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Sample records for cells express markers

  1. Expression of Stem Cell Markers in the Human Fetal Kidney

    PubMed Central

    Metsuyanim, Sally; Harari-Steinberg, Orit; Buzhor, Ella; Omer, Dorit; Pode-Shakked, Naomi; Ben-Hur, Herzl; Halperin, Reuvit; Schneider, David; Dekel, Benjamin

    2009-01-01

    In the human fetal kidney (HFK) self-renewing stem cells residing in the metanephric mesenchyme (MM)/blastema are induced to form all cell types of the nephron till 34th week of gestation. Definition of useful markers is crucial for the identification of HFK stem cells. Because wilms' tumor, a pediatric renal cancer, initiates from retention of renal stem cells, we hypothesized that surface antigens previously up-regulated in microarrays of both HFK and blastema-enriched stem-like wilms' tumor xenografts (NCAM, ACVRIIB, DLK1/PREF, GPR39, FZD7, FZD2, NTRK2) are likely to be relevant markers. Comprehensive profiling of these putative and of additional stem cell markers (CD34, CD133, c-Kit, CD90, CD105, CD24) in mid-gestation HFK was performed using immunostaining and FACS in conjunction with EpCAM, an epithelial surface marker that is absent from the MM and increases along nephron differentiation and hence can be separated into negative, dim or bright fractions. No marker was specifically localized to the MM. Nevertheless, FZD7 and NTRK2 were preferentially localized to the MM and emerging tubules (<10% of HFK cells) and were mostly present within the EpCAMneg and EpCAMdim fractions, indicating putative stem/progenitor markers. In contrast, single markers such as CD24 and CD133 as well as double-positive CD24+CD133+ cells comprise >50% of HFK cells and predominantly co-express EpCAMbright, indicating they are mostly markers of differentiation. Furthermore, localization of NCAM exclusively in the MM and in its nephron progenitor derivatives but also in stroma and the expression pattern of significantly elevated renal stem/progenitor genes Six2, Wt1, Cited1, and Sall1 in NCAM+EpCAM- and to a lesser extent in NCAM+EpCAM+ fractions confirmed regional identity of cells and assisted us in pinpointing the presence of subpopulations that are putative MM-derived progenitor cells (NCAM+EpCAM+FZD7+), MM stem cells (NCAM+EpCAM-FZD7+) or both (NCAM+FZD7+). These results and

  2. Expression of Stem Cell Markers in Primo Vessel of Rat

    PubMed Central

    Park, Eun Seok; Lee, Jeong Hoon; Kim, Won Jin; Heo, Jinbeom; Shin, Dong Myung; Leem, Chae Hun

    2013-01-01

    Accumulating line of evidence support that adult tissues contain a rare population of pluripotent stem cells (PSCs), which differentiate into all types of cells in our body. Bonghan microcell (primo microcells (PMCs)) discovered in 1960s was reported to have a pluripotency like a stem cell in vivo as well as in vitro condition. Here, we describe the detailed morphology and molecular features of PMCs. PMCs reside in Bonghan duct (primo vessel (PV)) reported as a corresponding structure of acupuncture points and meridian system. We found that PMCs were frequently observed in the liver surface of the rat between 300 g and 400 g from April to June, suggesting that the their detection frequency depends on the weight, the season, and the organ of rat. As reported, PMCs freshly isolated from PVs were spherical ~1-2 μm microsized cells. In contrast, a unique bithread or budding-shaped PMCs emerged during tissue culture around 8 days. RT-PCR analysis demonstrated that PVs-derived cells express the Oct4, the most important PSCs gene, in addition to several PSCs markers (Sox2, Stella, Rex1, and Klf4). Thus, we for the first time provide the evidence about Oct4-expressing stem-like characteristics for cells resident in PVs, a possible novel stem cell enriched niche. PMID:23983780

  3. Expression of stem cell markers in primo vessel of rat.

    PubMed

    Park, Eun Seok; Lee, Jeong Hoon; Kim, Won Jin; Heo, Jinbeom; Shin, Dong Myung; Leem, Chae Hun

    2013-01-01

    Accumulating line of evidence support that adult tissues contain a rare population of pluripotent stem cells (PSCs), which differentiate into all types of cells in our body. Bonghan microcell (primo microcells (PMCs)) discovered in 1960s was reported to have a pluripotency like a stem cell in vivo as well as in vitro condition. Here, we describe the detailed morphology and molecular features of PMCs. PMCs reside in Bonghan duct (primo vessel (PV)) reported as a corresponding structure of acupuncture points and meridian system. We found that PMCs were frequently observed in the liver surface of the rat between 300 g and 400 g from April to June, suggesting that the their detection frequency depends on the weight, the season, and the organ of rat. As reported, PMCs freshly isolated from PVs were spherical ~1-2  μ m microsized cells. In contrast, a unique bithread or budding-shaped PMCs emerged during tissue culture around 8 days. RT-PCR analysis demonstrated that PVs-derived cells express the Oct4, the most important PSCs gene, in addition to several PSCs markers (Sox2, Stella, Rex1, and Klf4). Thus, we for the first time provide the evidence about Oct4-expressing stem-like characteristics for cells resident in PVs, a possible novel stem cell enriched niche.

  4. Objective analysis of cancer stem cell marker expression using immunohistochemistry.

    PubMed

    Miller, T J; McCoy, M J; Hemmings, C; Bulsara, M K; Iacopetta, B; Platell, C F

    2017-01-01

    Analysis of immunohistochemical expression is often a subjective and semiquantitative process that can lead to the inconsistent reporting of results. To assess the effect that region selection and quantification method have on results, five different cancer stem cell markers were used in this study to compare tissue scoring with digital analysis methods that used three different tissue annotation methods. Samples of tumour and normal mucosa were used from 10 consecutive stage II colon cancer patients and stained for the putative cancer stem cell markers ALDH1, CD44v6, CD133, Lgr5 and SOX2. Tissue scoring was found to have considerably different results to digital analysis with the three different digital methods harbouring concordant results overall. However, SOX2 on normal tissue and CD133 on tumour and normal tissue produced discordant results which could be attributed to the different regions of tissue that were analysed. It is important that quantification method and selection of analysis areas are considered as part of study design to ensure that reproducible and consistent results are reported in the literature.

  5. Inflammation increases cells expressing ZSCAN4 and progenitor cell markers in the adult pancreas

    PubMed Central

    Azuma, Sakiko; Yokoyama, Yukihiro; Yamamoto, Akiko; Kyokane, Kazuhiro; Niida, Shumpei; Ishiguro, Hiroshi; Ko, Minoru S. H.

    2013-01-01

    We have recently identified the zinc finger and SCAN domain containing 4 (Zscan4), which is transiently expressed and regulates telomere elongation and genome stability in mouse embryonic stem (ES) cells. The aim of this study was to examine the expression of ZSCAN4 in the adult pancreas and elucidate the role of ZSCAN4 in tissue inflammation and subsequent regeneration. The expression of ZSCAN4 and other progenitor or differentiated cell markers in the human pancreas was immunohistochemically examined. Pancreas sections of alcoholic or autoimmune pancreatitis patients before and under maintenance corticosteroid treatment were used in this study. In the adult human pancreas a small number of ZSCAN4-positive (ZSCAN4+) cells are present among cells located in the islets of Langerhans, acini, ducts, and oval-shaped cells. These cells not only express differentiated cell markers for each compartment of the pancreas but also express other tissue stem/progenitor cell markers. Furthermore, the number of ZSCAN4+ cells dramatically increased in patients with chronic pancreatitis, especially in the pancreatic tissues of autoimmune pancreatitis actively regenerating under corticosteroid treatment. Interestingly, a number of ZSCAN4+ cells in the pancreas of autoimmune pancreatitis returned to the basal level after 1 yr of maintenance corticosteroid treatment. In conclusion, coexpression of progenitor cell markers and differentiated cell markers with ZSCAN4 in each compartment of the pancreas may indicate the presence of facultative progenitors for both exocrine and endocrine cells in the adult pancreas. PMID:23599043

  6. Immortalized epithelial cells derived from human colon biopsies express stem cell markers and differentiate in vitro.

    PubMed

    Roig, Andres I; Eskiocak, Ugur; Hight, Suzie K; Kim, Sang Bum; Delgado, Oliver; Souza, Rhonda F; Spechler, Stuart J; Wright, Woodring E; Shay, Jerry W

    2010-03-01

    Long-term propagation of human colonic epithelial cells (HCEC) of adult origin has been a challenge; currently used HCEC lines are of malignant origin and/or contain multiple cytogenetic changes. We sought to immortalize human colon biopsy-derived cells expressing stem cell markers and retaining multilineage epithelial differentiation capability. We isolated and cultured cells from biopsy samples of 2 patients undergoing routine screening colonoscopy. Cells were immortalized by expression of the nononcogenic proteins cyclin-dependent kinase 4 (Cdk4) and the catalytic component of human telomerase (hTERT) and maintained for more than 1 year in culture. The actively proliferating HCECs expressed the mesenchymal markers vimentin and alpha-smooth muscle actin. Upon growth arrest, cells assumed a cuboidal shape, decreased their mesenchymal features, and expressed markers of colonic epithelial cells such as cytokeratin 18, zonula occludens-1, mucins-1 and -2, antigen A33, and dipeptidyl peptidase 4. Immortalized cells expressed stem cell markers that included LGR5, BMI1, CD29, and CD44. When placed in Matrigel in the absence of a mesenchymal feeder layer, individual cells divided and formed self-organizing, cyst-like structures; a subset of cells exhibited mucin-2 or polarized villin staining. We established immortalized HCECs that are capable of self-renewal and multilineage differentiation. These cells should serve as valuable reagents for studying colon stem cell biology, differentiation, and pathogenesis. Copyright 2010 AGA Institute. Published by Elsevier Inc. All rights reserved.

  7. Shared Cell Surface Marker Expression in Mesenchymal Stem Cells and Adult Sarcomas

    PubMed Central

    Wirths, Stefan; Malenke, Elke; Kluba, Torsten; Rieger, Simone; Müller, Martin R.; Schleicher, Sabine; Hann von Weyhern, Claus; Nagl, Florian; Fend, Falko; Vogel, Wichard; Mayer, Frank; Kanz, Lothar; Bühring, Hans-Jörg

    2013-01-01

    Advanced adult soft-tissue sarcomas (STSs) are rare tumors with a dismal prognosis and limited systemic treatment options. STSs may originate from mesenchymal stem cells (MSCs); the latter have mainly been isolated from adult bone marrow as plastic-adherent cells with differentiation capacity into mesenchymal tissues. Recently, a panel of antibodies has been established that allows for the prospective isolation of primary MSCs with high selectivity. Similar to cancer stem cells in other malignancies, sarcoma stem cells may bear immunophenotypic similarity with the corresponding precursor, that is, MSCs. We therefore set out to establish the expression pattern of MSC markers in sarcoma cell lines and primary tumor samples by flow cytometry. In addition, fibroblasts from different sources were examined. The results document a significant amount of MSC markers shared by sarcoma cells. The expression pattern includes uniformly expressed markers, as well as MSC markers that only stained subpopulations of sarcoma cells. Expression of W5C5, W8B2 (tissue nonspecific alkaline phosphatase [TNAP]), CD344 (frizzled-4), and CD271 marked subpopulations displaying increased proliferation potential. Moreover, CD271+ cells displayed in vitro doxorubicin resistance and an increased capacity to form spheres under serum-free conditions. Interestingly, another set of antigens, including the bona fide progenitor cell markers CD117 and CD133, were not expressed. Comparative expression patterns of novel MSC markers in sarcoma cells, as well as fibroblasts and MSCs, are presented. Our data suggest a hierarchical cytoarchitecture of the most common adult type sarcomas and introduce W5C5, TNAP, CD344, and CD271 as potential sarcoma progenitor cell markers. PMID:23283492

  8. Human amniotic epithelial cells differentiate into cells expressing germ cell specific markers when cultured in medium containing serum substitute supplement

    PubMed Central

    2012-01-01

    Background Human amniotic epithelial cells (hAECs) maintain the plasticity of pregastrulation embryonic cells, having the potential to differentiate into all three germ layers. The potential of these cells to differentiate into cells expressing germ cell specific markers has never been described before. Methods In the present study, hAECs were cultured in medium containing serum substitute supplement (SSS). Gene and protein expression of germ cell and oocyte specific markers was assessed by reverse transcription-polymerase chain reaction (RT-PCR), immunofluorescence staining and flow activated cell sorter analysis (FACS) in hAECs at different time points during the differentiation into cells expressing germ cell specific markers. Results When cultured with SSS, already at passage 1, hAECs start to express the germ cell specific genes C-KIT, DAZL, VASA and ZP3 and at passage 5 large round cells, resembling oocytes, appeared. The cells express the germ cell specific marker DAZL, the oocyte specific markers GDF9 and ZP3 and the meiosis specific markers DMC1 and SCP3 at the protein level. Conclusions From our preliminary results we can conclude that hAECs have the potential to differentiate into cells expressing germ cell specific markers. PMID:23241213

  9. Expression of epithelial markers by human umbilical cord stem cells. A topographical analysis.

    PubMed

    Garzón, I; Alfonso-Rodríguez, C A; Martínez-Gómez, C; Carriel, V; Martin-Piedra, M A; Fernández-Valadés, R; Sánchez-Quevedo, M C; Alaminos, M

    2014-12-01

    Human umbilical cord stem cells have inherent differentiation capabilities and potential usefulness in regenerative medicine. However, the epithelial differentiation capability and the heterogeneity of these cells have not been fully explored to the date. We analyzed the expression of several undifferentiation and epithelial markers in cells located in situ in different zones of the umbilical cord -in situ analysis- and in primary ex vivo cell cultures of Wharton's jelly stem cells by microarray and immunofluorescence. Our results demonstrated that umbilical cord cells were heterogeneous and had intrinsic capability to express in situ stem cell markers, CD90 and CD105 and the epithelial markers cytokeratins 3, 4, 7, 8, 12, 13, 19, desmoplakin and zonula occludens 1 as determined by microarray and immunofluorescence, and most of these markers remained expressed after transferring the cells from the in situ to the ex vivo cell culture conditions. However, important differences were detected among some cell types in the umbilical cord, with subvascular zone cells showing less expression of stem cell markers and cells in Wharton's jelly and the amnioblastic zones showing the highest expression of stem cells and epithelial markers. These results suggest that umbilical cord mesenchymal cells have intrinsic potential to express relevant epithelial markers, and support the idea that they could be used as alternative cell sources for epithelial tissue engineering. Copyright © 2014 Elsevier Ltd. All rights reserved.

  10. Expression of Stem Cell Markers in Preinvasive Tubal Lesions of Ovarian Carcinoma

    PubMed Central

    Chene, G.; Ouellet, V.; Rahimi, K.; Barres, V.; Meunier, L.; De Ladurantaye, M.; Provencher, D.; Mes-Masson, A. M.

