structures reveals the potential role of the dislocations on charge transport in CdTe, particularly in terms of charge trapping and recombination.« less

Biotemplated preparation of CdS nanoparticles/bacterial cellulose hybrid nanofibers for photocatalysis application.

PubMed

Yang, Jiazhi; Yu, Junwei; Fan, Jun; Sun, Dongping; Tang, Weihua; Yang, Xuejie

2011-05-15

In this work, we describe a novel facile and effective strategy to prepare micrometer-long hybrid nanofibers by deposition of CdS nanoparticles onto the substrate of hydrated bacterial cellulose nanofibers (BCF). Hexagonal phase CdS nanocrystals were achieved via a simple hydrothermal reaction between CdCl(2) and thiourea at relatively low temperature. The prepared pristine BCF and the CdS/BCF hybrid nanofibers were characterized by transmission electron microscopy (TEM), X-ray diffraction (XRD), thermogravimetric analysis (TGA), UV-vis absorption spectroscopy (UV-vis), and X-ray photoelectron spectroscopy (XPS). The results reveal that the CdS nanoparticles were homogeneously deposited on the BCF surface and stabilized via coordination effect. The CdS/BCF hybrid nanofibers demonstrated high-efficiency photocatalysis with 82% methyl orange (MO) degradation after 90 min irradiation and good recyclability. The results indicate that the CdS/BCF hybrid nanofibers are promising candidate as robust visible light responsive photocatalysts. Copyright © 2011 Elsevier B.V. All rights reserved.

Chondrogenic induction of mesenchymal stromal/stem cells from Wharton's jelly embedded in alginate hydrogel and without added growth factor: an alternative stem cell source for cartilage tissue engineering.

PubMed

Reppel, Loïc; Schiavi, Jessica; Charif, Naceur; Leger, Léonore; Yu, Hao; Pinzano, Astrid; Henrionnet, Christel; Stoltz, Jean-François; Bensoussan, Danièle; Huselstein, Céline

2015-12-30

Due to their intrinsic properties, stem cells are promising tools for new developments in tissue engineering and particularly for cartilage tissue regeneration. Although mesenchymal stromal/stem cells from bone marrow (BM-MSC) have long been the most used stem cell source in cartilage tissue engineering, they have certain limits. Thanks to their properties such as low immunogenicity and particularly chondrogenic differentiation potential, mesenchymal stromal/stem cells from Wharton's jelly (WJ-MSC) promise to be an interesting source of MSC for cartilage tissue engineering. In this study, we propose to evaluate chondrogenic potential of WJ-MSC embedded in alginate/hyaluronic acid hydrogel over 28 days. Hydrogels were constructed by the original spraying method. Our main objective was to evaluate chondrogenic differentiation of WJ-MSC on three-dimensional scaffolds, without adding growth factors, at transcript and protein levels. We compared the results to those obtained from standard BM-MSC. After 3 days of culture, WJ-MSC seemed to be adapted to their new three-dimensional environment without any detectable damage. From day 14 and up to 28 days, the proportion of WJ-MSC CD73(+), CD90(+), CD105(+) and CD166(+) decreased significantly compared to monolayer marker expression. Moreover, WJ-MSC and BM-MSC showed different phenotype profiles. After 28 days of scaffold culture, our results showed strong upregulation of cartilage-specific transcript expression. WJ-MSC exhibited greater type II collagen synthesis than BM-MSC at both transcript and protein levels. Furthermore, our work highlighted a relevant result showing that WJ-MSC expressed Runx2 and type X collagen at lower levels than BM-MSC. Once seeded in the hydrogel scaffold, WJ-MSC and BM-MSC have different profiles of chondrogenic differentiation at both the phenotypic level and matrix synthesis. After 4 weeks, WJ-MSC, embedded in a three-dimensional environment, were able to adapt to their environment and express specific cartilage-related genes and matrix proteins. Today, WJ-MSC represent a real alternative source of stem cells for cartilage tissue engineering.

Differentiation of Mesenchymal Stem Cells Derived from Pancreatic Islets and Bone Marrow into Islet-Like Cell Phenotype

PubMed Central

Zanini, Cristina; Bruno, Stefania; Mandili, Giorgia; Baci, Denisa; Cerutti, Francesco; Cenacchi, Giovanna; Izzi, Leo; Camussi, Giovanni; Forni, Marco

2011-01-01

Background Regarding regenerative medicine for diabetes, accessible sources of Mesenchymal Stem Cells (MSCs) for induction of insular beta cell differentiation may be as important as mastering the differentiation process itself. Methodology/Principal Findings In the present work, stem cells from pancreatic islets (human islet-mesenchymal stem cells, HI-MSCs) and from human bone marrow (bone marrow mesenchymal stem cells, BM-MSCs) were cultured in custom-made serum-free medium, using suitable conditions in order to induce differentiation into Islet-like Cells (ILCs). HI-MSCs and BM-MSCs were positive for the MSC markers CD105, CD73, CD90, CD29. Following this induction, HI-MSC and BM-MSC formed evident islet-like structures in the culture flasks. To investigate functional modifications after induction to ILCs, ultrastructural analysis and immunofluorescence were performed. PDX1 (pancreatic duodenal homeobox gene-1), insulin, C peptide and Glut-2 were detected in HI-ILCs whereas BM-ILCs only expressed Glut-2 and insulin. Insulin was also detected in the culture medium following glucose stimulation, confirming an initial differentiation that resulted in glucose-sensitive endocrine secretion. In order to identify proteins that were modified following differentiation from basal MSC (HI-MSCs and BM-MSCs) to their HI-ILCs and BM-ILCs counterparts, proteomic analysis was performed. Three new proteins (APOA1, ATL2 and SODM) were present in both ILC types, while other detected proteins were verified to be unique to the single individual differentiated cells lines. Hierarchical analysis underscored the limited similarities between HI-MSCs and BM-MSCs after induction of differentiation, and the persistence of relevant differences related to cells of different origin. Conclusions/Significance Proteomic analysis highlighted differences in the MSCs according to site of origin, reflecting spontaneous differentiation and commitment. A more detailed understanding of protein assets may provide insights required to master the differentiation process of HI-MSCs to functional beta cells based only upon culture conditioning. These findings may open new strategies for the clinical use of BM-MSCs in diabetes. PMID:22194812

Plasma-soluble CD30 in childhood tuberculosis: effects of disease severity, nutritional status, and vitamin A therapy.

PubMed

Hanekom, W A; Hussey, G D; Hughes, E J; Potgieter, S; Yogev, R; Check, I J

1999-03-01

Plasma-soluble CD30 (sCD30) is the result of proteolytic splicing from the membrane-bound form of CD30, a putative marker of type 2 cytokine-producing cells. We measured sCD30 levels in children with tuberculosis, a disease characterized by prominent type 1 lymphocyte cytokine responses. We postulated that disease severity and nutritional status would alter cytokine responses and therefore sCD30 levels. Samples from South African children enrolled prospectively at the time of diagnosis of tuberculosis were analyzed. (Patients were originally enrolled in a randomized, double-blind placebo-controlled study of the effects of oral vitamin A supplementation on prognosis of tuberculosis.) Plasma samples collected at the time of diagnosis and 6 and 12 weeks later (during antituberculosis therapy) were analyzed. sCD30 levels were measured by enzyme immunoassay. The 91 children included in the study demonstrated high levels of sCD30 at diagnosis (median, 98 U/liter; range, 11 to 1,569 U/liter). Although there was a trend toward higher sCD30 levels in more severe disease (e.g., culture-positive disease or miliary disease), this was not statistically significant. Significantly higher sCD30 levels were demonstrated in the presence of nutritional compromise: the sCD30 level was higher in patients with a weight below the third percentile for age, in those with clinical signs of kwashiorkor, and in those with a low hemoglobin content. There was minimal change in the sCD30 level after 12 weeks of therapy, even though patients improved clinically. However, changes in sCD30 after 12 weeks differed significantly when 46 patients (51%) who received vitamin A were compared with those who had received a placebo. Vitamin A-supplemented children demonstrated a mean (+/- standard error of the mean) decrease in sCD30 by a factor of 0.99 +/- 0.02 over 12 weeks, whereas a factor increase of 1.05 +/- 0.02 was demonstrated in the placebo group (P = 0.02). We conclude that children with tuberculosis had high sCD30 levels, which may reflect the presence of a type 2 cytokine response. Nutritional compromise was associated with higher sCD30 levels. Vitamin A therapy resulted in modulation of sCD30 levels over time.

Plasma-Soluble CD30 in Childhood Tuberculosis: Effects of Disease Severity, Nutritional Status, and Vitamin A Therapy

PubMed Central

Hanekom, W. A.; Hussey, G. D.; Hughes, E. J.; Potgieter, S.; Yogev, R.; Check, I. J.

1999-01-01

Plasma-soluble CD30 (sCD30) is the result of proteolytic splicing from the membrane-bound form of CD30, a putative marker of type 2 cytokine-producing cells. We measured sCD30 levels in children with tuberculosis, a disease characterized by prominent type 1 lymphocyte cytokine responses. We postulated that disease severity and nutritional status would alter cytokine responses and therefore sCD30 levels. Samples from South African children enrolled prospectively at the time of diagnosis of tuberculosis were analyzed. (Patients were originally enrolled in a randomized, double-blind placebo-controlled study of the effects of oral vitamin A supplementation on prognosis of tuberculosis.) Plasma samples collected at the time of diagnosis and 6 and 12 weeks later (during antituberculosis therapy) were analyzed. sCD30 levels were measured by enzyme immunoassay. The 91 children included in the study demonstrated high levels of sCD30 at diagnosis (median, 98 U/liter; range, 11 to 1,569 U/liter). Although there was a trend toward higher sCD30 levels in more severe disease (e.g., culture-positive disease or miliary disease), this was not statistically significant. Significantly higher sCD30 levels were demonstrated in the presence of nutritional compromise: the sCD30 level was higher in patients with a weight below the third percentile for age, in those with clinical signs of kwashiorkor, and in those with a low hemoglobin content. There was minimal change in the sCD30 level after 12 weeks of therapy, even though patients improved clinically. However, changes in sCD30 after 12 weeks differed significantly when 46 patients (51%) who received vitamin A were compared with those who had received a placebo. Vitamin A-supplemented children demonstrated a mean (± standard error of the mean) decrease in sCD30 by a factor of 0.99 ± 0.02 over 12 weeks, whereas a factor increase of 1.05 ± 0.02 was demonstrated in the placebo group (P = 0.02). We conclude that children with tuberculosis had high sCD30 levels, which may reflect the presence of a type 2 cytokine response. Nutritional compromise was associated with higher sCD30 levels. Vitamin A therapy resulted in modulation of sCD30 levels over time. PMID:10066655

Prenatally fabricated autologous human living heart valves based on amniotic fluid derived progenitor cells as single cell source.

PubMed

Schmidt, Dörthe; Achermann, Josef; Odermatt, Bernhard; Breymann, Christian; Mol, Anita; Genoni, Michele; Zund, Gregor; Hoerstrup, Simon P

2007-09-11

A novel concept providing prenatally tissue engineered human autologous heart valves based on routinely obtained fetal amniotic fluid progenitors as single cell source is introduced. Fetal human amniotic progenitors were isolated from routinely sampled amniotic fluid and sorted using CD133 magnetic beads. After expansion and differentiation, cell phenotypes of CD133- and CD133+ cells were analyzed by immunohistochemistry and flowcytometry. After characterization, CD133- derived cells were seeded onto heart valve leaflet scaffolds (n=18) fabricated from rapidly biodegradable polymers, conditioned in a pulse duplicator system, and subsequently coated with CD133+ derived cells. After in vitro maturation, opening and closing behavior of leaflets was investigated. Neo-tissues were analyzed by histology, immunohistochemistry, and scanning electron microscopy (SEM). Extracellular matrix (ECM) elements and cell numbers were quantified biochemically. Mechanical properties were assessed by tensile testing. CD133- derived cells demonstrated characteristics of mesenchymal progenitors expressing CD44 and CD105. Differentiated CD133+ cells showed features of functional endothelial cells by eNOS and CD141 expression. Engineered heart valve leaflets demonstrated endothelialized tissue formation with production of ECM elements (GAG 80%, HYP 5%, cell number 100% of native values). SEM showed intact endothelial surfaces. Opening and closing behavior was sufficient under half of systemic conditions. The use of amniotic fluid as single cell source is a promising low-risk approach enabling the prenatal fabrication of heart valves ready to use at birth. These living replacements with the potential of growth, remodeling, and regeneration may realize the early repair of congenital malformations.

Army Generating Force Census Utilization. FY2008 Summer Study

DTIC Science & Technology

2009-10-01

TD 󈧻 -a ^ 01 3 3" 𔃽 3 QJ <j IH b rj •3 re .£ > OJ re a re (— ft- c g...73 £ rn H QJ -3 QJ J3 co Ŗ •*•’ ro 3 •_ .a - Si «o 2 g.| S c x > 2 2 O £ Q. QJ .£ o X ft- 3 TD O 3 co re QJ IH X T3 >H...co to 󈧻 — _>» TD c 5 < CO 75 CD .2 II CO o n LJ- LL U O • c CD E CD O) 03 c 03 CD o CL c

CD10-bearing fibroblasts may inhibit skin inflammation by down-modulating substance P.

PubMed

Xie, Lining; Takahara, Masakazu; Nakahara, Takeshi; Oba, Junna; Uchi, Hiroshi; Takeuchi, Satoshi; Moroi, Yoichi; Furue, Masutaka

2011-01-01

Substance P (SP) is a multipotent neuropeptide that affects the proliferation, activation and motility of keratinocytes and fibroblasts (Fbs). SP in pulmonary and synovial cells is degraded by CD10, a 90- to 110-kDa cell surface zinc-dependent metalloprotease. However, the expression and function of CD10 in human dermal Fbs have not yet been investigated in vivo and in vitro specifically with reference to SP. Our immunohistologic study revealed moderate to strong fibroblastic CD10 expression in the majority of psoriasis vulgaris (16/16), chronic eczema (15/16), lichen planus (18/20) and atopic dermatitis (4/5). Keratinocytes showed no CD10 expression in vivo and in vitro. Cultured Fbs constitutively expressed CD10 and SP. CD10 expression was augmented by external interleukin (IL)-1β and IL-22, but not by IL-8 and IL-17A in Fbs. SP production was enhanced in CD10 knockdown-Fbs (CD10ND-Fbs) compared with control-Fbs. In the presence of IL-1β or IL-22, the enhancement of SP production was more prominent in CD10ND-Fbs than in control-Fbs, suggesting the down-modulating activity of CD10 on SP in cytokine-mediated inflammation. In conclusion, fibroblastic CD10 expression may down-regulate skin inflammation by degrading SP or reducing its level in the dermal microenvironment.