    2015-01-01

    In order to better understand the ovarian serous carcinogenic process with tubal origin, we investigated the expression of stem cell markers in premalignant tubal lesions (serous tubal intraepithelial carcinoma or STIC). We found an increased stem cell marker density in the normal fallopian tube followed by a high CD117 and a low ALDH and CD44 expression in STICs raising the question of the role of the stem cell markers in the serous carcinogenic process. PMID:26504831

  11. High Throughput Gene Expression Analysis Identifies Reliable Expression Markers of Human Corneal Endothelial Cells

    PubMed Central

    Chng, Zhenzhi; Peh, Gary S. L.; Herath, Wishva B.; Cheng, Terence Y. D.; Ang, Heng-Pei; Toh, Kah-Peng; Robson, Paul; Mehta, Jodhbir S.; Colman, Alan

    2013-01-01

    Considerable interest has been generated for the development of suitable corneal endothelial graft alternatives through cell-tissue engineering, which can potentially alleviate the shortage of corneal transplant material. The advent of less invasive suture-less key-hole surgery options such as Descemet’s Stripping Endothelial Keratoplasty (DSEK) and Descemet’s Membrane Endothelial Keratoplasty (DMEK), which involve transplantation of solely the endothelial layer instead of full thickness cornea, provide further impetus for the development of alternative endothelial grafts for clinical applications. A major challenge for this endeavor is the lack of specific markers for this cell type. To identify genes that reliably mark corneal endothelial cells (CECs) in vivo and in vitro, we performed RNA-sequencing on freshly isolated human CECs (from both young and old donors), CEC cultures, and corneal stroma. Gene expression of these corneal cell types was also compared to that of other human tissue types. Based on high throughput comparative gene expression analysis, we identified a panel of markers that are: i) highly expressed in CECs from both young donors and old donors; ii) expressed in CECs in vivo and in vitro; and iii) not expressed in corneal stroma keratocytes and the activated corneal stroma fibroblasts. These were SLC4A11, COL8A2 and CYYR1. The use of this panel of genes in combination reliably ascertains the identity of the CEC cell type. PMID:23844023

  12. Stem cell marker nestin is expressed in plasma cells of multiple myeloma patients.

    PubMed

    Svachova, H; Pour, L; Sana, J; Kovarova, L; Raja, K R Muthu; Hajek, R

    2011-08-01

    Nestin is considered to be a characteristic marker of multipotent proliferative precursors found in some embryonic and fetal tissues. Its expression might be a suitable diagnostic and prognostic indicator of malignancy and a potential marker of cancer stem cells in solid tumors. Unexpectedly, nestin protein was detected in mature CD138(+)CD38(+) plasma cells of multiple myeloma patients and statistical analysis confirmed significant differences between myeloma patients and control group without hematological malignancy. Our results represent the first evidence of nestin expression in multiple myeloma. Further studies are required to elucidate the role of this protein in multiple myeloma. Copyright © 2011 Elsevier Ltd. All rights reserved.

  13. Identification of novel stem cell markers using gap analysis of gene expression data

    PubMed Central

    Krzyzanowski, Paul M; Andrade-Navarro, Miguel A

    2007-01-01

    We describe a method for detecting marker genes in large heterogeneous collections of gene expression data. Markers are identified and characterized by the existence of demarcations in their expression values across the whole dataset, which suggest the presence of groupings of samples. We apply this method to DNA microarray data generated from 83 mouse stem cell related samples and describe 426 selected markers associated with differentiation to establish principles of stem cell evolution. PMID:17875203

  14. Quantification of cells expressing mesenchymal stem cell markers in healthy and osteoarthritic synovial membranes.

    PubMed

    Hermida-Gómez, Tamara; Fuentes-Boquete, Isaac; Gimeno-Longas, Maria José; Muiños-López, Emma; Díaz-Prado, Silvia; de Toro, Francisco Javier; Blanco, Francisco Javier

    2011-02-01

    To quantify cells expressing mesenchymal stem cell (MSC) markers in synovial membranes from human osteoarthritic (OA) and healthy joints. Synovial membranes from OA and healthy joints were digested with collagenase and the isolated cells were cultured. Synovial membrane-derived cells were phenotypically characterized for differentiation experiments using flow cytometry to detect the expression of mesenchymal markers (CD29, CD44, CD73, CD90, CD105, CD117, CD166, and STRO-1) and hematopoietic markers (CD34 and CD45). Chondrogenesis was assessed by staining for proteoglycans and collagen type II, adipogenesis by using a stain for lipids, and osteogenesis by detecting calcium deposits. Coexpression of CD44, CD73, CD90, and CD105 was determined using immunofluorescence. Cells expressing MSC markers were diffusely distributed in OA synovial membranes; in healthy synovial membrane these cells were localized in the subintimal zone. More numerous MSC markers in OA synovial membranes were observed in cells also expressing the CD90 antigen. FACS analysis showed that more than 90% of OA synovial membrane-derived cells were positive for CD44, CD73, and CD90, and negative for CD34 and CD45. OA synovial membrane-derived cells were also positive for CD29 (85.23%), CD117 (72.35%), CD105 (45.5%), and STRO-1 (49.46%). Micropellet analyses showed that the culture of cells with transforming growth factor-ß3 stimulated proteoglycan and collagen type II synthesis. Synovial membranes from patients with OA contain more cells positive for CD44, CD90, and CD105 antigens than those from joints with undamaged cartilage.

  15. Expression pattern of embryonic stem cell markers in DFAT cells and ADSCs.

    PubMed

    Gao, Qian; Zhao, Lili; Song, Ziyi; Yang, Gongshe

    2012-05-01

    Mature adipocytes can revert to a more primitive phenotype and gain cell proliferative ability under the condition of ceiling method, named dedifferentiated fat cells (DFAT cells). These cells exhibit multilineage potential as adipose tissue-derived stromal cells (ADSCs). However, the stem molecular signature of DFAT cells and the difference distinct from ADSCs are still not sure. To study the molecular signature of DFAT cells better, highly purified mature adipocytes were obtained from rats and the purity was more than 98%, and about 98.6% were monocytes. These mature adipocytes dedifferentiated into fibroblast-like cells spontaneously by the ceiling culture method, these cells proliferated rapidly in vitro, grew in the same direction and formed vertex, and expressed extensively embryonic stem cell markers such as Oct4, Sox2, c-Myc, and Nanog, surface antigen SSEA-1, CD105, and CD31, moreover, these cells possessed ALP and telomerase activity. The expression level was Oct4 1.3%, Sox2 1.3%, c-Myc 1.2%, Nanog 1.2%, CD105 0.6%, CD31 0.6% and SSEA-1 0.4%, respectively, which was lower than that in ADSCs, but the purity of DFAT cells was much higher than that of ADSCs. In conclusion, DFAT cells is a highly purified stem cell population, and expressed some embryonic stem cell markers like ADSCs, which seems to be a good candidate source of adult stem cells for the future cell replacement therapy.

  16. Heterogeneous lineage marker expression in naive embryonic stem cells is mostly due to spontaneous differentiation

    PubMed Central

    Nair, Gautham; Abranches, Elsa; Guedes, Ana M. V.; Henrique, Domingos; Raj, Arjun

    2015-01-01

    Populations of cultured mouse embryonic stem cells (ESCs) exhibit a subfraction of cells expressing uncharacteristically low levels of pluripotency markers such as Nanog. Yet, the extent to which individual Nanog-negative cells are differentiated, both from ESCs and from each other, remains unclear. Here, we show the transcriptome of Nanog-negative cells exhibits expression of classes of genes associated with differentiation that are not yet active in cells exposed to differentiation conditions for one day. Long non-coding RNAs, however, exhibit more changes in expression in the one-day-differentiated cells than in Nanog-negative cells. These results are consistent with the concept that Nanog-negative cells may contain subpopulations of both lineage-primed and differentiated cells. Single cell analysis showed that Nanog-negative cells display substantial and coherent heterogeneity in lineage marker expression in progressively nested subsets of cells exhibiting low levels of Nanog, then low levels of Oct4, and then a set of lineage markers, which express intensely in a small subset of these more differentiated cells. Our results suggest that the observed enrichment of lineage-specific marker gene expression in Nanog-negative cells is associated with spontaneous differentiation of a subset of these cells rather than the more random expression that may be associated with reversible lineage priming. PMID:26292941

  17. Adult vascular smooth muscle cells in culture express neural stem cell markers typical of resident multipotent vascular stem cells.

    PubMed

    Kennedy, Eimear; Mooney, Ciaran J; Hakimjavadi, Roya; Fitzpatrick, Emma; Guha, Shaunta; Collins, Laura E; Loscher, Christine E; Morrow, David; Redmond, Eileen M; Cahill, Paul A

    2014-10-01

    Differentiation of resident multipotent vascular stem cells (MVSCs) or de-differentiation of vascular smooth muscle cells (vSMCs) might be responsible for the SMC phenotype that plays a major role in vascular diseases such as arteriosclerosis and restenosis. We examined vSMCs from three different species (rat, murine and bovine) to establish whether they exhibit neural stem cell characteristics typical of MVSCs. We determined their SMC differentiation, neural stem cell marker expression and multipotency following induction in vitro by using immunocytochemistry, confocal microscopy, fluorescence-activated cell sorting analysis and quantitative real-time polymerase chain reaction. MVSCs isolated from rat aortic explants, enzymatically dispersed rat SMCs and rat bone-marrow-derived mesenchymal stem cells served as controls. Murine carotid artery lysates and primary rat aortic vSMCs were both myosin-heavy-chain-positive but weakly expressed the neural crest stem cell marker, Sox10. Each vSMC line examined expressed SMC differentiation markers (smooth muscle α-actin, myosin heavy chain and calponin), neural crest stem cell markers (Sox10(+), Sox17(+)) and a glia marker (S100β(+)). Serum deprivation significantly increased calponin and myosin heavy chain expression and decreased stem cell marker expression, when compared with serum-rich conditions. vSMCs did not differentiate to adipocytes or osteoblasts following adipogenic or osteogenic inductive stimulation, respectively, or respond to transforming growth factor-β1 or Notch following γ-secretase inhibition. Thus, vascular SMCs in culture express neural stem cell markers typical of MVSCs, concomitant with SMC differentiation markers, but do not retain their multipotency. The ultimate origin of these cells might have important implications for their use in investigations of vascular proliferative disease in vitro.

  18. Human mesenchymal stem cells express neuronal markers after osteogenic and adipogenic differentiation.

    PubMed

    Foudah, Dana; Redondo, Juliana; Caldara, Cristina; Carini, Fabrizio; Tredici, Giovanni; Miloso, Mariarosaria

    2013-06-01

    Mesenchymal stem cells (MSCs) are multipotent cells that are able to differentiate into mesodermal lineages (osteogenic, adipogenic, chondrogenic), but also towards non-mesodermal derivatives (e.g. neural cells). Recent in vitro studies revealed that, in the absence of any kind of differentiation stimuli, undifferentiated MSCs express neural differentiation markers, but the literature data do not all concur. Considering their promising therapeutic potential for neurodegenerative diseases, it is very important to expand our knowledge about this particular biological property of MSCs. In this study, we confirmed the spontaneous expression of neural markers (neuronal, glial and progenitor markers) by undifferentiated human MSCs (hMSCs) and in particular, we demonstrated that the neuronal markers βIII-tubulin and NeuN are expressed by a very high percentage of hMSCs, regardless of the number of culture passages and the culture conditions. Moreover, the neuronal markers βIII-tubulin and NeuN are still expressed by hMSCs after in vitro osteogenic and adipogenic differentiation. On the other hand, chondrogenically differentiated hMSCs are negative for these markers. Our findings suggest that the expression of neuronal markers could be common to a wide range of cellular types and not exclusive for neuronal lineages. Therefore, the expression of neuronal markers alone is not sufficient to demonstrate the differentiation of MSCs towards the neuronal phenotype. Functional properties analysis is also required.

  19. Hematopoietic stem cell markers are expressed by ductal plate and bile duct cells in developing human liver.

    PubMed

    Blakolmer, K; Jaskiewicz, K; Dunsford, H A; Robson, S C

    1995-06-01

    The identification of ductal plate cells as likely progenitors for bile duct epithelium and hepatocytes and their possible reappearance as oval cells in the regenerating liver have generated much interest in their pluripotential capacities. We have examined the distribution of three hematopoietic stem cell markers, c-kit, CD34, and CD33 in addition to laminin, the standard cytokeratin markers CAM 5.2, CK 18, and CK 7 and the oval cell marker OV-6 in fetal liver during various stages of development. Hematopoietic stem cell markers were expressed in ductal plate cells in a pattern similar to the early cytokeratin markers CAM 5.2 and CK 18. Cells stained strongly for these early cytokeratin markers until 22 weeks. Thereafter, the expression of these markers decreased while positivity for CK 7 increased. Bile duct cells showed a distribution of hematopoietic and cytokeratin markers resembling that of ductal plate cells. Both ductal plate cells and bile duct cells expressed OV-6 strongly throughout development. This study showed similarity between hepatic and bile duct precursors and bone marrow stem cells. The comparable distribution of markers in bile duct epithelium and ductal plate cells may imply fewer transitional stages between ductal plate cells and bile duct epithelium than between the putative stem cells and hepatocytes.

  20. Correlation of cell surface marker expression with African swine fever virus infection.

    PubMed

    Lithgow, Pamela; Takamatsu, Haru; Werling, Dirk; Dixon, Linda; Chapman, Dave

    2014-01-31

    The expression of surface markers on African swine fever virus (ASFV) infected cells was evaluated to assess their involvement in infection. Previous findings indicated CD163 expression was correlated with ASFV susceptibility. However, in this study the expression of porcine CD163 on cell lines did not increase the infection rate of these cells indicating other factors are likely to be important in determining susceptibility to infection. On adherent porcine bone marrow (pBM) cells the expression of CD45 was strongly correlated with infection. CD163 and CD203a expression correlated at intermediate levels with infection, indicating cells expressing these markers could become infected but were not preferentially infected by the virus. Most of the cells expressing MHCII were infected, indicating that they may be preferentially infected although expression of MHCII was not essential for infection and a large percentage of the infected cells were MHCII negative. CD16 showed a marked decrease in expression following infection and significantly lower levels of infected cells were shown to express CD16. Altogether these results suggest CD163 may be involved in ASFV infection but it may not be essential; the results also highlight the importance of other cell markers which requiring further investigation.