Expression of CD73/ecto-5'-nucleotidase on human gingival fibroblasts and contribution to the inhibition of interleukin-1alpha-induced granulocyte-macrophage colony stimulating factor production.

PubMed

Nemoto, Eiji; Kunii, Ryotaro; Tada, Hiroyuki; Tsubahara, Taisuke; Ishihata, Hiroshi; Shimauchi, Hidetoshi

2004-02-01

CD73/5'-nucleotidase (5'-NT) is an ectoenzyme that participates in immune/inflammatory reactions. We examined the possible expression of CD73/5'-NT on human gingival fibroblasts (hGF), which are important to the immune/inflammatory system in periodontal tissue. We demonstrated that CD73/5'-NT was expressed on hGF by flow cytometry. We found that pre-treatment of hGF with 5'-AMP induced marked inhibition of granulocyte-macrophage colony-stimulating factor (GM-CSF) production from hGF upon stimulation with interleukin-1alpha (IL-1alpha) by enzyme-linked immunosorbent assay (ELISA). A specific inhibitor of 5'-NT, adenosine 5'-[alpha,beta-methylene] diphosphate blocked the inhibition of GM-CSF production, suggesting that adenosine converted from 5'-AMP acts on the inhibitory effects. The GM-CSF inhibition suggested that A3 receptor might be involved. The rank order of agonists was found to be (N6-benzyl-5'-N-ethylcarboxamidoadenosine) A3 receptor agonist > or = (2-chloroadenosine) non-selective agonist > (CGS-21680) A2A receptor agonist > adenosine > or = (N6-cyclohexyladenosine) A1 agonist. Further support for the main role of A3 receptor was the binding A3 antagonist [9-chloro-2-(2-furanyl)-5-([phenylacetyl]amino)[1,2,4]-triazolo[1,5-c]quinazdine] reversed the effect of adenosine, but no significant reverse was observed by A1 (1,3-dipropyl-8-cyclopentylxanthine), A2 [3,7-dimethyl-1-(2-propargyl)xanthine], A2A[8-(3-chlorostyryl)caffeine], and A2B (alloxazine) antagonists. The CD73/5'-NT expression was increased upon stimulation with gamma-interferon, but not other stimulants such as tumor necrosis factor-alpha, IL-4, lipopolysaccharide from Porphyromonas gingivalis and Escherichia coli, and fimbriae from P. gingivalis, and this increase was correlated with the enhanced GM-CSF inhibition by 5'-AMP but not adenosine. These findings suggested that CD73/5'-NT on hGF exerts an anti-inflammatory effects in periodontal disease by conversion from 5'-AMP to adenosine.

Interaction effects of As, Cd and Pb on their respective bioaccessibility with time in co-contaminated soils assessed by the Unified BARGE Method.

PubMed

Xia, Qing; Lamb, Dane; Peng, Cheng; Ng, Jack C

2017-02-01

Interaction effects of As, Cd and Pb on their respective bioaccessibility in co-contaminated soils were reported. In addition, the influence of aging time (up to 90 days) on potential interactions was also investigated. Experiments were carried out by spiking four diverse soils with single, binary or ternary mixtures of As, Cd and Pb. Soils were measured for bioaccessibility at different aging periods. Results demonstrate that bioaccessibility of As, Cd and Pb reached a steady state after soils were aged for 30 days. Bioaccessibility of As, Cd and Pb in soils spiked with binary mixtures of As, Cd and Pb were not affected by the other co-existing metal/metalloid. But when As, Cd and Pb were introduced together to acidic soils which lacked abundant binding sites, intestinal bioaccessibility of Cd was increased at the early stage of aging (7 to 30 days) whilst bioaccessibility of As and Pb remained unchanged. However, when Pb and As were added after Cd has been incubated in soil for 7 days, Cd intestinal bioaccessibility was not influenced by As and Pb. Therefore, a number of factors should be taken into consideration when estimating the bioaccessibility of mixed As, Cd and Pb, including the loadings of As, Cd and Pb in soils, the time for which they have been aged together and the time period between As, Cd and Pb entering the soils.

Predictive factors for long-term engraftment of autologous blood stem cells.

PubMed

Duggan, P R; Guo, D; Luider, J; Auer, I; Klassen, J; Chaudhry, A; Morris, D; Glück, S; Brown, C B; Russell, J A; Stewart, D A

2000-12-01

Data from 170 consecutive patients aged 19-66 years (median age 46 years) who underwent unmanipulated autologous blood stem cell transplant (ASCT) were analyzed to determine if total CD34+ cells/kg infused, CD34+ subsets (CD34+41+, CD34+90+, CD34+33-, CD34+38-, CD34+38-DR-), peripheral blood CD34+ cell (PBCD34+) count on first apheresis day, or various clinical factors were associated with low blood counts 6 months post ASCT. Thirty-four patients were excluded from analysis either because of death (n = 17) or re-induction chemotherapy prior to 6 months post ASCT (n = 13), or because of lack of follow-up data (n = 4). Of the remaining 136 patients, 46% had low WBC ( < 4 x 10(9)/l), 41% low platelets (<150 x 10(9)/l), and 34% low hemoglobin ( < 120 g/l) at a median of 6 months following ASCT. By Spearman's rank correlation, both the total CD34+ cell dose/kg and the PBCD34+ count correlated with 6 month blood counts better than any subset of CD34+ cells or any clinical factor. The PBCD34+ count was overall a stronger predictor of 6 month blood counts than was the total CD34+ cells/kg infused. Both factors retained their significance in multivariate analysis, controlling for clinical factors. In conclusion, subsets of CD34+ cells and clinical factors are inferior to the total CD34+ cell dose/kg and PBCD34+ count in predicting 6 month blood counts following ASCT.

CD90-positive cells, an additional cell population, produce laminin {alpha}2 upon transplantation to dy{sup 3k}/dy{sup 3k} mice

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

Fukada, So-ichiro; Yamamoto, Yukiko; Segawa, Masashi

2008-01-01

Laminin {alpha}2 is a component of skeletal and cardiac muscle basal lamina. A defect of the laminin {alpha}2 chain leads to severe congenital muscular dystrophy (MDC1A) in humans and dy/dy mice. Myogenic cells including myoblasts, myotubes, and myofibers in skeletal muscle are a possible source of the laminin {alpha}2 chain, and myogenic cells are thus proposed as a cell source for congenital muscular dystrophy therapy. However, we observed production of laminin {alpha}2 in non-myogenic cells of normal mice, and we could enrich these laminin {alpha}2-producing cells in CD90{sup +} cell fractions. Intriguingly, the number of CD90{sup +} cells increased dramaticallymore » during skeletal muscle regeneration in mice. This fraction did not include myogenic cells but exhibited a fibroblast-like phenotype. Moreover, these cells were resident in skeletal muscle, not derived from bone marrow. Finally, the production of laminin {alpha}2 in CD90{sup +} cells was not dependent on fusion with myogenic cells. Thus, CD90{sup +} cells are a newly identified additional cell fraction that increased during skeletal muscle regeneration in vivo and could be another cell source for therapy for lama2-deficient muscular dystrophy.« less

Depletion of CD52-positive cells inhibits the development of central nervous system autoimmune disease, but deletes an immune-tolerance promoting CD8 T-cell population. Implications for secondary autoimmunity of alemtuzumab in multiple sclerosis.

PubMed

von Kutzleben, Stephanie; Pryce, Gareth; Giovannoni, Gavin; Baker, David

2017-04-01

The objective was to determine whether CD52 lymphocyte depletion can act to promote immunological tolerance induction by way of intravenous antigen administration such that it could be used to either improve efficiency of multiple sclerosis (MS) inhibition or inhibit secondary autoimmunities that may occur following alemtuzumab use in MS. Relapsing experimental autoimmune encephalomyelitis was induced in ABH mice and immune cell depletion was therapeutically applied using mouse CD52 or CD4 (in conjunction with CD8 or CD20) depleting monoclonal antibodies. Immunological unresponsiveness was then subsequently induced using intravenous central nervous system antigens and responses were assessed clinically. A dose-response of CD4 monoclonal antibody depletion indicated that the 60-70% functional CD4 T-cell depletion achieved in perceived failed trials in MS was perhaps too low to even stop disease in animals. However, more marked (~75-90%) physical depletion of CD4 T cells by CD4 and CD52 depleting antibodies inhibited relapsing disease. Surprisingly, in contrast to CD4 depletion, CD52 depletion blocked robust immunological unresponsiveness through a mechanism involving CD8 T cells. Although efficacy was related to the level of CD4 T-cell depletion, the observations that CD52 depletion of CD19 B cells was less marked in lymphoid organs than in the blood provides a rationale for the rapid B-cell hyper-repopulation that occurs following alemtuzumab administration in MS. That B cells repopulate in the relative absence of T-cell regulatory mechanisms that promote immune tolerance may account for the secondary B-cell autoimmunities, which occur following alemtuzumab treatment of MS. © 2016 The Authors. Immunology Published by John Wiley & Sons Ltd.

Social and immunological differences among uninfected Brazilians exposed or unexposed to human immunodeficiency virus-infected partners.

PubMed

Silva, Maria Luiza; Melo, Victor Hugo; Aleixo, Agdemir Waléria; Aleixo, Lúcia Fernandes; Pascoal-Xavier, Marcelo Antônio; Silva, Rafaela Oliveira; Ferreira, Laís Alves; Domingos, Willian Cunha; Greco, Dirceu Bartolomeu

2014-09-01

Understanding the social conditions and immunological characteristics that allow some human immunodeficiency virus (HIV)-exposed patients to remain uninfected represents an on-going challenge. In this study, the socio-demographic and sexual behaviour characteristics and immune activation profiles of uninfected individuals exposed to HIV-infected partners were investigated. A confidential and detailed questionnaire was administered and venous blood was tested using HIV-1/enzyme immunoassays, plasma HIV-1 RNA levels/bDNA and immunophenotyping/flow cytometry to determine the frequencies of CD4 and CD8 T cells expressing activation markers. The data analysis showed significant differences (p < 0.05) for immune parameters in individuals who were uninfected, albeit exposed to HIV-infected partners, compared with unexposed individuals. In particular, the exposed, uninfected individuals had a higher frequency (median, minimum-maximum) of CD4⁺HLA-DR⁺ (4.2, 1.8-6.1), CD8⁺HLA-DR⁺ (4.6, 0.9-13.7), CD4⁺CD45RO⁺ (27.5, 14.2-46.6), CD4⁺CD45RO⁺CD62L⁺ (46.7, 33.9-67.1), CD8⁺CD45RA⁺HLA-DR⁺ (12.1, 3.4-35.8) and CD8⁺CD45RO⁺HLA-DR⁺ (9.0, 3.2-14.8) cells, a decreased percentage of CD8⁺CD28⁺ cells (11.7, 4.5-24.0) and a lower cell-surface expression of Fcγ-R/CD16 on monocytes (56.5, 22.0-130.0). The plasma HIV-1 RNA levels demonstrated detectable RNA virus loads in 57% of the HIV-1⁺ female partners. These findings demonstrate an activation profile in both CD4 and CD8 peripheral T cells from HIV-1 exposed seronegative individuals of serodiscordant couples from a referral centre in Belo Horizonte, state of Minas Gerais.

Lymphoblastic lymphoma.

PubMed

Cortelazzo, Sergio; Ponzoni, Maurilio; Ferreri, Andrés J M; Hoelzer, Dieter

2011-09-01

Lymphoblastic lymphoma (LBL) is a neoplasm of immature B cells committed to the B-(B-LBL) or T-cell lineage (T-LBL) that accounts for approximately 2% of all lymphomas. From a histopathological point of view, blasts may be encountered in tissue biopsy and/or bone marrow (BM). In tissue sections, LBL is generally characterized by a diffuse or, as in lymph nodes and less commonly, paracortical pattern. Although histological features are usually sufficient to distinguish lymphoblastic from mature B- or T-cell neoplasms, a differential diagnosis with blastoid variant of mantle cell lymphoma, Burkitt lymphoma or myeloid leukemia may arise in some cases. Of greater importance is the characterization of immunophenotype by flow cytometry. In B-LBL, tumour cells are virtually always positive for B cell markers CD19, CD79a and CD22. They are positive for CD10, CD 24, PAX5, and TdT in most cases, while the expression of CD20 and the lineage independent stem cell antigen CD34 is variable and CD45 may be absent. Surface immunoglobulin is usually absent. In T-LBL, neoplastic cells are usually TdT positive and variably express CD1a, CD2, CD3, CD4, CD5, CD7 and CD8. The only reliable lineage-specific is surface CD3. Most B-LBL have clonal rearrangements of the Ig heavy chain or less frequently of light chain genes. T-cell receptor γ or β chain gene rearrangements may be seen in a significant number of cases, but rearrangements are not helpful for lineage assignment. LBL occurs more commonly in children than in adults, mostly in males. Although 80% of precursor B-cell neoplasms present as acute leukemias, with BM and peripheral blood (PB) involvement, a small proportion present with a mass lesion and have <25% blasts in the BM. Unlike precursor T-LBL, mediastinal masses and involvement of BM are rare, but lymph nodes and extranodal sites are more frequently involved. T-LBL patients, compared to those with B-LBL, show younger age, a higher rate of mediastinal tumours or BM involvement. Patients are usually males in their teens to twenties and present with lymphadenopathy in cervical, supraclavicular and axillary regions, or with a mediastinal mass. In most patients the mediastinal mass is anterior, bulky, and associated with pleural effusions, superior vena cava syndrome, tracheal obstruction, and pericardial effusions. They frequently present with advanced disease, B symptoms and elevated serum LDH levels. Abdominal involvement (liver and spleen) is unusual. LBL is highly aggressive, but frequently curable with current therapy. The prognosis in all age groups has dramatically improved with the use of intensive ALL-type chemotherapy regimes, with a disease-free survival of 73-90% in children and 45-72% in adults. Intensive intrathecal chemotherapy prophylaxis is required to reduce the CNS relapse incidence, while the role of prophylactic cranial irradiation is unclear. Consolidation mediastinal irradiation may decrease mediastinal relapse. Patients with adverse prognostic features should be considered for high-dose chemotherapy and SCT. Autologous SCT has been shown to produce similar good results as chemotherapy alone, and allogeneic SCT is likely to be a more appropriate option for patients who are beyond first remission or with more advanced disease. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

Electrodeposition of CdTe thin film from acetate-based ionic liquid bath

NASA Astrophysics Data System (ADS)