  1. Hexavalent chromium induces expression of mesenchymal and stem cell markers in renal epithelial cells

    PubMed Central

    Li, Wei‐Jen; Yang, Cheng‐Lin; Kuo, Ting‐Wei

    2015-01-01

    Cr(VI) causes severe kidney damage. The patient's renal function could gradually recover by spontaneous kidney regeneration. The molecular effect of Cr(VI) on recovery of kidney cells, however, has not been clearly elucidated. Here we show that Cr(VI) induces expression of mesenchymal and stem cell markers, cell markers, such as paxillin, vimentin, α‐SMA, nanog, and CD133 of HK‐2 cells. Moreover, Cr(VI) activates epithelial‐to‐mesenchymal transition (EMT). By revealing that levels of dihydrodiol dehydrogenase were promptly reduced following Cr(VI) challenge, our data suggested that DDH could be involved in a Cr(VI)‐related oxidation to generate massive reactive oxygen species and H2O2, and to create intracellular hypoxia, which then increased levels of SUMO‐1 activating enzyme subunit 2, and sumoylation of eukaryotic elongation factor‐2, to mediate the subsequent molecular and cellular responses, e.g., expression of mesenchymal and stem cell markers. Pretreatment with vitamin C reduced Cr(VI)‐related cellular effects. However, no evident effect was observed when vitamin C was added following Cr(VI) challenge. © 2015 The Authors. Molecular Carcinogenesis published by Wiley Periodicals, Inc. PMID:25620490

  2. Immortalized bovine mammary epithelial cells express stem cell markers and differentiate in vitro.

    PubMed

    Hu, Han; Zheng, Nan; Gao, Haina; Dai, Wenting; Zhang, Yangdong; Li, Songli; Wang, Jiaqi

    2016-08-01

    The bovine mammary epithelial cell is a secretory cell, and its cell number and secretory activity determine milk production. In this study, we immortalized a bovine mammary epithelial cell line by SV40 large T antigen gene using a retrovirus based on Chinese Holstein primary mammary epithelial cells (CMEC) cultured in vitro. An immortalized bovine mammary epithelial cell line surpassed the 50-passage mark and was designated the CMEC-H. The immortalized mammary epithelial cells grew in close contact with each other and exhibited the typical cobblestone morphology characteristic with obvious boundaries. The telomerase expression of CMEC-H has consistently demonstrated the presence of telomerase activity as an immortalized cell line, but the cell line never induced tumor formation in nude mice. CMEC-H expressed epithelial (cytokeratins CK7, CK8, CK18, and CK19), mesenchymal (vimentin), and stem/progenitor (CD44 and p63) cell markers. The induced expression of milk proteins, αS1 -casein, β-casein, κ-casein, and butyrophilin, indicated that CMEC-H maintained the synthesis function of the mammary epithelial cells. The established immortalized bovine mammary epithelial cell line CMEC-H is capable of self-renewal and differentiation and can serve as a valuable reagent for studying the physiological mechanism of the mammary gland.

  3. Morphology and expression status investigations of specific surface markers on B-cell chronic lymphocytic leukemia cells.

    PubMed

    Niu, Suli; Chan, Ryan; Berini, Pierre; Wang, Chen; Zou, Shan

    2013-11-01

    The morphology of cells and expression status of specific surface markers [cluster of differentiation (CD)], such as CD5, CD19, CD20, CD38, and CD45, have long been considered as the essential indicators for the diagnosis and prognosis of B-cell chronic lymphocytic leukemia (B-CLL). Clinically, it is difficult to simultaneously obtain cell morphology and distribution of surface markers with flow cytometry, especially for some surrogate markers such as CD38. Here, as an alternative and complementary prognostic method, fluorescence microscopy and image processing method are introduced to directly visualize the cells from patients and to quantitatively determine the expression status of surface markers. In this study, the morphological parameters of B-CLL cells were measured to establish the correlation between the cellular morphology and the surface marker expression. It was clear that the CD38+ and CD38- B-CLL cells from the same CD38+ patients had hardly any size differences; however, an increase in perimeter was observed for CD38- patients. Moreover, the expression level of the receptors on the cell was independent of the cell size. There was no evidence showing that the expression intensities of CD19 and CD38 were related to each other for the CD38+ B-CLL cells. On the same cells, CD5 was more selectively expressed on the cell membrane; however, the expression patterns suggested that the cell membrane of CD38- B-CLL cells contained the least expression level of CD19.

  4. Chondrogenic potential of subpopulations of cells expressing mesenchymal stem cell markers derived from human synovial membranes.

    PubMed

    Arufe, M C; De la Fuente, A; Fuentes, I; de Toro, F J; Blanco, F J

    2010-11-01

    In this study we analyzed the chondrogenic potential of subpopulations of mesenchymal stem cells (MSCs) derived from human synovial membranes enriched for CD73, CD106, and CD271 markers. Subpopulations of human synovial membrane MSCs enriched for CD73, CD106, and CD271 markers were isolated using a cytometry sorter and characterized by flow cytometry for MSC markers. The expression of Sox9, Nanog, and Runx2 genes by these cells was measured by reverse transcriptase-polymerase chain reaction. The chondrogenesis of each subpopulation was assessed by culturing the cells in a defined medium to produce spontaneous spheroid formation and differentiation towards chondrocyte-like cells. The examination of the spheroids by histological and immunohistochemical analyses for collagen type II (COL2), aggrecan, collagen type I (COL1), metalloprotease 13 (MMP13), and collagen type X (COLX) levels were performed to assess their chondrogenesis capacity. The adipogenesis and osteogenesis potential of each subpopulation was determined using commercial media; the resulting cells were stained with oil red O or red alizarin to test the degree of differentiation. The subpopulations had different profiles of cells positive for the MSC markers CD44, CD69, CD73, CD90, and CD105 and showed different expression levels of the genes Sox9, Nanog, and Runx2 involved in chondrogenesis, undifferentiation, and osteoblastogenesis, respectively. Immunohistochemical analysis demonstrated that COL1, COL2, COLX, MMP13, and aggrecan were expressed in the spheroids as soon as 14 days of culture. The CD271(+) subpopulation expressed the highest levels of COL2 staining compared to the other subpopulations. CD105 and Runx2 were shown by immunohistochemistry and genetic analysis to have significantly higher expression CD271(+) subpopulation than the other subpopulations. Spheroids formed from CD271-enriched and CD73-enriched MSCs from normal human synovial membranes mimic the native cartilage extracellular

  5. The expression of retinal cell markers in human retinal pigment epithelial cells and their augmentation by the synthetic retinoid fenretinide

    PubMed Central

    Vugler, Anthony A.; Yu, Lu; Semo, Maayan; Coffey, Pete; Moss, Stephen E.; Greenwood, John

    2011-01-01

    Purpose In several species the retinal pigment epithelium (RPE) has the potential to transdifferentiate into retinal cells to regenerate functional retinal tissue after injury. However, this capacity for regeneration is lost in mammals. The synthetic retinoic acid derivative, fenretinide [N(4-hydroxyphenyl) retinamide], induces a neuronal-like phenotype in the human adult retinal pigment epithelial cell line (ARPE-19). These changes are characterized by the appearance of neural-like processes and the expression of neuronal markers not normally associated with RPE cells. Here we assess whether fenretinide can induce a neuroretinal cell phenotype in ARPE-19 cells, by examining retinal cell marker expression. Methods ARPE-19 cells were treated daily with culture medium containing either 3 μM fenretinide or dimethyl sulfoxide as a control for 7 days. Cells were processed for immunocytochemistry, western blotting, and for analysis by PCR to examine the expression of a panel of RPE, neural, and retinal-associated cellular markers, including classical and non-canonical opsins. Results Treatment with fenretinide for 7 days induced the formation of neuronal-like processes in ARPE-19 cells. Fenretinide induced the expression of the cone long wavelength sensitive opsin (OPN1lw) but not rhodopsin (RHO), while decreasing the expression of RPE cell markers. Many of the neuronal and retinal specific markers examined were expressed in both control and fenretinide treated cells, including those involved in photoreceptor cell development and the multipotency of neural retinal progenitor cells. Interestingly, ARPE-19 cells also expressed both photoreceptor specific and non-specific canonical opsins. Conclusions The expression of retinal-associated markers and loss of RPE cell markers in control ARPE-19 cells suggests that these cells might have dedifferentiated from an RPE cell phenotype under standard culture conditions. The expression of molecules, such as the transcription

  6. The expression of retinal cell markers in human retinal pigment epithelial cells and their augmentation by the synthetic retinoid fenretinide.

    PubMed

    Carr, Amanda-Jayne; Vugler, Anthony A; Yu, Lu; Semo, Maayan; Coffey, Pete; Moss, Stephen E; Greenwood, John

    2011-01-01

    In several species the retinal pigment epithelium (RPE) has the potential to transdifferentiate into retinal cells to regenerate functional retinal tissue after injury. However, this capacity for regeneration is lost in mammals. The synthetic retinoic acid derivative, fenretinide [N(4-hydroxyphenyl) retinamide], induces a neuronal-like phenotype in the human adult retinal pigment epithelial cell line (ARPE-19). These changes are characterized by the appearance of neural-like processes and the expression of neuronal markers not normally associated with RPE cells. Here we assess whether fenretinide can induce a neuroretinal cell phenotype in ARPE-19 cells, by examining retinal cell marker expression. ARPE-19 cells were treated daily with culture medium containing either 3 μM fenretinide or dimethyl sulfoxide as a control for 7 days. Cells were processed for immunocytochemistry, western blotting, and for analysis by PCR to examine the expression of a panel of RPE, neural, and retinal-associated cellular markers, including classical and non-canonical opsins. Treatment with fenretinide for 7 days induced the formation of neuronal-like processes in ARPE-19 cells. Fenretinide induced the expression of the cone long wavelength sensitive opsin (OPN1lw) but not rhodopsin (RHO), while decreasing the expression of RPE cell markers. Many of the neuronal and retinal specific markers examined were expressed in both control and fenretinide treated cells, including those involved in photoreceptor cell development and the multipotency of neural retinal progenitor cells. Interestingly, ARPE-19 cells also expressed both photoreceptor specific and non-specific canonical opsins. The expression of retinal-associated markers and loss of RPE cell markers in control ARPE-19 cells suggests that these cells might have dedifferentiated from an RPE cell phenotype under standard culture conditions. The expression of molecules, such as the transcription factors paired box 6 gene (PAX6), sex

  7. Expression of pluripotent stem cell markers in mouse uterine tissue during estrous cycle

    PubMed Central

    Choobineh, Kolsum; Zavareh, Saeed; Salehnia, Mojdeh; Ghorbanian, Mohamad Taghi; paylakhi, Seyed Hassan

    2016-01-01

    It was assumed that uterine stem cells are responsible for the unique regenerative capacity of uterine. Therefore, the aim of the present study was to investigate the expression of the pluripotent stem cell markers in the mice uterine tissue during different stages of estrous cycles. Twelve virgin female NMRI mice (6 to 8 weeks old) were considered at proestrus, estrus, metestrus and diestrus according to the cell types observed in the vaginal smear and underwent hysterectomy operation. Quantitative real-time polymerase chain reaction (PCR) and immunohistochemical staining for pluripotent stem cell markers (SOX2, OCT4, KLF4, and NANOG) were performed. Immunofluorescence staining revealed that expression and localization of the pluripotency markers SOX2, OCT4, KLF4, and NANOG at the protein level were not different throughout estrous cycle. Also, mRNA of pluripotency markers was detected in all tested samples. However, there were no significant differences in their genes expression at each stage and during the estrous cycle. Different hormonal profile during the estrous cycle could not affect expression of pluripotent stem cell markers in uterine tissue. PMID:27872713

  8. Defining the expression of marker genes in equine mesenchymal stromal cells.

    PubMed

    Guest, Deborah J; Ousey, Jennifer C; Smith, Matthew Rw

    2008-01-01

    Mesenchymal stromal (MS) cells have been derived from multiple sources in the horse including bone marrow, adipose tissue and umbilical cord blood. To date these cells have been investigated for their differentiation potential and are currently being used to treat damage to horse musculoskeletal tissues. However, no work has been done in horse MS cells to examine the expression profile of proteins and cell surface antigens that are expressed in human MS cells. The identification of such profiles in the horse will allow the comparison of putative MS cells isolated from different laboratories and different tissues. At present it is difficult to ascertain whether equivalent cells are being used in different reports. Here, we report on the expression of a range of markers used to define human MS cells. Using immunocytochemistry we show that horse MS cells homogenously express collagens, alkaline phosphatase activity, CD44, CD90 and CD29. In contrast, CD14, CD79α and the embryonic stem cell markers Oct-4, SSEA (stage specific embryonic antigen) -1, -3, -4, TRA (tumor rejection antigen) -1-60 and -1-81 are not expressed. The MS cells also express MHC class I antigens but do not express class II antigens, although they are inducible by treatment with interferon gamma (IFN-γ).

  9. Stromal cell markers are differentially expressed in the synovial tissue of patients with early arthritis.

    PubMed

    Choi, Ivy Y; Karpus, Olga N; Turner, Jason D; Hardie, Debbie; Marshall, Jennifer L; de Hair, Maria J H; Maijer, Karen I; Tak, Paul P; Raza, Karim; Hamann, Jörg; Buckley, Christopher D; Gerlag, Danielle M; Filer, Andrew

    2017-01-01

    Previous studies have shown increased expression of stromal markers in synovial tissue (ST) of patients with established rheumatoid arthritis (RA). Here, ST expression of stromal markers in early arthritis in relationship to diagnosis and prognostic outcome was studied. ST from 56 patients included in two different early arthritis cohorts and 7 non-inflammatory controls was analysed using immunofluorescence to detect stromal markers CD55, CD248, fibroblast activation protein (FAP) and podoplanin. Diagnostic classification (gout, psoriatic arthritis, unclassified arthritis (UA), parvovirus associated arthritis, reactive arthritis and RA), disease outcome (resolving vs persistent) and clinical variables were determined at baseline and after follow-up, and related to the expression of stromal markers. We observed expression of all stromal markers in ST of early arthritis patients, independent of diagnosis or prognostic outcome. Synovial expression of FAP was significantly higher in patients developing early RA compared to other diagnostic groups and non-inflammatory controls. In RA FAP protein was expressed in both lining and sublining layers. Podoplanin expression was higher in all early inflammatory arthritis patients than controls, but did not differentiate diagnostic outcomes. Stromal marker expression was not associated with prognostic outcomes of disease persistence or resolution. There was no association with clinical or sonographic variables. Stromal cell markers CD55, CD248, FAP and podoplanin are expressed in ST in the earliest stage of arthritis. Baseline expression of FAP is higher in early synovitis patients who fulfil classification criteria for RA over time. These results suggest that significant fibroblast activation occurs in RA in the early window of disease.