Waldiya, Manmohansingh; Bhagat, Dharini; Mukhopadhyay, Indrajit

2018-05-01

CdTe being a direct band gap semiconductor, is mostly used in photovoltaics. Here we present, the synthesis of CdTe thin film on fluorine doped tin oxide (FTO) substrate potentiostatically using 1-butyl-3-methylimidazolium acetate ([Bmim][Ac]) ionic liquid (IL) bath at 90 °C. Major advantages of using electrodeposition involves process simplicity, large scalability & economic viability. Some of the benefits offered by IL electrolytic bath are low vapour pressure, wide electrochemical window, and good ionic mobility. Cd(CH3COO)2 (anhydrous) and TeO2 were used as the source precursors. The IL electrolytic bath temperature was kept at 90 °C for deposition, owing to the limited solubility of TeO2 in [Bmim][Ac] IL at room temperature. Cathodic electrodeposition was carried out using a three electrode cell setup at a constant potential of -1.20 V vs. platinum (Pt) wire. The CdTe/FTO thin film were annealed in argon (Ar) atmosphere. Optical study of nanostructured CdTe film were done using UV-Vis-IR and Raman spectroscopy. Raman analysis confirms the formation of CdTe having surface optics (SO) mode at 160.6 cm-1 and transverse optics (TO) mode at 140.5 cm-1. Elemental Te peaks at 123, 140.5 and 268 cm-1 were also observed. The optical band gap of Ar annealed CdTe thin film were found to be 1.47 eV (absorbance band edge ˜ 846 nm). The optimization of deposition parameters using acetate-based IL electrolytic bath to get nearly stoichiometric CdTe thin film is currently being explored.

Identification of Three Molecular and Functional Subtypes in Canine Hemangiosarcoma through Gene Expression Profiling and Progenitor Cell Characterization

PubMed Central

Gorden, Brandi H.; Kim, Jong-Hyuk; Sarver, Aaron L.; Frantz, Aric M.; Breen, Matthew; Lindblad-Toh, Kerstin; O'Brien, Timothy D.; Sharkey, Leslie C.; Modiano, Jaime F.; Dickerson, Erin B.

2015-01-01

Canine hemangiosarcomas have been ascribed to an endothelial origin based on histologic appearance; however, recent findings suggest that these tumors may arise instead from hematopoietic progenitor cells. To clarify this ontogenetic dilemma, we used genome-wide expression profiling of primary hemangiosarcomas and identified three distinct tumor subtypes associated with angiogenesis (group 1), inflammation (group 2), and adipogenesis (group 3). Based on these findings, we hypothesized that a common progenitor may differentiate into the three tumor subtypes observed in our gene profiling experiment. To investigate this possibility, we cultured hemangiosarcoma cell lines under normal and sphere-forming culture conditions to enrich for tumor cell progenitors. Cells from sphere-forming cultures displayed a robust self-renewal capacity and exhibited genotypic, phenotypic, and functional properties consistent with each of the three molecular subtypes seen in primary tumors, including expression of endothelial progenitor cell (CD133 and CD34) and endothelial cell (CD105, CD146, and αvβ3 integrin) markers, expression of early hematopoietic (CD133, CD117, and CD34) and myeloid (CD115 and CD14) differentiation markers in parallel with increased phagocytic capacity, and acquisition of adipogenic potential. Collectively, these results suggest that canine hemangiosarcomas arise from multipotent progenitors that differentiate into distinct subtypes. Improved understanding of the mechanisms that determine the molecular and phenotypic differentiation of tumor cells in vivo could change paradigms regarding the origin and progression of endothelial sarcomas. PMID:24525151

Development and validation of a fluorescent microsphere immunoassay for soluble CD30 testing.

PubMed

Pavlov, Igor; Martins, Thomas B; Delgado, Julio C

2009-09-01

Testing for soluble CD30 (sCD30), an indicator of Th2 immune response, is a useful prognostic marker in solid organ transplantation, lymphoproliferative disorders, autoimmunity, and various parasitic diseases. In this study we report the development and validation of a fluorescent microsphere immunoassay for the detection of sCD30 in serum, plasma, and culture supernatants. The dynamic range of this assay is 1 to 400 ng/ml, and the rate of recovery of various concentrations of recombinant sCD30 ranges from 97 to 116% (average recovery, 105%). The test showed a high degree of precision in both intra-assay and interassay studies (coefficients of variation, as high as 7% and 8%, respectively), with a sensitivity of 1 ng/ml. The normal reference range calculated for a cohort of 151 healthy individuals was 1 to 29 ng/ml. The clinical usefulness of the sCD30 fluorescent microsphere immunoassay was demonstrated by showing that levels of sCD30 have a positive correlation with specimens containing high titers of anti-double-stranded DNA antibodies and high titers of immunoglobulin G against Leishmania species. Given the multiplexing potential of the sCD30 fluorescent microsphere immunoassay reported in this study, it is expected that testing of sCD30 concentrations along with those of other cytokines will become an important diagnostic tool for selected immunological and inflammatory diseases where Th2-type cytokine responses have been reported.

Development and Validation of a Fluorescent Microsphere Immunoassay for Soluble CD30 Testing▿

PubMed Central

Pavlov, Igor; Martins, Thomas B.; Delgado, Julio C.

2009-01-01

Testing for soluble CD30 (sCD30), an indicator of Th2 immune response, is a useful prognostic marker in solid organ transplantation, lymphoproliferative disorders, autoimmunity, and various parasitic diseases. In this study we report the development and validation of a fluorescent microsphere immunoassay for the detection of sCD30 in serum, plasma, and culture supernatants. The dynamic range of this assay is 1 to 400 ng/ml, and the rate of recovery of various concentrations of recombinant sCD30 ranges from 97 to 116% (average recovery, 105%). The test showed a high degree of precision in both intra-assay and interassay studies (coefficients of variation, as high as 7% and 8%, respectively), with a sensitivity of 1 ng/ml. The normal reference range calculated for a cohort of 151 healthy individuals was 1 to 29 ng/ml. The clinical usefulness of the sCD30 fluorescent microsphere immunoassay was demonstrated by showing that levels of sCD30 have a positive correlation with specimens containing high titers of anti-double-stranded DNA antibodies and high titers of immunoglobulin G against Leishmania species. Given the multiplexing potential of the sCD30 fluorescent microsphere immunoassay reported in this study, it is expected that testing of sCD30 concentrations along with those of other cytokines will become an important diagnostic tool for selected immunological and inflammatory diseases where Th2-type cytokine responses have been reported. PMID:19605595

[Effects of Rice Cultivar and Typical Soil Improvement Measures on the Uptake of Cd in Rice Grains].

PubMed

Wang, Mei-e; Peng, Chi; Chen, Wei-ping

2015-11-01

Cadmium pollution of rice is a big problem in agricultural food safety. The accident "Cd rice" occurred last year in Youxian County, Hunan Province caused serious social panic. In this study, trials on "Cd rice" controlling techniques specific to the Cd pollution in paddy soil in Youxian were investigated. It was suggested that the average Cd contents in rice grains of the rice variety "Zhu Liang You 06" in Datongqiao and Wangling were 0.167 and 0.127 mg x kg(-1), respectively, which were only equal to 20% of the contents of other varieties. The trials for stabilizing agents revealed that treatments of lime and mineral fertilizer decreased Cd contents in rice grains to 20-30% of the control. Plastic film-mulched treatment decreased the rice grain Cd to 50%. And combined treatment of plastic film-mulched and biochar and silicon foliar-fertilizer decreased 80% of rice Cd content. Single treatments of silicon foliar-fertilizer and combined treatment of silicon foliar-fertilizer and topdressing fertilizer decreased more than 90% of Cd content. Results of BCR revealed that the percentage of cationic exchangeable and/or carbonate associated Cd fraction was more than 55% for most of the soil samples. Lime treatment significantly decreased the percentage of cationic exchangeable and/or carbonate and oxides of Fe and Mn associated Cd and increased the crystalline structure of clay minerals associated Cd. The change rate reached about 20%. Our results suggested concentration of soil Cd and pH were the two significant factors impacting the uptake of Cd by rice grains.