  10. Keratins 17 and 19 expression as prognostic markers in oral squamous cell carcinoma.

    PubMed

    Coelho, B A; Peterle, G T; Santos, M; Agostini, L P; Maia, L L; Stur, E; Silva, C V M; Mendes, S O; Almança, C C J; Freitas, F V; Borçoi, A R; Archanjo, A B; Mercante, A M C; Nunes, F D; Carvalho, M B; Tajara, E H; Louro, I D; Silva-Conforti, A M A

    2015-11-25

    Five-year survival rates for oral squamous cell carcinoma (OSCC) are 30% and the mortality rate is 50%. Immunohistochemistry panels are used to evaluate proliferation, vascularization, apoptosis, HPV infection, and keratin expression, which are important markers of malignant progression. Keratins are a family of intermediate filaments predominantly expressed in epithelial cells and have an essential role in mechanical support and cytoskeleton formation, which is essential for the structural integrity and stability of the cell. In this study, we analyzed the expressions of keratins 17 and 19 (K17 and K19) by immunohistochemistry in tumoral and non-tumoral tissues from patients with OSCC. The results show that expression of these keratins is higher in tumor tissues compared to non-tumor tissues. Positive K17 expression correlates with lymph node metastasis and multivariate analysis confirmed this relationship, revealing a 6-fold increase in lymph node metastasis when K17 is expressed. We observed a correlation between K17 expression with disease-free survival and disease-specific death in patients who received surgery and radiotherapy. Multivariate analysis revealed that low expression of K17 was an independent marker for early disease relapse and disease-specific death in patients treated with surgery and radiotherapy, with an approximately 4-fold increased risk when compared to high K17 expression. Our results suggest a potential role for K17 and K19 expression profiles as tumor prognostic markers in OSCC patients.

  11. Differential Expression of Stem Cell Markers and Vascular Endothelial Growth Factor in Human Retinoblastoma Tissue

    PubMed Central

    Kim, Martha; Kim, Jeong Hun; Kim, Jin Hyoung; Kim, Dong Hun

    2010-01-01

    Purpose To investigate the relationship between vascular endothelial growth factor (VEGF) and the cancer stem cell-vascular niche complex in human retinoblastoma tissue. Methods Six human retinoblastoma specimens primarily enucleated for Reese-Ellsworth classification stage 5a were stained to detect cancer stem cell markers, including ABCG2 for the stem cell marker and MCM2 for the neural stem cell marker, as well as to detect VEGF for the angiogenic cytokine. Using immunofluorescence, the expression of these proteins was analyzed, and their relative locations noted. Results In non-neoplastic retina of tumor-bearing eyes, ABCG2 and MCM2 were sporadically expressed in the ganglion cell layer and the inner nuclear layer, whereas VEGF was sporadically expressed in inner retina where retinal vessels are abundantly distributed. In the tumor, ABCG2 was strongly expressed out of Wintersteiner rosettes, whereas MCM2 and VEGF were strongly stained in the rosettes. Interestingly, the outer portion of the rosettes was positive for MCM2, and the inner portion of the rosettes was positive for VEGF. Conclusions Our data demonstrated that MCM2 and VEGF are strongly expressed in the rosettes of the tumor, which were far from the area of ABCG2-positive cells. Although VEGF might not directly contribute to the cancer stem cell-vascular niche complex, it could play some role in the differentiation of tumor cells to build up the rosettes. PMID:20157412

  12. Differential expression of stem cell markers and vascular endothelial growth factor in human retinoblastoma tissue.

    PubMed

    Kim, Martha; Kim, Jeong Hun; Kim, Jin Hyoung; Kim, Dong Hun; Yu, Young Suk

    2010-02-01

    To investigate the relationship between vascular endothelial growth factor (VEGF) and the cancer stem cell-vascular niche complex in human retinoblastoma tissue. Six human retinoblastoma specimens primarily enucleated for Reese-Ellsworth classification stage 5a were stained to detect cancer stem cell markers, including ABCG2 for the stem cell marker and MCM2 for the neural stem cell marker, as well as to detect VEGF for the angiogenic cytokine. Using immunofluorescence, the expression of these proteins was analyzed, and their relative locations noted. In non-neoplastic retina of tumor-bearing eyes, ABCG2 and MCM2 were sporadically expressed in the ganglion cell layer and the inner nuclear layer, whereas VEGF was sporadically expressed in inner retina where retinal vessels are abundantly distributed. In the tumor, ABCG2 was strongly expressed out of Wintersteiner rosettes, whereas MCM2 and VEGF were strongly stained in the rosettes. Interestingly, the outer portion of the rosettes was positive for MCM2, and the inner portion of the rosettes was positive for VEGF. Our data demonstrated that MCM2 and VEGF are strongly expressed in the rosettes of the tumor, which were far from the area of ABCG2-positive cells. Although VEGF might not directly contribute to the cancer stem cell-vascular niche complex, it could play some role in the differentiation of tumor cells to build up the rosettes.

  13. Expression and localization of epithelial stem cell and differentiation markers in equine skin, eye and hoof.

    PubMed

    Linardi, Renata L; Megee, Susan O; Mainardi, Sarah R; Senoo, Makoto; Galantino-Homer, Hannah L

    2015-08-01

    The limited characterization of equine skin, eye and hoof epithelial stem cell (ESC) and differentiation markers impedes the investigation of the physiology and pathophysiology of these tissues. To characterize ESC and differentiation marker expression in epithelial tissues of the equine eye, haired skin and hoof capsule. Indirect immunofluorescence microscopy and immunoblotting were used to detect expression and tissue localization of keratin (K) isoforms K3, K10, K14 and K124, the transcription factor p63 (a marker of ESCs) and phosphorylated p63 [pp63; a marker of ESC transition to transit-amplifying (TA) cell] in epithelial tissues of the foot (haired skin, hoof coronet and hoof lamellae) and the eye (limbus and cornea). Expression of K14 was restricted to the basal layer of epidermal lamellae and to basal and adjacent suprabasal layers of the haired skin, coronet and corneal limbus. Coronary and lamellar epidermis was negative for both K3 and K10, which were expressed in the cornea/limbus epithelium and haired skin epidermis, respectively. Variable expression of p63 with relatively low to high levels of phosphorylation was detected in individual basal and suprabasal cells of all epithelial tissues examined. To the best of the author's knowledge, this is the first report of the characterization of tissue-specific keratin marker expression and the localization of putative epithelial progenitor cell populations, including ESCs (high p63 expression with low pp63 levels) and TA cells (high expression of both p63 and pp63), in the horse. These results will aid further investigation of epidermal and corneal epithelial biology and regenerative therapies in horses. © 2015 ESVD and ACVD.

  14. Expression and localization of epithelial stem cell and differentiation markers in equine skin, eye and hoof

    PubMed Central

    Linardi, Renata L.; Megee, Susan O.; Mainardi, Sarah R.; Senoo, Makoto; Galantino-Homer, Hannah L.

    2015-01-01

    Background The limited characterization of equine skin, eye and hoof epithelial stem cell (ESC) and differentiation markers impedes the investigation of the physiology and pathophysiology of these tissues. Hypothesis/Objectives To characterize ESC and differentiation marker expression in epithelial tissues of the equine eye, haired skin and hoof capsule. Methods Indirect immunofluorescence microscopy and immunoblotting were utilized to detect expression and tissue localization of keratin (K) isoforms K3, K10, K14, and K124, the transcription factor p63 (a marker of ESCs) and phosphorylated p63 (pp63, a marker of ESC to transit-amplifying (TA) cell transition) in epithelial tissues of the foot (haired skin, hoof coronet and hoof lamellae) and the eye (limbus and cornea). Results K14 expression was restricted to the basal layer of epidermal lamellae, and to basal and adjacent suprabasal layers of the haired skin, coronet and corneal limbus. Coronary and lamellar epidermis was negative for both K3 and K10, which were expressed in the cornea/limbus epithelium and haired skin epidermis, respectively. Variable expression of p63 with relatively low to high levels of phosphorylation was detected in individual basal and suprabasal cells of all epithelial tissues examined. Conclusions This is the first report of the characterization of tissue-specific keratin marker expression and the localization of putative epithelial progenitor cell populations, including ESCs (high p63 expression with low pp63 levels) and TA cells (high expression of both p63 and pp63), in the horse. These results will aid further investigation of epidermal and corneal epithelial biology and regenerative therapies in horses. PMID:25963063

  15. A method for cell type marker discovery by high-throughput gene expression analysis of mixed cell populations.

    PubMed

    Andrade-Navarro, Miguel A; Kanji, Femina; Palii, Carmen G; Brand, Marjorie; Atkins, Harold; Perez-Iratxeta, Carol

    2013-10-03

    Gene transcripts specifically expressed in a particular cell type (cell-type specific gene markers) are useful for its detection and isolation from a tissue or other cell mixtures. However, finding informative marker genes can be problematic when working with a poorly characterized cell type, as markers can only be unequivocally determined once the cell type has been isolated. We propose a method that could identify marker genes of an uncharacterized cell type within a mixed cell population, provided that the proportion of the cell type of interest in the mixture can be estimated by some indirect method, such as a functional assay. We show that cell-type specific gene markers can be identified from the global gene expression of several cell mixtures that contain the cell type of interest in a known proportion by their high correlation to the concentration of the corresponding cell type across the mixtures. Genes detected using this high-throughput strategy would be candidate markers that may be useful in detecting or purifying a cell type from a particular biological context. We present an experimental proof-of-concept of this method using cell mixtures of various well-characterized hematopoietic cell types, and we evaluate the performance of the method in a benchmark that explores the requirements and range of validity of the approach.

  16. Molecular beacon imaging of tumor marker gene expression in pancreatic cancer cells.

    PubMed

    Yang, Lily; Cao, Zehong; Lin, Yiming; Wood, William C; Staley, Charles A

    2005-05-01

    We have developed a fluorescence imaging-based approach to detect expression of tumor marker genes in pancreatic cancer cells using molecular beacons (MBs). MBs are short hairpin oligonucleotide probes that bind to specific oligonucleotide sequences and produce fluorescent signals. MBs targeting transcripts of two tumor marker genes, mutant K-ras and survivin, were synthesized and their specificity in detection of the expression of those genes in pancreatic cancer cells was examined. We found that K-ras MBs differentially bind to mutant K-ras mRNAs, resulting in strong fluorescent signals in pancreatic cancer cells with specific mutant K-ras genes but not in normal cells or cancer cells expressing either wild type or a different mutation of the K-ras gene. Additionally, MBs targeting survivin mRNA produced a bright fluorescent signal specifically in pancreatic cancer cells. We also demonstrated that MBs labeled with different fluorophores could detect survivin and mutant K-ras mRNAs simultaneously in single cancer cells. Furthermore, we showed that survivin and K-ras MBs have a high specificity in identifying cancer cells on frozen sections of pancreatic cancer tissues. In conclusion, molecular beacon-based imaging of expression of tumor marker genes has potential for the development of novel approaches for the detection of pancreatic cancer cells.

  17. Effect of radiation on the expression of osteoclast marker genes in RAW264.7 cells.

    PubMed

    Yang, Bing; Zhou, Hui; Zhang, Xiao-Dong; Liu, Zheng; Fan, Fei-Yue; Sun, Yuan-Ming

    2012-04-01

    Cancer radiation therapy can cause skeletal complications, such as osteopenia and osteoporosis. To understand the mechanism responsible for the skeletal complications, the expression profiles of osteoclast marker genes in RAW264.7 cells were observed. Osteoclast formation was established by RAW264.7 cells that were treated with the receptor activator of nuclear factor (NF)-κB ligand (RANKL) and detected using immunochemistry and morphological observations. Quantitative real-time polymerase chain reaction was used to assess the expression of a panel of osteoclast markers, including the receptor activator of NF-κB (RANK), tartrate-resistant acid phosphatase (TRAP), integrin β3 and the calcitonin receptor (CTR). RANKL-induced osteoclasts were TRAP-positive and multinucleated, and displayed a distinct morphology. RANKL-induced osteoclast precursor cells had increased TRAP and RANK expression and decreased CTR expression compared to the control cells not treated with RANKL. RAW264.7 cells irradiated with 2-Gy γ-rays had upregulated integrin β3 and RANK expression and downregulated CTR expression compared to the control RAW264.7 cells. The effect of radiation on RANKL-induced osteoclast differentiation enhanced the expression of CTR and inhibited the expression of RANK and TRAP. Therefore, radiation damage from 2-Gy γ-rays can promote the activities of osteoclast precursor cells, but not those of osteoclasts.

  18. Effect of radiation on the expression of osteoclast marker genes in RAW264.7 cells

    PubMed Central

    YANG, BING; ZHOU, HUI; ZHANG, XIAO-DONG; LIU, ZHENG; FAN, FEI-YUE; SUN, YUAN-MING

    2012-01-01

    Cancer radiation therapy can cause skeletal complications, such as osteopenia and osteoporosis. To understand the mechanism responsible for the skeletal complications, the expression profiles of osteoclast marker genes in RAW264.7 cells were observed. Osteoclast formation was established by RAW264.7 cells that were treated with the receptor activator of nuclear factor (NF)-κB ligand (RANKL) and detected using immunochemistry and morphological observations. Quantitative real-time polymerase chain reaction was used to assess the expression of a panel of osteoclast markers, including the receptor activator of NF-κB (RANK), tartrate-resistant acid phosphatase (TRAP), integrin β3 and the calcitonin receptor (CTR). RANKL-induced osteoclasts were TRAP-positive and multinucleated, and displayed a distinct morphology. RANKL-induced osteoclast precursor cells had increased TRAP and RANK expression and decreased CTR expression compared to the control cells not treated with RANKL. RAW264.7 cells irradiated with 2-Gy γ-rays had upregulated integrin β3 and RANK expression and downregulated CTR expression compared to the control RAW264.7 cells. The effect of radiation on RANKL-induced osteoclast differentiation enhanced the expression of CTR and inhibited the expression of RANK and TRAP. Therefore, radiation damage from 2-Gy γ-rays can promote the activities of osteoclast precursor cells, but not those of osteoclasts. PMID:22294242

  19. Expression of functionally relevant cell surface markers in dibutyltin-exposed human natural killer cells.

    PubMed

    Odman-Ghazi, Sabah O; Hatcher, Frank; Whalen, Margaret M

    2003-07-25

    Butyltin (BT) compounds are known for their worldwide contamination. Dibutyltin (DBT) is used as a stabilizer in plastic products, and as a deworming agent in poultry. Poultry products have been shown to contain measurable levels of DBT. Drinking water has also been reported to contain BTs due to leaching from PVC pipes. We, and others, have found measurable levels of DBT in human blood. BTs appear to increase the risk of cancer and other viral infections in exposed individuals. In previous studies we have shown that the tumor killing function of natural killer (NK) lymphocytes was greatly diminished after as little as a 1 h exposure to DBT and the inhibition continued even after removal of the compound. We also showed that there was a significant decrease in NK cell lysis of K562 target cells after an exposure to 1.5 microM DBT for 24 h. This 24 h exposure also decreased the ability of NK cells to bind to tumor cells. Loss of binding function was not seen when NK cells were exposed to 5-10 microM DBT for 1 h. However, NK cells exposed to 5 microM DBT for 1 h and then incubated in DBT-free media for 24, 48, or 96 h, showed a significant loss of tumor-binding function within 24 h. The effects of DBT exposure on seven cell surface molecules that are involved in NK-cell interactions with target cells were investigated. The results indicated that the exposure of NK cells to 1.5 microM DBT for 24 h decreased the expression of CD2, CD11a, CD16, CD11c. There was no decrease in expression of any of the markers studied when NK cells were exposed to 5 microM DBT for 1 h, consistent with the fact that a 1-h exposure had no effect on the ability of NK cells to bind tumor cells. However, when NK cells were exposed to 5 microM DBT for 1 h followed by 24, 48 or 96 h incubations in DBT-free media there was decreased expression of several of the cells surface molecules with the most dramatic decreases being in CD16 and CD56.