Cadmium availability in rice paddy fields from a mining area: The effects of soil properties highlighting iron fractions and pH value.

PubMed

Yu, Huan-Yun; Liu, Chuanping; Zhu, Jishu; Li, Fangbai; Deng, Dong-Mei; Wang, Qi; Liu, Chengshuai

2016-02-01

Cadmium (Cd) availability can be significantly affected by soil properties. The effect of pH value on Cd availability has been confirmed. Paddy soils in South China generally contain high contents of iron (Fe). Thus, it is hypothesized that Fe fractions, in addition to pH value, may play an important role in the Cd bioavailability in paddy soil and this requires further investigation. In this study, 73 paired soil and rice plant samples were collected from paddy fields those were contaminated by acid mine drainage containing Cd. The contents of Fe in the amorphous and DCB-extractable Fe oxides were significantly and negatively correlated with the Cd content in rice grain or straw (excluding DCB-extractable Fe vs Cd in straw). In addition, the concentration of HCl-extractable Fe(II) derived from Fe(III) reduction was positively correlated with the Cd content in rice grain or straw. These results suggest that soil Fe redox could affect the availability of Cd in rice plant. Contribution assessment of soil properties to Cd accumulation in rice grain based on random forest (RF) and stochastic gradient boosting (SGB) showed that pH value should be the most important factor and the content of Fe in the amorphous Fe oxides should be the second most important factor in affecting Cd content in rice grain. Overall, compared with the studies from temperate regions, such as Europe and northern China, Fe oxide exhibited its unique role in the bioavailability of Cd in the reddish paddy soil from our study area. The exploration of practical remediation strategies for Cd from the perspective of Fe oxide may be promising. Copyright © 2015 Elsevier Ltd. All rights reserved.

Combined influence of adjuvant therapy and interval after surgery on peripheral CD4+ T lymphocytes in patients with esophageal squamous cell carcinoma

PubMed Central

LING, YANG; FAN, LIEYING; DONG, CHUNLEI; ZHU, JING; LIU, YONGPING; NI, YAN; ZHU, CHANGTAI; ZHANG, CHANGSONG

2010-01-01

The aim of this study was to investigate possible differences in cellular immunity between chemo- and/or radiotherapy groups during a long interval after surgery in esophageal squamous cell carcinoma (ESCC) patients. Cellular immunity was assessed as peripheral lymphocyte subsets in response to chemotherapy (CT), radiotherapy (RT) and CT+RT by flow cytometric analysis. There were 139 blood samples obtained at different time points relative to surgery from 73 patients with ESCC. The changes in the absolute and relative proportions of lymphocyte phenotypes were significant among the adjuvant therapy groups. There were significant differences in the absolute counts of CD4+ and CD8+ T cells among the interval groups, and a lower CD4/CD8 ratio was found in patients following a prolonged interval. RT alone had a profound effect on the absolute counts of CD3+, CD4+ and CD8+ T cells compared with the other groups. CD4+ T cells exhibited a decreasing trend during a long interval, leading to a prolonged T-cell imbalance after surgery. Univariate analysis revealed that the interaction of the type of adjuvant therapy and the interval after surgery was correlated only with the percentage of CD4+ T cells. The percentage of CD4+ T cells can be used as an indicator of the cellular immunity after surgery in ESCC patients. However, natural killer cells consistently remained suppressed in ESCC patients following adjuvant therapy after surgery. These findings confirm an interaction between adjuvant therapy and the interval after surgery on peripheral CD4+ T cells, and implies that adjuvant therapy may have selective influence on the cellular immunity of ESCC patients after surgery. PMID:23136603

Identification of contact sites between ankyrin and band 3 in the human erythrocyte membrane.

PubMed

Grey, Jesse L; Kodippili, Gayani C; Simon, Katya; Low, Philip S

2012-08-28

The red cell membrane is stabilized by a spectrin/actin-based cortical cytoskeleton connected to the phospholipid bilayer via multiple protein bridges. By virtue of its interaction with ankyrin and adducin, the anion transporter, band 3 (AE1), contributes prominently to these bridges. In a previous study, we demonstrated that an exposed loop comprising residues 175-185 of the cytoplasmic domain of band 3 (cdB3) constitutes a critical docking site for ankyrin on band 3. In this paper, we demonstrate that an adjacent loop, comprising residues 63-73 of cdB3, is also essential for ankyrin binding. Data that support this hypothesis include the following. (1) Deletion or mutation of residues within the latter loop abrogates ankyrin binding without affecting cdB3 structure or its other functions. (2) Association of cdB3 with ankyrin is inhibited by competition with the loop peptide. (3) Resealing of the loop peptide into erythrocyte ghosts alters membrane morphology and stability. To characterize cdB3-ankyrin interaction further, we identified their interfacial contact sites using molecular docking software and the crystal structures of D(3)D(4)-ankyrin and cdB3. The best fit for the interaction reveals multiple salt bridges and hydrophobic contacts between the two proteins. The most important ion pair interactions are (i) cdB3 K69-ankyrin E645, (ii) cdB3 E72-ankyrin K611, and (iii) cdB3 D183-ankyrin N601 and Q634. Mutation of these four residues on ankyrin yielded an ankyrin with a native CD spectrum but little or no affinity for cdB3. These data define the docking interface between cdB3 and ankyrin in greater detail.

Identification of contact sites between ankyrin and band 3 in the human erythrocyte membrane

PubMed Central

Grey, Jesse L.; Kodippili, Gayani C.; Simon, Katya; Low, Philip S.

2012-01-01

The red cell membrane is stabilized by a spectrin/actin-based cortical cytoskeleton connected to the phospholipid-bilayer via multiple protein bridges. By virtue of its interaction with ankyrin and adducin, the anion transporter, band 3 (AE1), contributes prominently to these bridges. In a previous study, we demonstrated that an exposed loop comprising residues 175–185 of the cytoplasmic domain of band 3 (cdB3) constitutes a critical docking site for ankyrin on band 3. In this paper, we demonstrate that an adjacent loop, comprising residues 63–73 of cdB3, is also essential for ankyrin binding. Data in support of this hypothesis include: 1) deletion or mutation of residues within the latter loop abrogates ankyrin binding without affecting cdB3 structure or its other functions, 2) association of cdB3 with ankyrin is inhibited by competition with the loop peptide, and 3) resealing of the loop peptide into erythrocyte ghosts alters membrane morphology and stability. To characterize cdB3-ankyrin interaction further, we identified their interfacial contact sites using molecular docking software and the crystal structures of D3D4-ankyrin and cdB3. The best fit for the interaction reveals multiple salt bridges and hydrophobic contacts between the two proteins. The most important ion pair interactions are: i) cdB3 K69 to ankyrin E645, ii) cdB3 E72 to ankyrin K611, and iii) cdB3 D183 to ankyrin N601 and Q634. Mutation of the above four residues on ankyrin yielded an ankyrin with native CD spectrum, but little or no affinity for cdB3. These data define the docking interface between cdB3 and ankyrin in greater detail. PMID:22861190

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

Effects of soil acidification and liming on the phytoavailability of cadmium in paddy soils of central subtropical China.

PubMed

Zhu, Hanhua; Chen, Cheng; Xu, Chao; Zhu, Qihong; Huang, Daoyou

2016-12-01

Intensive and paired soil and rice grain survey and multiple-field liming experiments were conducted to assess soil acidification in the past 30 years, quantify the relationships of Cd phytoavailability with soil acidity, and determine efficacies of liming on soil acidity and Cd phytoavailability in paddy soils of central subtropical China at a regional scale. Soil pH, total and extractable Cd (Cd tot and Cd ext ), rice grain Cd were determined, and all measured data were analyzed separately in groups of 0.1 pH units intervals. Paddy soil pH averagely declined at 0.031 unit yr -1 between 1980s and 2014 (P < 0.01). Piecewise means of log Cd transfer ratio kept around -0.062 between soil pH 4.0 and 5.5 and around -1.31 between pH 6.9 and 7.3, whereas linearly decreased by a factor of 0.76 with pH 5.5-6.9, and by a factor of 1.38 with pH 7.3-8.2 (P < 0.01), respectively. Similar responses to soil pH were observed for soil Cd ext to Cd tot ratio. However, the former exhibited a lag effect to soil acidification in the acidic soils and a leading effect in alkaline soils. Liming increased soil pH by 0.50 units, and decreased rice grain Cd by 35.3% and log Cd transfer ratio by a factor of 0.76 (P < 0.01). The piecewise relationship based on the survey precisely predicted the changes in Cd transfer ratio across the multiple-field liming experiments. In conclusion, soil acidification occurred and accelerated in the past 30 years, and piecewise-linearly increased Cd phytoavailability of paddy soils in central subtropical China. Mitigating soil acidification, i.e. liming, should be preferentially implemented to minimize Cd phytoavailability. Copyright © 2016 Elsevier Ltd. All rights reserved.

Energy dispersive X-ray fluorescence determination of cadmium in uranium matrix using Cd Kα line excited by continuum

NASA Astrophysics Data System (ADS)

Dhara, Sangita; Misra, N. L.; Aggarwal, S. K.; Venugopal, V.

2010-06-01

An energy dispersive X-ray fluorescence method for determination of cadmium (Cd) in uranium (U) matrix using continuum source of excitation was developed. Calibration and sample solutions of cadmium, with and without uranium were prepared by mixing different volumes of standard solutions of cadmium and uranyl nitrate, both prepared in suprapure nitric acid. The concentration of Cd in calibration solutions and samples was in the range of 6 to 90 µg/mL whereas the concentration of Cd with respect to U ranged from 90 to 700 µg/g of U. From the calibration solutions and samples containing uranium, the major matrix uranium was selectively extracted using 30% tri-n-butyl phosphate in dodecane. Fixed volumes (1.5 mL) of aqueous phases thus obtained were taken directly in specially designed in-house fabricated leak proof Perspex sample cells for the energy dispersive X-ray fluorescence measurements and calibration plots were made by plotting Cd Kα intensity against respective Cd concentration. For the calibration solutions not having uranium, the energy dispersive X-ray fluorescence spectra were measured without any extraction and Cd calibration plots were made accordingly. The results obtained showed a precision of 2% (1 σ) and the results deviated from the expected values by < 4% on average.

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

Pretargeted Radioimmunotherapy Using Anti-CD45 Monoclonal Antibodies to Deliver Radiation to Murine Hematolymphoid Tissues and Human Myeloid Leukemia

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

Pagel, John M.; Matthews, Dana C.; Kenoyer, Aimee L.

2009-01-01

The efficacy of radioimmunotherapy (RIT) for treatment of patients with hematological malignancies frequently fails because of disease recurrence. We therefore conducted pretargeted RIT studies to augment the efficacy in mice of therapy using a pretargeted anti-human (h)CD45 antibody (Ab)-streptavidin (SA) conjugate followed by delivery of a biotinylated clearing agent and radiolabeled-DOTA-biotin. Tumor-to-blood ratios at 24 hours were 20:1 using pretargeted anti-hCD45 RIT and <1:1 with conventional RIT. In vivo imaging studies confirmed that the pretargeted RIT approach provided high-contrast tumor images with minimal blood-pool activity, whereas directly-labeled anti-hCD45 Ab produced distinct tumor images but the blood pool retained a largemore » amount of labeled antibody for a prolonged time. Therapy experiments demonstrated that 90Y-DOTA-biotin significantly prolonged survival of mice treated pretargeted with anti-hCD45 Ab-SA compared to mice treated with conventional RIT using 90Y-labeled anti-hCD45 Ab at the maximally tolerated dose (400 µCi). Since human CD45 antigens are confined to xenograft tumor cells in this model, and all murine tissues are devoid of hCD45 and will not bind anti-hCD45 Ab, we also compared one-step and pretargeted RIT using an anti-murine (m)CD45 Ab (A20 ) in a model where the target antigen is present on normal hematopoietic tissues. After 24 hours, 27.3 ± 2.8% of the injected dose of radionuclide was delivered per gram (% ID/g) of lymph node using 131I-A20-Ab compared with 40.0 ± 5.4% ID/g for pretargeted 111In-DOTA-biotin (p value). These data suggest that multi-step pretargeted methods for delivering RIT are superior to conventional RIT when targeting CD45 for the treatment of leukemia and may allow for the intensification of therapy, while minimizing toxicities.« less

Effect of CdS nanocrystals on charge transport mechanism in poly(3-hexylthiophene)

NASA Astrophysics Data System (ADS)

Khan, Mohd Taukeer; Almohammedi, Abdullah

2017-08-01

The present manuscript demonstrates the optical and electrical characteristics of poly(3-hexylthiophene) (P3HT) and cadmium sulphide (CdS) hybrid nanocomposites. Optical results suggest that there is a formation of charge transfer complex (CTC) between host P3HT and guest CdS nanocrystals (NCs). Electrical properties of P3HT and P3HT-CdS thin films have been studied in hole only device configurations at different temperatures (290 K-150 K), and results were analysed by the space charge limited conduction mechanism. Density of traps and characteristic trap energy increase on incorporation of inorganic NCs in the polymer matrix, which might be due to the additional favourable energy states created by CdS NCs in the band gap of P3HT. These additional trap states assist charge carriers to move quicker which results in enhancement of hole mobility from 7 × 10-6 to 5.5 × 10-5 cm2/V s in nanocomposites. These results suggest that the P3HT-CdS hybrid system has desirable optical and electrical properties for its applications to photovoltaics devices.

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.

CD4+CD28null T Cells are related to previous cytomegalovirus infection but not to accelerated atherosclerosis in ANCA-associated vasculitis.

PubMed

Slot, Marjan C; Kroon, Abraham A; Damoiseaux, Jan G M C; Theunissen, Ruud; Houben, Alfons J H M; de Leeuw, Peter W; Tervaert, Jan Willem Cohen

2017-05-01

Previous studies have suggested an increased risk for cardiovascular events in antineutrophil cytoplasmic antibodies (ANCA)-associated vasculitis (AAV). We analyzed the presence of atherosclerotic damage in patients with AAV in relation to the presence of CD4 + CD28 null T cells and antibodies against cytomegalovirus (CMV) and human Heat-Shock Protein 60 (hHSP60). In this cross-sectional study, patients with inactive AAV were compared with healthy controls (HC). Carotid intima-media thickness (IMT) and aortic pulse-wave velocity (PWV) were measured. In addition, CD4 + CD28 null T cells, anti-CMV, and anti-hHSP60 levels were determined. Forty patients with AAV were included. Patients' spouses were recruited as HC (N = 38). CD4 + CD28 null T cells are present in patients with AAV in a higher percentage (median 3.1, range 0.01-85) than in HC (0.28, 0-36, P < 0.0001). No significant difference in IMT (mm) between patients and controls was detected (mean 0.77 ± standard deviation 0.15 and 0.73 ± 0.11, respectively, P = 0.20). PWV standardized for MAP was increased in AAV patients (9.80 ± 2.50 m/s, compared to 8.72 ± 1.68 in HC, P = 0.04). There was a strong association between a previous CMV infection and the presence and percentage of CD4 + CD28 null T cells (0.33 vs 13.8, P < 0.001). There was no relationship between CD4 + CD28 null T cells and/or a previous CMV infection and IMT or PWV. There was no relation between anti-hHSP60 and CD4 + CD28 null T cells. Increased PWV values suggest atherosclerotic damage in patients with AAV. Plaque size, as determined by IMT, did not differ. CD4 + CD28 null T cells are increased in AAV and related to the previous CMV infection.