  20. Expression of the Memory Marker CD45RO on Helper T Cells in Macaques

    PubMed Central

    Valentine, Michael; Song, Kejing; Maresh, Grace A.; Mack, Heather; Huaman, Maria Cecilia; Polacino, Patricia; Ho, On; Cristillo, Anthony; Kyung Chung, Hye; Hu, Shiu-Lok; Pincus, Seth H.

    2013-01-01

    Background In humans it has been reported that a major site of the latent reservoir of HIV is within CD4+ T cells expressing the memory marker CD45RO, defined by the mAb UCHL1. There are conflicting reports regarding the expression of this antigen in macaques, the most relevant animal species for studying HIV pathogenesis and testing new therapies. There is now a major effort to eradicate HIV reservoirs and cure the infection. One approach is to eliminate subsets of cells housing the latent reservoir, using UCHL1 to target these cells. So that such studies may be performed in macaques, it is essential to determine expression of CD45RO. Methods We have used immunofluorescence and flow cytometry to study cell surface expression of CD45RO on lymphocytes from PBMC, lymphoid, and GI organs of rhesus, pigtailed, and cynomolgus macaques. Both direct and indirect immunofluorescence experiments were performed. Findings CD45RO is expressed on a subset of CD4+ lymphocytes of all pigtailed, a fraction of rhesus, and neither of the cynomolgus macaques studied. The binding of UCHL1 to macaque cells was of lower avidity than to human cells. This could be overcome by forming UCHL1 multimers. Directly conjugating fluors to UCHL1 can inhibit UCHL1 binding to macaque cells. Patterns of UCHL1 expression differ somewhat in macaques and humans, and from that of other memory markers often used in macaques. Conclusions CD45RO, defined with mAb UCHL1, is well expressed on CD4+ cells in pigtailed macaques. Using tissues recovered from latently infected pigtailed macaques we are determining whether UCHL1, or other memory markers, can define the cellular locus of the reservoir. The low avidity of this interaction could limit the utility of UCHL1, in its conventional form, to eliminate cells in vivo and test this approach in macaque models of HIV infection. PMID:24023920

  1. Expression of phenotypic markers of mast cells, macrophages and dendritic cells in gallbladder mucosa with calculous cholecystitis.

    PubMed

    Kasprzak, A A; Szmyt, M; Malkowski, W; Surdyk-Zasada, J; Przybyszewska, W; Szmeja, J; Helak-Łapaj, C; Seraszek-Jaros, A; Kaczmarek, E

    2013-12-01

    The study aimed at quantitative analysis of expression involving markers of mast cells (tryptase), monocytes/macrophages (CD68 molecule) and dendritic cells (S100 protein) in gallbladder mucosa with acute and chronic calculous cholecystitis. Routinely prepared tissue material from the patients with acute (ACC) (n = 16) and chronic calculous cholecystitis (CCC) (n = 55) was evaluated. Three cellular markers were localized by immunocytochemistry. Their expression was quantified using spatial visualization technique. The expression of tryptase was similar in acute and chronic cholecystitis. CD68 expression in ACC was significantly higher than in the CCC group. Expression of S100 protein was significantly higher in CCC as compared to the ACC group. No significant correlations were disclosed between expression of studied markers and grading in the gallbladder wall. A weak negative correlation was noted between expression of CD68 and number of gallstones in the CCC group. The spatial visualization technique allowed for a credible quantitative evaluation of expression involving markers of mast cells (MCs), monocytes/macrophages (Mo/Ma) and dendritic cells (DCs) in gallbladder mucosa with ACC and CCC. For the first time mucosal expression of S100 protein-positive DCs was evaluated in calculous cholecystitis. The results point to distinct functions of studied cell types in the non-specific immune response in calculous cholecystitis.

  2. Distinct expression profile of stem cell markers, LGR5 and LGR6, in basaloid skin tumors.

    PubMed

    Jang, Bo Gun; Lee, Cheol; Kim, Hye Sung; Shin, Myung Soo; Cheon, Min Seok; Kim, Jae Wang; Kim, Woo Ho

    2017-03-01

    Mammalian epidermis, which is composed of hair follicles, sebaceous glands, and interfollicular epidermis, is maintained by discrete stem cells. In vivo lineage tracing demonstrated that murine LGR5 cells are mainly responsible for hair follicle regeneration whereas LGR6 cells generate sebaceous glands and interfollicular epidermis. However, little is known about their expression in the human skin tumors. In this study, we investigated the expression profile of LGR5 and LGR6 in a variety of human skin tumors including basaloid tumors with follicular differentiation (94 basal cell carcinomas, 18 trichoepitheliomas, 3 basaloid follicular hamartomas, and 12 pilomatricomas) and tumors with ductal differentiation (7 eccrine poromas, 8 hidradenomas, and 5 spiradenomas). LGR5 expression was highest in basal cell carcinomas (BCCs) followed by trichoepitheliomas (TEs) and basaloid follicular hamartomas. LGR6 had the same expression pattern as LGR5, even though its expression was lower. Interestingly, LGR6 expression was detected in stromal cells around the tumor and papillary mesenchymal bodies of TEs but not in stromal cells of BCCs, suggesting different characteristics of tumor-associated fibroblasts between TEs and BCCs. It was unexpected to find that pilomatricomas exclusively expressed LGR6, and its expression was limited to the basaloid cells. Notably, LGR6-positive cells were observed in sweat gland ductal cells in normal skin. This might explain, in part, the finding that LGR6 expression was relatively higher in basaloid tumors with ductal differentiation than in those with follicular differentiation. In particular, spiradenomas displayed the same distribution pattern of LGR6 as normal sweat glands, suggesting the possibility of LGR6-positive cells as tumor stem cells. In conclusion, we documented the different expression patterns of stem cell markers, LGR5 and LGR6 in various skin tumors. These data may provide important insights to understand the origin and

  3. Induction of expression of two phenotypic markers of pulmonary type II cells in a cultured cell line

    SciTech Connect

    Henderson, R.F.; Waide, J.J.; Scott, G.G.

    1994-11-01

    The functions of pulmonary type II cells, such as synthesis of pulmonary surfactant and metabolism of inhaled xenobiotics, can be studied in primary isolates of lung cells. However, isolated type II cells, when cultured, quickly lose the phenotypic expressions characteristics of type II cells, including surfactant lipid and protein synthesis and alkaline phosphatase (AP) activity. A cultured cell line that maintained expression of type II cell markers of differentiation would be advantageous for the study of such functions as surfactant synthesis and secretion. Such a cell line would allow generation of a large number of homogeneous cells for study. The purpose of the current study was to induce markers of differentiated type II cells in a cultured cell line to facilitate studies of factors that control surfactant synthesis and secretion.

  4. Following damage, the majority of bone marrow-derived airway cells express an epithelial marker

    PubMed Central

    MacPherson, Heather; Keir, Pamela A; Edwards, Carol J; Webb, Sheila; Dorin, Julia R

    2006-01-01

    Background Adult-derived bone marrow stem cells are capable of reconstituting the haematopoietic system. However there is ongoing debate in the literature as to whether bone marrow derived cells have the ability to populate other tissues and express tissue specific markers. The airway has been an organ of major interest and was one of the first where this was demonstrated. We have previously demonstrated that the mouse airway can be repopulated by side population bone marrow transplanted cells. Here we investigate the frequency and phenotypic nature of these bone marrow derived cells. Methods Female mice were engrafted with male whole bone marrow or side population (SP) cells and subjected to detergent-induced damage after 3 months. Donor cells were identified by Y chromosome fluorescence in situ hybridisation and their phenotype was assessed by immunohistochemistry on the same sections. Slides were visualised by a combination of widefield and deconvolved microscopy and whole cells were analysed on cytospin preparations. Results The frequencies of engraftment of male cells in the airway of mice that show this (9/10), range from 1.0 – 1.6% with whole marrow and 0.6 – 1.5% with SP cells. Undamaged controls have only between 0.1 and 0.2% male cells in the trachea. By widefield microscopy analysis we find 60.2% (53/88) of male donor derived cells express cytokeratins as a marker of epithelial cells. These results were reinforced using deconvolved microscopy and scored by two independent investigators. In addition cytospin analysis of cells dissociated from the damaged trachea of engrafted mice also reveals donor derived Y chromosome positive cells that are immunopositive for cytokeratin. Using cytokeratin and the universal haematopoietic marker CD45 immunohistochemistry, we find the donor derived cells fall into four phenotypic classes. We do not detect cytokeratin positive cells in whole bone marrow using cytokeratin immunostaining and we do not detect any

  5. [Expression of CD48 as a live marker to distinguish division of hematopoietic stem cells].

    PubMed

    Yang, Xin; Zhang, Yu; Peng, Lu-Yun; Pang, Ya-Kun; Dong, Fang; Ji, Qing; Xu, Jing; Cheng, Tao; Yuan, Wei-Ping; Gao, Ying-Dai

    2014-06-01

    AF488(+) cells was significantly higher than that of AF488(-) cells (P < 0.05). The proliferation ability of AF488(-) cells was also significantly higher than AF488(+) cells (P < 0.05). It is concluded that the expression of CD48 can distinguish cell division of hematopoietic stem cells and can be used as a live marker for the loss of stemness. In comparison with the Numb protein staining, this method can be used in living cells, thus provides greater convenience for subsequent cell culture studies and cell transplantation experiments.

  6. Chlorogenic acid regulates apoptosis and stem cell marker-related gene expression in A549 human lung cancer cells.

    PubMed

    Yamagata, Kazuo; Izawa, Yuri; Onodera, Daiki; Tagami, Motoki

    2017-09-05

    Previous studies indicated that chlorogenic acid, a compound present in many fruits and vegetables, has anti-cancer activities. We report that chlorogenic acid regulates the expression of apoptosis-related genes and self-renewal-related stem cell markers in cancer cells. The lung cancer cell line A549 was cultured with or without chlorogenic acid. The presence of chlorogenic acid decreased cell proliferation as measured by MTT activity. Polymerase chain reaction (PCR) showed that treatment of cells with chlorogenic acid reduced the expression of BCL2 but increased that of both BAX and CASP3. Chlorogenic acid enhanced annexin V expression as measured using fluorescently labeled annexin V. Chlorogenic acid also induced p38 MAPK and JNK gene expression. Meanwhile, several agents, including SB203580 (p38 MAP kinase inhibitor), N-acetylcysteine (antioxidant inhibitor), dipyridamole (phosphodiesterase inhibitor), and apocynin (NADPH-oxidase inhibitor) blocked chlorogenic acid-induced BAX gene expression. Chlorogenic acid reduced gene expression levels of stem cell-associated markers NANOG, POU5F1, and SOX2. Together these results indicate that chlorogenic acid affects the expression of apoptosis-related genes that are part of oxidative stress and p38 MAP-dependent pathways, as well as genes encoding stem cell markers. In conclusion, chlorogenic acid may contribute to the polyphenolic anti-cancer effect associated with consumption of vegetables and fruits.

  7. Differential expression of distinct surface markers in early endothelial progenitor cells and monocyte-derived macrophages.

    PubMed

    Cheng, Shu-Meng; Chang, Shing-Jyh; Tsai, Tsung-Neng; Wu, Chun-Hsien; Lin, Wei-Shing; Lin, Wen-Yu; Cheng, Cheng-Chung

    2013-01-01

    Bone marrow-derived endothelial progenitor cells (EPCs) play a fundamental role in postnatal angiogenesis. Currently, EPCs are defined as early and late EPCs based on their biological properties and their time of appearance during in vitro culture. Reports have shown that early EPCs share common properties and surface markers with adherent blood cells, especially CD14+ monocytes. Distinguishing early EPCs from circulating monocytes or monocyte-derived macrophages (MDMs) is therefore crucial to obtaining pure endothelial populations before they can be applied as part of clinical therapies. We compared the gene expression profiles of early EPCs, blood cells (including peripheral blood mononuclear cells, monocytes, and MDMs), and various endothelial lineage cells (including mature endothelial cells, late EPCs, and CD133+ stem cells). We found that early EPCs expressed an mRNA profile that showed the greatest similarity to MDMs than any other cell type tested. The functional significance of this molecular profiling data was explored by Gene Ontology database search. Novel plasma membrane genes that might potentially be novel isolation biomarkers were also pinpointed. Specifically, expression of CLEC5A was high in MDMs, whereas early EPCs expressed abundant SIGLEC8 and KCNE1. These detailed mRNA expression profiles and the identified functional modules will help to develop novel cell isolation approaches that will allow EPCs to be purified; these can then be used to target cardiovascular disease, tumor angiogenesis, and various ischemia-related diseases.

  8. Expression analysis of KDEL-CysEPs programmed cell death markers during reproduction in Arabidopsis.

    PubMed

    Zhou, Liang-Zi; Höwing, Timo; Müller, Benedikt; Hammes, Ulrich Z; Gietl, Christine; Dresselhaus, Thomas

    2016-09-01

    CEP cell death markers. Programmed cell death (PCD) is essential for proper plant growth and development. Plant-specific papain-type KDEL-tailed cysteine endopeptidases (KDEL-CysEPs or CEPs) have been shown to be involved in PCD during vegetative development as executors for the last step in the process. The Arabidopsis genome encodes three KDEL-CysEPs: AtCEP1, AtCEP2 and AtCEP3. With the help of fluorescent fusion reporter lines, we report here a detailed expression analysis of KDEL-CysEP (pro)proteins during reproductive processes, including flower organ and germline development, fertilization and seed development. AtCEP1 is highly expressed in different reproductive tissues including nucellus cells of mature ovule and the connecting edge of anther and filament. After fertilization, AtCEP1 marks integument cell layers of the seeds coat as well as suspensor and columella cells of the developing embryo. Promoter activity of AtCEP2 is detected in the style of immature and mature pistils, in other floral organs including anther, sepal and petal. AtCEP2 mainly localizes to parenchyma cells next to xylem vessels. Although there is no experimental evidence to demonstrate that KDEL-CysEPs are involved in PCD during fertilization, the expression pattern of AtCEPs, which were previously shown to represent cell death markers during vegetative development, opens up new avenues to investigate PCD in plant reproduction.

  9. The expression of neurogenic markers after neuronal induction of chorion-derived mesenchymal stromal cells.

    PubMed

    Manochantr, Sirikul; Marupanthorn, Kulisara; Tantrawatpan, Chairat; Kheolamai, Pakpoom

    2015-06-01

    Chorion is a tissue of early embryologic period that is discarded after delivery. It might be the potential source of mesenchymal stromal cells (MSCs) that can be used for research and eventually for therapeutic studies. At present, the biological properties and the differentiation capacity of chorion-derived MSCs are still poorly characterised. The objective of this study is to characterise and explore the differentiating potential of chorion-derived MSCs towards the neuronal lineages. Chorionic membrane was digested with enzyme and cultured in Dulbecco's Modified Eagle's medium supplemented with 10% fetal bovine serum. The expression of MSC markers was examined using flow cytometry. The adipogenic, osteogenic and neurogenic differentiation were examined by culturing in appropriate induction media. The expression of neuronal markers was determined by immunofluorescence and quantitative real time-PCR. Chorion-derived MSCs were easily expanded up to 20 passages. They were positive for MSC markers (CD73, CD90 and CD105), and negative for haematopoietic markers (CD34 and CD45). Chorion-derived MSCs could differentiate into several mesodermal-lineages including adipocytes and osteoblasts. Moreover, chorion-derived MSCs could differentiate into neuronal-like cells as characterised by cell morphology and the presence of neural markers including MAP-2, glial fibrillary acidic protein (GFAP) and beta-tubulin III. Chorion-derived MSCs can be readily obtained and expanded in culture. These cells also have transdifferentiation capacity as evidenced by their neuronal differentiation potential. Therefore, chorion can be used as an alternative source of MSCs for stem cell therapy in nervous system disorders.

  10. Prognostic value of changes in the expression of stem cell markers in the peripheral blood of patients with colon cancer.

    PubMed

    Padín-Iruegas, Maria-Elena; Herranz-Carnero, Michel; Aguin-Losada, Santiago; Brozos-Vazquez, Elena; Anido-Herranz, U; Antunez-Lopez, Jose-Ramon; Ruibal-Morell, Alvaro; López-López, Rafael

    2013-06-01

    Cancer stem cells play an important role in carcinogenesis and resistance to treatment and may lead to metastasis. The isolation of circulating stem cells involves cell sorting based on the presence of cell surface markers. Many surface markers such as CD133, c-Kit, SOX, OCT4 and TWIST have been reported. In the present study, we determined the expression of different stem cell markers and their variation in expression at different stages of the treatment process. Samples of EDTA blood were collected from metastatic colorectal cancer patients, and circulating cancer stem cells were isolated for the analysis of the expression of stem cell markers using RT-PCR. These findings were correlated with the response to therapy. All statistical analyses were performed using the GraphPad Prism 5.03 software. Significant differences were found in the expression levels of the markers CD133, SOX2, OCT4 and TWIST1. No differences were found in c-Kit expression. Correlation in the expression levels of most of the markers was observed. Expression of CD133, OCT4, SOX2 and TWIST1 had a predictive value for colon cancer behavior. Evaluation of this stem cell gene expression panel may be useful for predicting the response during the process of treatment, and the relative easy access to samples facilitates this method. Moreover the correlation between CD133 and TWIST1 expression may be associated with tumor regrowth and metastatic relapse.

  11. Association of expression levels of pluripotency/stem cell markers with the differentiation outcome of Wharton's jelly mesenchymal stem cells into insulin producing cells.

    PubMed

    Kassem, Dina H; Kamal, Mohamed M; El-Kholy, Abd El-Latif G; El-Mesallamy, Hala O

    2016-08-01

    Recently, there has been much attention towards generation of insulin producing cells (IPCs) from stem cells, especially from Wharton's jelly mesenchymal stem cells (WJ-MSCs). However, generation of mature IPCs remains a challenge. Assessment of generation of IPCs was usually done by examining β-cell markers, however, assessment of pluripotency/stem cell markers drew less attention. Therefore, the purpose of this study was to investigate the levels of pluripotency/stem cell markers during differentiation of WJ-MSCs into IPCs and the association of these levels with differentiation outcomes. WJ-MSCs were isolated, characterized then induced to differentiate into IPCs using three different protocols namely A, B and C. Differentiated IPCs were assessed by the expression of pluripotency/stem cell markers, together with β-cell markers using qRT-PCR, and functionally by measuring glucose stimulated insulin secretion. Differentiated cells from protocol A showed lowest expression of pluripotency/stem cell markers and relatively best GSIS. However, protocol B showed concomitant expression of pluripotency/stem cell and β-cell markers with relatively less insulin secretion as compared to protocol A. Protocol C failed to generate glucose-responsive IPCs. In conclusion, sustained expression of pluripotency/stem cell markers could be associated with the incomplete differentiation of WJ-MSCs into IPCs. A novel finding for which further investigations are warranted.

  12. Expression of epithelial-mesenchymal transition markers at the invasive front of oral squamous cell carcinoma

    PubMed Central

    COSTA, Liana Cristina Melo Carneiro; LEITE, Camila Ferreira; CARDOSO, Sérgio Vitorino; LOYOLA, Adriano Mota; de FARIA, Paulo Rogério; SOUZA, Paulo Eduardo Alencar; HORTA, Martinho Campolina Rebello

    2015-01-01

    Oral squamous cell carcinoma (OSCC) is one of the most common malignances. In epithelial-mesenchymal transition (EMT), epithelial cells switch to mesenchymal-like cells exhibiting high mobility. This migratory phenotype is significant during tumor invasion and metastasis. Objective : The aim of this study is to evaluate the expression of the EMT markers E-cadherin, N-cadherin and vimentin in OSCC. Material and Methods : Immunohistochemical detection of E-cadherin, N-cadherin and vimentin was performed on 20 OSCC samples. Differences in the expression of each protein at the invasive front (IF) and in the central/superficial areas (CSA) of the tumor were assessed. Differences in the expression of each protein at the IF of both histologically high- and low-invasive OSCCs were evaluated. Associations among expression of proteins at the IF were assessed. Correlations between the expression levels of each protein at the IF and the tumor stage and clinical nodal status were also evaluated. Results : Reduced expression of E-cadherin was detected in 15 samples (75%). E-cadherin expression was reduced at the IF when compared to the CSA and in high-invasive tumors when compared to low-invasive tumors. All samples were negative for N-cadherin, even though one sample showed an inconspicuous expression. Positive expression of vimentin was observed in 6 samples (30%). Nevertheless, there was no difference in vimentin expression between the IF and the CSA regions or between the low- and high-invasive tumors. Furthermore, no association was observed among protein expression levels at the IF. Finally, no correlations were observed between each protein’s expression levels and tumor stage or clinical nodal status. Conclusions : Reduced E-cadherin expression at the IF and its association with histological invasiveness suggest that this protein is a noteworthy EMT marker in OSCC. Although vimentin was also detected as an EMT marker, its expression was neither limited to the IF nor was

  13. A not cytotoxic nickel concentration alters the expression of neuronal differentiation markers in NT2 cells.

    PubMed

    Ceci, Claudia; Barbaccia, Maria Luisa; Pistritto, Giuseppa

    2015-03-01

    Nickel, a known occupational/environmental hazard, may cross the placenta and reach appreciable concentrations in various fetal organs, including the brain. The aim of this study was to investigate whether nickel interferes with the process of neuronal differentiation. Following a 4 week treatment with retinoic acid (10μM), the human teratocarcinoma-derived NTera2/D1 cell line (NT2 cells) terminally differentiate into neurons which recapitulate many features of human fetal neurons. The continuous exposure of the differentiating NT2 cells to a not cytotoxic nickel concentration (10μM) increased the expression of specific neuronal differentiation markers such as neural cell adhesion molecule (NCAM) and microtubule associated protein 2 (MAP2). Furthermore, nickel exposure increased the expression of hypoxia-inducible-factor-1α (HIF-1α) and induced the activation of the AKT/PKB kinase pathway, as shown by the increase of P(Ser-9)-GSK-3β, the inactive form of glycogen synthase kinase-3β (GSK-3β). Intriguingly, by the end of the fourth week the expression of tyrosine hydroxylase (TH) protein, a marker of dopaminergic neurons, was lower in nickel-treated than in control cultures. Thus, likely by partially mimicking hypoxic conditions, a not-cytotoxic nickel concentration appears to alter the process of neuronal differentiation and hinder the expression of the dopaminergic neuronal phenotype. Taken together, these results suggest that nickel, by altering normal brain development, may increase susceptibility to neuro-psychopathology later in life.

  14. The mycotoxin deoxynivalenol inhibits the cell surface expression of activation markers in human macrophages.

    PubMed

    Waché, Yann J; Hbabi-Haddioui, Laila; Guzylack-Piriou, Laurence; Belkhelfa, Haouaria; Roques, Christine; Oswald, Isabelle P

    2009-08-21

    Deoxynivalenol (DON) is the most prevalent trichothecene mycotoxin in crops in Europe and North America. It exhibits several toxic effects including impaired growth and immune dysregulation. Macrophages play pivotal role in the host defense; upon activation, they express several specific cell surface receptors that are important in adhesion and cell signaling. Several studies have demonstrated that DON can affect macrophages, however, very few data are available concerning the effect of DON on human macrophages, and the effect on macrophage cell surface receptors is unknown. In the present study, human blood monocytes, differentiated in vitro into macrophages, were activated with IFN-gamma, in the presence or absence of low concentrations of DON. The expression of CD11c, CD13, CD14, CD18, CD33, CD35, CD54, CD119 and HLA-DP/DQ/DR was analyzed by flow cytometry. As expected, macrophage activation by IFN-gamma upregulated the expression of CD54, CD14, CD119 and HLA-DP/DQ/DR. Incubation with DON decrease the cell surface expression of these activation markers in a dose-dependent manner. When cells were treated with 5muM DON, the mean fluorescence intensity measured for the expression of these receptors was the same as that observed in non-activated macrophages. This inhibitory effect of DON was only observed when the mycotoxin was applied before the activation signal. Taken together, our results suggest that low concentration of DON alter macrophage activation as measured by the expression of cell surface markers. This may have implications for human health when consuming DON contaminated feed.

  15. Adipose derived mesenchymal stem cells express keratinocyte lineage markers in a co-culture model.

    PubMed

    Irfan-Maqsood, M; Matin, M M; Heirani-Tabasi, A; Bahrami, M; Naderi-Meshkin, H; Mirahmadi, M; Hassanzadeh, H; Sanjar Moussavi, N; Raza-Shah, H; Raeesolmohaddeseen, M; Bidkhori, H; Bahrami, A R

    2016-04-30

    Cutaneous wound healing is a complex type of biological event involving proliferation, differentiation, reprograming, trans/de-differentiation, recruitment, migration, and apoptosis of a number of cells (keratinocytes, fibroblasts, endothelial cells, nerve cells and stem cells) to regenerate a multi-layered tissue that is damaged by either internal or external factors. The exact regeneration mechanism of damaged skin is still unknown but the epithelial and other kinds of stem cells located in skin play crucial roles in the healing process. In this work, a co-culture model composed of adipose derived mesenchymal stem cells and keratinocytes was developed to understand the cellular differentiation behaviour in wound healing. Human mesenchymal stem cells were isolated from waste lipoaspirates. Keratinocytes were isolated from neonatal rats skin as well from human adult skin. Both types of cells were cultured and their culturing behaviour was observed microscopically under regular intervals of time. The identity of both cells was confirmed by flow cytometry and qRT-PCR. Cells were co-cultured under the proposed co-culturing model and the model was observed for 7, 14 and 21 days. The cellular behaviour was studied based on change in morphology, colonization, stratification, migration and expression of molecular markers. Expression of molecular markers was studied at transcriptional level and change in cellular morphology and migration capabilities was observed under the invert microscope regularly. Successfully isolated and characterized mesenchymal stem cells were found to express keratinocyte lineage markers i.e. K5, K10, K14, K18, K19 and Involucrin when co-cultured with keratinocytes after 14 and 21 days. Their expression was found to increase by increasing the time span of cell culturing. The keratinocyte colonies started to disappear after 10 days of culturing which might be due to stratification process initiated by possibly transdifferentiated stem cells. It can

  16. Differential Expression of Stem Cell Markers in Ocular Surface Squamous Neoplasia

    PubMed Central

    Mishra, Dilip Kumar; Veena, Uppala; Kaliki, Swathi; Kethiri, Abhinav Reddy; Sangwan, Virender S.; Ali, Mohammed Hasnat; Naik, Milind N.; Singh, Vivek

    2016-01-01

    Ocular Surface Squamous Neoplasm (OSSN) is the neoplasia arising from the conjunctiva, cornea and limbus. OSSN ranges from mild, moderate, severe dysplasia, carcinoma in situ (CIS) to squamous cell carcinoma (SCC). Recent findings on cancer stem cells theory indicate that population of stem-like cell as in neoplasia determines its heterogeneity and complexity leading to varying tumor development of metastatic behavior and recurrence. Cancer stem cell markers are not much explored in the cases of OSSN. In the present study, we aim to evaluate the expression of stem cells using stem cell markers mainly p63, ABCG2, c-KIT (CD117) and CD44 in OSSN tissue, which could have prognostic significance. The present study tries for the first time to explore expression of these stem markers in the cases of OSSN. These cases are subdivided into two groups. One group comprises of carcinoma in situ (n = 6) and the second group comprises of invasive carcinoma (n = 6). The mean age at presentation was 52 years; with 53 years for CIS group and 52 years for SCC group. From each group section from the paraffin block were taken for the IHC staining of p63, c-Kit, ABCG2 and CD44. Our experiments show high expression of P63 and CD44 in the cases of CIN and SCC. Both CIS and SCC displayed positive staining with p63, with more than 80% cells staining positive. However minimal expression of c-kit in both CIN and SCC. But surprisingly we got high expression of ABCG2 in cases of carcinoma in situ as compared to that of invasive squamous cell carcinoma. More than 50% of cells showed CD44 positivity in both CIS and SCC groups. Our results show for the first time that these four stem cells especially the limbal epithelium stem cells play a vital role in the genesis of OSSN but we need to explore more cases before establishing its clinical and biological significance. PMID:27584160

  17. Differences of cell surface marker expression between bone marrow- and kidney-derived murine mesenchymal stromal cells and fibroblasts.