Leaching of cadmium and tellurium from cadmium telluride (CdTe) thin-film solar panels under simulated landfill conditions

PubMed Central

Ramos-Ruiz, Adriana; Wilkening, Jean V.; Field, James A.; Sierra-Alvarez, Reyes

2017-01-01

A crushed non-encapsulated CdTe thin-film solar cell was subjected to two standardized batch leaching tests (i.e., Toxicity Characteristic Leaching Procedure (TCLP) and California Waste Extraction Test (WET)) and to a continuous-flow column test to assess cadmium (Cd) and tellurium (Te) dissolution under conditions simulating the acidic- and the methanogenic phases of municipal solid waste landfills. Low levels of Cd and Te were solubilized in both batch leaching tests (<8.2% and <3.6% of added Cd and Te, respectively). On the other hand, over the course of 30 days, 73% of the Cd and 21% of the Te were released to the synthetic leachate of a continuous-flow column simulating the acidic landfill phase. The dissolved Cd concentration was 3.24-fold higher than the TCLP limit (1 mg L-1), and 650-fold higher than the maximum contaminant level established by the US-EPA for this metal in drinking water (0.005 mg L-1). In contrast, the release of Cd and Te to the effluent of the continuous-flow column simulating the methanogenic phase of a landfill was negligible. The remarkable difference in the leaching behavior of CdTe in the columns is related to different aqueous pH and redox conditions promoted by the microbial communities in the columns, and is in agreement with thermodynamic predictions. PMID:28472709

Hydrous ferric oxide: evaluation of Cd-HFO surface complexation models combining Cd(K) EXAFS data, potentiometric titration results, and surface site structures identified from mineralogical knowledge.

PubMed

Spadini, Lorenzo; Schindler, Paul W; Charlet, Laurent; Manceau, Alain; Vala Ragnarsdottir, K

2003-10-01

The surface properties of ferrihydrite were studied by combining wet chemical data, Cd(K) EXAFS data, and a surface structure and protonation model of the ferrihydrite surface. Acid-base titration experiments and Cd(II)-ferrihydrite sorption experiments were performed within 3<-log[H(+)]<10.5 and 0.5<[Cd(t)]<12 mM in 0.3 M NaClO(4) at 25 degrees C, where [Cd(t)] refers to total Cd concentration. Measurements at -5.5triple bond Fe-OH(-1/2),logk((int))=-8.29, assuming the existence of a unique intrinsic microscopic constant, logk((int)), and consequently the existence of a single significant type of acid-base reactive functional groups. The surface structure model indicates that these groups are terminal water groups. The Cd(II) data were modeled assuming the existence of a single reactive site. The model fits the data set at low Cd(II) concentration and up to 50% surface coverage. At high coverage more Cd(II) ions than predicted are adsorbed, which is indicative of the existence of a second type of site of lower affinity. This agrees with the surface structure and protonation model developed, which indicates comparable concentrations of high- and low-affinity sites. The model further shows that for each class of low- and high-affinity sites there exists a variety of corresponding Cd surface complex structure, depending on the model crystal faces on which the complexes develop. Generally, high-affinity surface structures have surface coordinations of 3 and 4, as compared to 1 and 2 for low-affinity surface structures.

Hepatitis B virus PreS1 facilitates hepatocellular carcinoma development by promoting appearance and self-renewal of liver cancer stem cells.

PubMed

Liu, Zhixin; Dai, Xuechen; Wang, Tianci; Zhang, Chengcheng; Zhang, Wenjun; Zhang, Wei; Zhang, Qi; Wu, Kailang; Liu, Fang; Liu, Yingle; Wu, Jianguo

2017-08-01

Hepatitis B virus (HBV) is a major etiologic agent of hepatocellular carcinoma (HCC). However, the molecular mechanism by which HBV infection contributes to HCC development is not fully understood. Here, we initially showed that HBV stimulates the production of cancer stem cells (CSCs)-related markers (CD133, CD117 and CD90) and CSCs-related genes (Klf4, Sox2, Nanog, c-Myc and Oct4) and facilitates the self-renewal of CSCs in human hepatoma cells. Cellular and clinical studies revealed that HBV facilitates hepatoma cell growth and migration, enhances white blood cell (WBC) production in the sera of patients, stimulates CD133 and CD117 expression in HCC tissues, and promotes the CSCs generation of human hepatoma cells and clinical cancer tissues. Detailed studies revealed that PreS1 protein of HBV is required for HBV-mediated CSCs generation. PreS1 activates CD133, CD117 and CD90 expression in normal hepatocyte derived cell line (L02) and human hepatoma cell line (HepG2 and Huh-7); facilitates L02 cells migration, growth and sphere formation; and finally enhances the abilities of L02 cells and HepG2 cells to induce tumorigeneses in nude mice. Thus, PreS1 acts as a new oncoprotein to play a key role in the appearance and self-renewal of CSCs during HCC development. Copyright © 2017 Elsevier B.V. All rights reserved.

Using a modification of the Clavien-Dindo system accounting for readmissions and multiple interventions: defining quality for pancreaticoduodenectomy.

PubMed

Baker, Marshall S; Sherman, Karen L; Stocker, Susan J; Hayman, Amanda V; Bentrem, David J; Prinz, Richard A; Talamonti, Mark S

2014-09-01

The Clavien-Dindo system (CD) does not change the grade assigned a complication when multiple readmissions or interventions are required to manage a complication. We apply a modification of CD accounting for readmissions and interventions to pancreaticoduodenectomy (PD). PDs done between 1999 and 2009 were reviewed. CD grade IIIa complications requiring more than one intervention and II and IIIa complications requiring significantly prolonged lengths of stay including all 90-day readmissions were classified severe-adverse-postoperative-outcomes (SAPO). CD IIIb, IV, and V complications were also classified SAPOs. All other complications were considered minor-adverse-postoperative-outcomes (MAPO). Four-hundred forty three of 490 PD patients (90.4%) had either no complication or a complication of low to moderate CD grade (I, II, IIIa). When reclassified by the new metric, 92 patient-outcomes (19%) were upgraded from CD II or IIIa to SAPO. One-hundred thirty nine patients (28.4%) had a SAPO. Multivariable regression identified age >75 years, pylorus preservation and operative blood loss >1,500 ml as predictors of SAPO. Age was not associated with poor outcome using the unmodified CD system. Established systems may under-grade the severity of some complications following PD. We define a procedure-specific modification of CD accounting for readmissions and multiple interventions. Using this modification, advanced age, pylorus preservation, and significant blood loss are associated with poor outcome. © 2014 Wiley Periodicals, Inc.