    PubMed

    Cakiroglu, F; Osbahr, J W; Kramer, J; Rohwedel, J

    2016-10-31

    Mesenchymal stromal cells (MSC) are undifferentiated, multipotent adult cells with regenerative properties. They are particularly relevant for therapeutic approaches due to the simplicity of their isolation and cultivation. Since MSC show an expression pattern of cell surface marker, which is almost identical to fibroblasts, many attempts have been made to address the similarities and differences between MSC and fibroblasts. In this study we aimed to isolate murine MSC from bone marrow (BM) and kidney to characterize them in comparison to fibroblasts. Cells were isolated from murine kidney, BM and abdominal skin by plastic adherence and subsequently characterized by analysing their capability to build colony-forming unit-fibroblasts (CFU-F), their morphology, their proliferation, expression of telomerase activity and cell surface antigens as well as their differentiation capacity. Plastic adherent cells from the 3 mouse tissues showed similar morphology, proliferation profiles and CFU-F building capacities. However, while MSC from BM and kidney differentiated into the adipogenic, chondrogenic and osteogenic direction, fibroblasts were not able to do so efficiently. In addition, a tendency for lower expression of telomerase was found in the fibroblast population. Proliferating cells from kidney and BM expressed the MSC-specific cell surface markers CD105 and Sca-1 on a significantly higher and CD117 on a significantly lower level compared to fibroblasts and were thereby distinguishable from fibroblasts. Furthermore, we found that certain CD markers were specifically expressed on a higher level, either in BM-derived cells or fibroblasts. This study demonstrates that murine MSC isolated from different organs express certain specific markers, which enable their discrimination.

  18. Prominin-1 (CD133) Expression in the Prostate and Prostate Cancer: A Marker for Quiescent Stem Cells.

    PubMed

    Pellacani, Davide; Oldridge, Emma E; Collins, Anne T; Maitland, Norman J

    2013-01-01

    The origin and phenotype of stem cells in human prostate cancer remains a subject of much conjecture. In this scenario, CD133 has been successfully used as a stem cell marker in both normal prostate and prostate cancer. However, cancer stem cells have been identified without the use of this marker, opening up the possibility of a CD133 negative cancer stem cell. In this chapter, we review the current literature regarding prostate cancer stem cells, with specific reference to the expression of CD133 as a stem cell marker to identify and purify stem cells in normal prostate epithelium and prostate cancer.

  19. Phenotypic heterogeneity, novel diagnostic markers, and target expression profiles in normal and neoplastic human mast cells.

    PubMed

    Valent, Peter; Cerny-Reiterer, Sabine; Herrmann, Harald; Mirkina, Irina; George, Tracy I; Sotlar, Karl; Sperr, Wolfgang R; Horny, Hans-Peter

    2010-09-01

    Mast cells (MC) are specialized immune cells that play a key role in anaphylactic reactions. Growth, differentiation, and function of these cells are regulated by a complex network of cytokines, surface receptors, signaling molecules, the microenvironment, and the genetic background. A number of previous and more recent data suggest that MC are heterogeneous in terms of cytokine-regulation, expression of cytoplasmic and cell surface antigens, and response to ligands. MC heterogeneity is often organ-specific and is considered to be related to MC plasticity, disease-associated factors, and the maturation stage of the cells. The stem cell factor (SCF) receptor KIT (CD117) is expressed on all types of MC independent of maturation and activation-status. In systemic mastocytosis (SM), KIT is often expressed in MC in a mutated and constitutively activated form. In these patients, MC aberrantly display CD2 and CD25, diagnostic markers of neoplastic MC in all SM variants. In advanced SM, MC co-express substantial amounts of CD30, whereas CD2 expression on MC may be decreased compared to indolent SM. Other surface molecules, such as CD63 or CD203c, are overexpressed on neoplastic MC in SM, and are further upregulated upon cross-linking of the IgE receptor. Some of the cell surface antigens expressed on MC or their progenitors may serve as therapeutic targets in the future. These targets include CD25, CD30, CD33, CD44, and CD117/KIT. The current article provides an overview on cell surface antigens and target receptors expressed by MC in physiologic and reactive tissues, and in patients with SM, with special reference to phenotypic heterogeneity and clinical implications.

  20. A conditional transgenic mouse line for targeted expression of the stem cell marker LGR5.

    PubMed

    Norum, Jens Henrik; Bergström, Åsa; Andersson, Agneta Birgitta; Kuiper, Raoul V; Hoelzl, Maria A; Sørlie, Therese; Toftgård, Rune

    2015-08-15

    LGR5 is a known marker of embryonic and adult stem cells in several tissues. In a mouse model, Lgr5+ cells have shown tumour-initiating properties, while in human cancers, such as basal cell carcinoma and colon cancer, LGR5 expression levels are increased: however, the effect of increased LGR5 expression is not fully understood. To study the effects of elevated LGR5 expression levels we generated a novel tetracycline-responsive, conditional transgenic mouse line expressing human LGR5, designated TRELGR5. In this transgenic line, LGR5 expression can be induced in any tissue depending on the expression pattern of the chosen transcriptional regulator. For the current study, we used transgenic mice with a tetracycline-regulated transcriptional transactivator linked to the bovine keratin 5 promoter (K5tTA) to drive expression of LGR5 in the epidermis. As expected, expression of human LGR5 was induced in the skin of double transgenic mice (K5tTA;TRELGR5). Inducing LGR5 expression during embryogenesis and early development resulted in macroscopically and microscopically detectable phenotypic changes, including kink tail, sparse fur coat and enlarged sebaceous glands. The fur and sebaceous gland phenotypes were reversible upon discontinued expression of transgenic LGR5, but this was not observed for the kink tail phenotype. There were no apparent phenotypic changes if LGR5 expression was induced at three weeks of age. The results demonstrate that increased expression of LGR5 during embryogenesis and the neonatal period alter skin development and homeostasis.

  1. FGFR Family Members Protein Expression as Prognostic Markers in Oral Cavity and Oropharyngeal Squamous Cell Carcinoma.

    PubMed

    Koole, Koos; Clausen, Martijn J A M; van Es, Robert J J; van Kempen, Pauline M W; Melchers, Lieuwe J; Koole, Ron; Langendijk, Johannes A; van Diest, Paul J; Roodenburg, Jan L N; Schuuring, Ed; Willems, Stefan M

    2016-08-01

    Fibroblast growth factor receptor family member proteins (FGFR1-4) have been identified as promising novel therapeutic targets and prognostic markers in a wide spectrum of solid tumors. The present study investigates the expression and prognostic value of four FGFR family member proteins in a large multicenter oral cavity squamous cell carcinoma (OCSCC) and oropharyngeal squamous cell carcinoma (OPSCC) cohort. Protein expression of FGFR1-4 was determined by immunohistochemistry on tissue microarrays containing 951 formalin-fixed paraffin embedded OCSCC and OPSCC tissues from the University Medical Center Utrecht and University Medical Center Groningen. Protein expression was correlated to overall survival using Cox regression models, and bootstrapping was performed as internal validation. FGFR proteins were highly expressed in 39-64 % of OCSCC and 63-79 % of OPSCC. Seventy-three percent (299/412) of OCSCC and 85 % (305/357) of OPSCC highly co-expressed two or more FGFR family member proteins. FGFR1 protein was more frequently highly expressed in human papillomavirus (HPV)-negative OPSCC than HPV-positive OPSCC (82 vs. 65 %; p = 0.008). Furthermore, protein expression of FGFR family members was not related to overall survival in OCSCC or OPSCC (p > 0.05). FGFR family members are frequently highly expressed in OCSCC and OPSCC. These FGFR family member proteins are therefore potential targets for novel therapies that are urgently required to improve survival of OCSCC and OPSCC patients.

  2. Differential Expression of Stem Cell Markers in Human Adamantinomatous Craniopharyngioma and Pituitary Adenoma.

    PubMed

    Chang, Claudia Veiga; Araujo, Ricardo Vieira; Cirqueira, Cinthya Santos; Cani, Carolina Maria Gomes; Matushita, Hamilton; Cescato, Valter Angelo Sperling; Fragoso, Maria Candida Barisson Villares; Bronstein, Marcello Delano; Zerbini, Maria Claudia Nogueira; Mendonca, Berenice Bilharinho; Carvalho, Luciani Renata

    2017-01-01

    Although craniopharyngioma (CP) is histologically benign, it is a pituitary tumour that grows rapidly and often recurs. Adamantinomatous CP (ACP) was associated with an activating mutation in β-catenin, and it has been postulated that pituitary stem cells might play a role in oncogenesis in human ACP. Stem cells have also been identified in pituitary adenoma. Our aim was to characterize the expression pattern of ABCG2, CD44, DLL4, NANOG, NOTCH2, POU5F1/OCT4, SOX2, and SOX9 stem cell markers in human ACP and pituitary adenoma. We studied 33 patients (9 ACP and 24 adenoma) using real-time quantitative PCR (RT-qPCR) and immunohistochemistry. SOX9 was up-regulated in ACP, exhibiting positive immunostaining in the epithelium and stroma, with the highest expression in patients with recurrence. CD44 was overexpressed in ACP as confirmed by immunohistochemistry. SOX2 did not significantly differ among the tumour types. The RT-qPCR array showed an increased expression of MKI67,OCT4/POU5F1, and DLL4 in all tumours. NANOG was decreased in ACP. ABCG2 was down-regulated in most of the tumours. NOTCH2 was significantly decreased in the adenomas. Our results confirm the presence of stem cell markers in human pituitary tumours as well as the different expression patterns of ACP and adenoma. These findings suggest that ACP may originate from a more undifferentiated cell cluster. Additionally, SOX9 immunodetection in the stroma and the highest expression levels related to the relapse of patients suggest a contribution to the aggressive behaviour and high recurrence of this tumour type. © 2016 S. Karger AG, Basel.

  3. Cell cycle markers have different expression and localization patterns in neuron-like PC12 cells and primary hippocampal neurons.

    PubMed

    Negis, Yesim; Unal, Aysegul Yildiz; Korulu, Sirin; Karabay, Arzu

    2011-06-01

    Neuron-like PC12 cells are extensively used in place of neurons in published studies. Aim of this paper has been to compare mRNA and protein expressions of cell cycle markers; cyclinA, B, D, E; Cdk1, 2 and 4; and p27 in post-mitotic primary hippocampal neurons, mitotically active PC12 cells and NGF-differentiated post-mitotic PC12 cells. Contrary to PC12 cells, in neurons, the presence of all these markers was detected only at mRNA level; except for cyclinA, cyclinE and Cdk4, which were detectable also at protein levels. In both NGF-treated PC12 cells and neurons, cyclinE was localized only in the nucleus. In NGF-treated PC12 cells cyclinD and Cdk4 were localized in the nucleus while, in neurons cyclinD expression was not detectable; Cdk4 was localized in the cytoplasm. In neurons, cyclinA was nuclear, whereas in NGF-treated PC12 cells, it was localized in the cell body and along the processes. These results suggest that PC12 cells and primary neurons are different in terms of cell cycle protein expressions and localizations. Thus, it may not be very appropriate to use these cells as neuronal model system in order to understand neuronal physiological activities, upstream of where may lie cell cycle activation triggered events.

  4. Bax expression measured by AQUAnalysis is an independent prognostic marker in oral squamous cell carcinoma

    PubMed Central

    2012-01-01

    Background Resistance to apoptosis is a hallmark of cancer and proteins regulating apoptosis have been proposed as prognostic markers in several malignancies. However, the prognostic impact of apoptotic markers has not been consistently demonstrated in oral squamous cell carcinoma (OSCC). This inconsistency in reported associations between apoptotic proteins and prognosis can be partly attributed to the intrinsic low resolution and misclassification associated with manual, semi-quantitative methods of biomarker expression measurement. The aim of this study was to examine the association between apoptosis-regulating proteins and clinical outcomes in oral squamous cell carcinoma (OSCC) using the quantitative fluorescence immunohistochemistry (IHC) based AQUAnalysis technique. Methods Sixty-nine OSCC patients diagnosed between 1998–2005 in Calgary, Alberta, Canada were included in the study. Clinical data were obtained from the Alberta Cancer Registry and chart review. Tissue microarrays (TMAs) were assembled from triplicate cores of formalin-fixed paraffin embedded pre-treatment tumour tissue. Bax, Bcl-2 and Bcl-XL protein expression was quantified using fluorescent IHC and AQUA technology in normal oral cavity squamous epithelium (OCSE) and OSCC tumour samples. Survival was analyzed using Kaplan-Meier plots and the Cox proportional hazard model. Results Bax expression was predominantly nuclear in OCSE and almost exclusively cytoplasmic in OSCC. No similar differences in localization were observed for Bcl-2 or Bcl-XL. Only Bax expression associated with disease-specific survival (DSS), with 5-year survival estimates of 85.7% for high Bax versus 50.3% for low Bax (p = 0.006), in univariate analysis. High Bax expression was also significantly associated with elevated Ki67 expression, indicating that increased proliferation might lead to an improved response to radiotherapy in patients with elevated Bax expression. In multivariate analyses, Bax protein expression

  5. T cells in chronic lymphocytic leukemia display dysregulated expression of immune checkpoints and activation markers

    PubMed Central

    Palma, Marzia; Gentilcore, Giusy; Heimersson, Kia; Mozaffari, Fariba; Näsman-Glaser, Barbro; Young, Emma; Rosenquist, Richard; Hansson, Lotta; Österborg, Anders; Mellstedt, Håkan

    2017-01-01

    Chronic lymphocytic leukemia is characterized by impaired immune functions largely due to profound T-cell defects. T-cell functions also depend on co-signaling receptors, inhibitory or stimulatory, known as immune checkpoints, including cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) and programmed death-1 (PD-1). Here we analyzed the T-cell phenotype focusing on immune checkpoints and activation markers in chronic lymphocytic leukemia patients (n=80) with different clinical characteristics and compared them to healthy controls. In general, patients had higher absolute numbers of CD3+ cells and the CD8+ subset was particularly expanded in previously treated patients. Progressive patients had higher numbers of CD4+ and CD8+ cells expressing PD-1 compared to healthy controls, which was more pronounced in previously treated patients (P=0.0003 and P=0.001, respectively). A significant increase in antigen-experienced T cells was observed in patients within both the CD4+ and CD8+ subsets, with a significantly higher PD-1 expression. Higher numbers of CD4+ and CD8+ cells with intracellular CTLA-4 were observed in patients, as well as high numbers of proliferating (Ki67+) and activated (CD69+) CD4+ and CD8+ cells, more pronounced in patients with active disease. The numbers of Th1, Th2, Th17 and regulatory T cells were substantially increased in patients compared to controls (P<0.05), albeit decreasing to low levels in pre-treated patients. In conclusion, chronic lymphocytic leukemia T cells display increased expression of immune checkpoints, abnormal subset distribution, and a higher proportion of proliferating cells compared to healthy T cells. Disease activity and previous treatment shape the T-cell profile of chronic lymphocytic leukemia patients in different ways. PMID:27927767

  6. T cells in chronic lymphocytic leukemia display dysregulated expression of immune checkpoints and activation markers.

    PubMed

    Palma, Marzia; Gentilcore, Giusy; Heimersson, Kia; Mozaffari, Fariba; Näsman-Glaser, Barbro; Young, Emma; Rosenquist, Richard; Hansson, Lotta; Österborg, Anders; Mellstedt, Håkan

    2017-03-01

    Chronic lymphocytic leukemia is characterized by impaired immune functions largely due to profound T-cell defects. T-cell functions also depend on co-signaling receptors, inhibitory or stimulatory, known as immune checkpoints, including cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) and programmed death-1 (PD-1). Here we analyzed the T-cell phenotype focusing on immune checkpoints and activation markers in chronic lymphocytic leukemia patients (n=80) with different clinical characteristics and compared them to healthy controls. In general, patients had higher absolute numbers of CD3(+) cells and the CD8(+) subset was particularly expanded in previously treated patients. Progressive patients had higher numbers of CD4(+) and CD8(+) cells expressing PD-1 compared to healthy controls, which was more pronounced in previously treated patients (P=0.0003 and P=0.001, respectively). A significant increase in antigen-experienced T cells was observed in patients within both the CD4(+) and CD8(+) subsets, with a significantly higher PD-1 expression. Higher numbers of CD4(+) and CD8(+) cells with intracellular CTLA-4 were observed in patients, as well as high numbers of proliferating (Ki67(+)) and activated (CD69(+)) CD4(+) and CD8(+) cells, more pronounced in patients with active disease. The numbers of Th1, Th2, Th17 and regulatory T cells were substantially increased in patients compared to controls (P<0.05), albeit decreasing to low levels in pre-treated patients. In conclusion, chronic lymphocytic leukemia T cells display increased expression of immune checkpoints, abnormal subset distribution, and a higher proportion of proliferating cells compared to healthy T cells. Disease activity and previous treatment shape the T-cell profile of chronic lymphocytic leukemia patients in different ways. Copyright© Ferrata Storti Foundation.

  7. Fusion-derived epithelial cancer cells express hematopoietic markers and contribute to stem cell and migratory phenotype in ovarian carcinoma.

    PubMed

    Ramakrishnan, Mallika; Mathur, Sandeep R; Mukhopadhyay, Asok

    2013-09-01

    For a long time, the external milieu of cancer cells was considered to be of secondary importance when compared with its intrinsic properties. That has changed now as the microenvironment is considered to be a major contributing factor toward the progression of tumor. In this study, we show that in human and mouse epithelial ovarian carcinoma and mouse lung carcinoma, the interaction between tumor-infiltrating hematopoietic cells and epithelial cancer cells results in their fusion. Intriguingly, even after the fusion event, cancer cells retain the expression of the pan-hematopoietic marker (CD45) and various markers of hematopoietic lineage, including those of hematopoietic stem cells, indicating that the hematopoietic genome is not completely reprogrammed. This observation may have implications on the bone marrow contribution to the cancer stem cell population. Interestingly, it was seen that in both cancer models, the expression of chemokine receptor CXCR4 was largely contributed to by the fused compartment of cancer cells. We hypothesize that the superior migratory potential gained by the cancer cells due to the fusion helps in its dissemination to various secondary organs upon activation of the CXCR4/CXCL12 axis. We are the first to report the presence of a hemato-epithelial cancer compartment, which contributes to stem cell markers and CXCR4 in epithelial carcinoma. This finding has repercussions on CXCR4-based therapeutics and opens new avenues in discovering novel molecular targets against fusion and metastasis.

  8. Increased expression of differentiation markers can accompany laminin-induced attachment of small cell lung cancer cells.

    PubMed Central

    Giaccone, G.; Broers, J.; Jensen, S.; Fridman, R. I.; Linnoila, R.; Gazdar, A. F.

    1992-01-01

    We investigated the interaction between human lung cancer cells, laminin, and several differentiating agents. When grown on laminin coated substrate eight out of 11 small cell lung cancer (SCLC) cell lines exhibited attachment to laminin and three had extensive outgrowth of long neurite-like processes. Of seven non-small cell lung cancer cell lines, selected for their in vitro anchorage-independent growth, attachment was observed in only three cell lines, and process formation was far less extensive than in SCLC cell lines. Among several differentiating agents, only dcAMP, which alone induced attachment and some process formation, increased laminin-mediated attachment and process formation of two SCLC cell lines, NCI-N417 a variant cell line, and NCI-H345, a classic cell line. The expression of several neuroendocrine and neuronal markers was investigated in these two SCLC cell lines. The expression of the light subunit of neurofilaments increased in NCI-N417 within 3 to 4 days of seeding, while NCI-H345 exhibited approximately 5 fold increase in expression of the GRP gene and a 3 fold increase expression of the beta-actin gene. The expression of a number of other neuroendocrine and neuronal markers did not change following growth on laminin. The doubling times remained unchanged independent of the presence of and attachment to laminin while topoisomerase II gene expression levels in NCI-N417 cells decreased approximately 5 fold when cells were growing on laminin. Images Figure 2 Figure 3 Figure 4 Figure 5 PMID:1325826

  9. ERK activation and expression of neuronal cell cycle markers in the hippocampus after entorhinal cortex lesion.

    PubMed

    Hernández-Ortega, Karina; Arias, Clorinda

    2012-11-01

    Current findings suggest that neuronal cell death is frequently associated with the aberrant expression of cell cycle-regulatory proteins in postmitotic neurons. Aberrant cell cycle reentry has been implicated in diverse neurodegenerative conditions, including Alzheimer's disease (AD). Previously we reported that the appearance of cell cycle markers in postmitotic neurons of the entorhinal cortex (EC) after excitotoxic hippocampal damage is associated with the expression of phospho-tau and amyloid precursor protein (APP). However, the question of the signaling pathway involved in this cell cycle reentry remains unresolved. Differentiated neurons use the molecular mechanisms initially acquired to direct cell proliferation, such as the Ras-extracellular signal-regulated kinase (ERK1/2) pathway, to regulate synaptic plasticity. In this work we explored whether ERK1/2-related signaling might contribute to the cell cycle reentry in hippocampal neurons after a unilateral EC lesion. We showed that, within the first 24 hr after hippocampal deafferentation, numerous neurons expressed phospho-ERK1/2, concomitantly with the gradual increases in cyclin D1 and cyclin B immunoreactivity in the dentate gyrus and hilus. Several of these immunopositive cells to phospho-ERK1/2 and cyclin B in hippocampus are postmitotic neurons, insofar as they are positive to NeuN. The intracisternal administration of U0126 (an MEK inhibitor), previous to the excitotoxic lesion, decreased the activation of ERK1/2 and the expression of cyclin D1 and cyclin B in the hippocampus. The present findings support the notion that ERK1/2 plays a role in cell cycle reactivation in mature neurons efferently connected to the lesion site. Copyright © 2012 Wiley Periodicals, Inc.

  10. MGFM: a novel tool for detection of tissue and cell specific marker genes from microarray gene expression data.

    PubMed

    El Amrani, Khadija; Stachelscheid, Harald; Lekschas, Fritz; Kurtz, Andreas; Andrade-Navarro, Miguel A

    2015-08-28

    Identification of marker genes associated with a specific tissue/cell type is a fundamental challenge in genetic and cell research. Marker genes are of great importance for determining cell identity, and for understanding tissue specific gene function and the molecular mechanisms underlying complex diseases. We have developed a new bioinformatics tool called MGFM (Marker Gene Finder in Microarray data) to predict marker genes from microarray gene expression data. Marker genes are identified through the grouping of samples of the same type with similar marker gene expression levels. We verified our approach using two microarray data sets from the NCBI's Gene Expression Omnibus public repository encompassing samples for similar sets of five human tissues (brain, heart, kidney, liver, and lung). Comparison with another tool for tissue-specific gene identification and validation with literature-derived established tissue markers established functionality, accuracy and simplicity of our tool. Furthermore, top ranked marker genes were experimentally validated by reverse transcriptase-polymerase chain reaction (RT-PCR). The sets of predicted marker genes associated with the five selected tissues comprised well-known genes of particular importance in these tissues. The tool is freely available from the Bioconductor web site, and it is also provided as an online application integrated into the CellFinder platform ( http://cellfinder.org/analysis/marker ). MGFM is a useful tool to predict tissue/cell type marker genes using microarray gene expression data. The implementation of the tool as an R-package as well as an application within CellFinder facilitates its use.

  11. Patterns of Expression of Vaginal T-Cell Activation Markers during Estrogen-Maintained Vaginal Candidiasis.

    PubMed

    Al-Sadeq, Ameera; Hamad, Mawieh; Abu-Elteen, Khaled

    2008-12-15

    : The immunosuppressive activity of estrogen was further investigated by assessing the pattern of expression of CD25, CD28, CD69, and CD152 on vaginal T cells during estrogen-maintained vaginal candidiasis. A precipitous and significant decrease in vaginal fungal burden toward the end of week 3 postinfection was concurrent with a significant increase in vaginal lymphocyte numbers. During this period, the percentage of CD3+, CD3+CD4+, CD152+, and CD28+ vaginal T cells gradually and significantly increased. The percentage of CD3+ and CD3+CD4+ cells increased from 43% and 15% at day 0 to 77% and 40% at day 28 postinfection. Compared with 29% CD152+ vaginal T cells in naive mice, > 70% of vaginal T cells were CD152+ at day 28 postinfection. In conclusion, estrogen-maintained vaginal candidiasis results in postinfection time-dependent changes in the pattern of expression of CD152, CD28, and other T-cell markers, suggesting that T cells are subject to mixed suppression and activation signals.

  12. Patterns of Expression of Vaginal T-Cell Activation Markers during Estrogen-Maintained Vaginal Candidiasis

    PubMed Central

    2008-01-01

    The immunosuppressive activity of estrogen was further investigated by assessing the pattern of expression of CD25, CD28, CD69, and CD152 on vaginal T cells during estrogen-maintained vaginal candidiasis. A precipitous and significant decrease in vaginal fungal burden toward the end of week 3 postinfection was concurrent with a significant increase in vaginal lymphocyte numbers. During this period, the percentage of CD3+, CD3+CD4+, CD152+, and CD28+ vaginal T cells gradually and significantly increased. The percentage of CD3+ and CD3+CD4+ cells increased from 43% and 15% at day 0 to 77% and 40% at day 28 postinfection. Compared with 29% CD152+ vaginal T cells in naive mice, > 70% of vaginal T cells were CD152+ at day 28 postinfection. In conclusion, estrogen-maintained vaginal candidiasis results in postinfection time-dependent changes in the pattern of expression of CD152, CD28, and other T-cell markers, suggesting that T cells are subject to mixed suppression and activation signals. PMID:20525139

  13. FAS ligand expression in inflammatory infiltrate lymphoid cells as a prognostic marker in oral squamous cell carcinoma.

    PubMed

    Peterle, G T; Santos, M; Mendes, S O; Carvalho-Neto, P B; Maia, L L; Stur, E; Agostini, L P; Silva, C V M; Trivilin, L O; Nunes, F D; Carvalho, M B; Tajara, E H; Louro, I D; Silva-Conforti, A M A

    2015-09-22

    Currently, the most important prognostic factor in oral squamous cell carcinoma (OSCC) is the presence of regional lymph node metastases, which correlates with a 50% reduction in life expectancy. We have previously observed that expression of hypoxia genes in the tumor inflammatory infiltrate is statistically related to prognosis in OSCC. FAS and FASL expression levels in OSCC have previously been related to patient survival. The present study analyzed the relationship between FASL expression in the inflammatory infiltrate lymphoid cells and clinical variables, tumor histology, and prognosis of OSCC. Strong FASL expression was significantly associated with lymph node metastases (P = 0.035) and disease-specific death (P = 0.014), but multivariate analysis did not confirm FASL expression as an independent death risk factor (OR = 2.78, 95%CI = 0.81-9.55). Disease-free and disease-specific survival were significantly correlated with FASL expression (P = 0.016 and P = 0.005, respectively). Multivariate analysis revealed that strong FASL expression is an independent marker for earlier disease relapse and disease-specific death, with approximately 2.5-fold increased risk compared with weak expression (HR = 2.24, 95%CI = 1.08-4.65 and HR = 2.49, 95%CI = 1.04-5.99, respectively). Our results suggest a potential role for this expression profile as a tumor prognostic marker in OSCC patients.

  14. Radiation Dose-effects on Cell Cycle, Apoptosis, and Marker Expression of Ataxia Telangiectasia-Heterozygous Human Breast Epithelial Cells

    NASA Technical Reports Server (NTRS)

    Cruz, A.; Bors, K.; Jansen, H.; Richmond, R.

    2003-01-01

    Ataxia-telangiectasia (A-T) is a radiation-sensitive genetic condition. AT-heterozygous human mammary epithelial cells (HMEC) were irradiated using a Cs137 source in order to compare cell cycle, apoptosis, and marker expression responses across 3 radiation doses. No differences in cell cycle and apoptosis were found with any of the radiation doses used (30, 60, and 90 rads) compared with the unirradiated control (0 rad). At the same doses, however, differences were found in marker expression, such as keratin 18 (kl8), keratin 14 (k14), insulin-like growth factor I receptor (IGF-IR), and connexin 43 (cx43). This may indicate that radiation sensitivity in the heterozygous state may be initiated through signal transduction responses.

  15. Radiation Dose-effects on Cell Cycle, Apoptosis, and Marker Expression of Ataxia Telangiectasia-Heterozygous Human Breast Epithelial Cells

    NASA Technical Reports Server (NTRS)

    Cruz, A.; Bors, K.; Jansen, H.; Richmond, R.

    2003-01-01

    Ataxia-telangiectasia (A-T) is a radiation-sensitive genetic condition. AT-heterozygous human mammary epithelial cells (HMEC) were irradiated using a Cs137 source in order to compare cell cycle, apoptosis, and marker expression responses across 3 radiation doses. No differences in cell cycle and apoptosis were found with any of the radiation doses used (30, 60, and 90 rads) compared with the unirradiated control (0 rad). At the same doses, however, differences were found in marker expression, such as keratin 18 (kl8), keratin 14 (k14), insulin-like growth factor I receptor (IGF-IR), and connexin 43 (cx43). This may indicate that radiation sensitivity in the heterozygous state may be initiated through signal transduction responses.

  16. Human osteoblasts derived from mesenchymal stem cells express adipogenic markers upon coculture with bone marrow adipocytes.

    PubMed

    Clabaut, Aline; Delplace, Séverine; Chauveau, Christophe; Hardouin, Pierre; Broux, Odile

    2010-07-01

    In osteoporosis, bone loss is accompanied by greater adiposity in the marrow. Given the cellular proximity within the bone marrow, we wondered whether adipocytes might have a paracrine impact on osteoblast differentiation. To test this hypothesis, we cocultured adipocytes with osteoblasts derived from mesenchymal stem cells (MSCs) in the absence of direct cell contact and then analyzed gene expression changes in the osteoblastic population by using real-time reverse transcription polymerase chain reaction. We found that, upon coculture, MSC-derived osteoblasts showed appearance of adipogenic (lipoprotein lipase, leptin) and decrease of osteogenic (osteocalcin) mRNA markers. Our results indicate that in vitro, MSC-derived adipocytes are capable of inducing MSC-derived osteoblasts to differentiate to an adipocyte phenotype. These new data suggest that (i) transdifferentiation of committed osteoblasts into adipocytes may contribute to the increase in marrow fat content at the expense of bone-forming cells and (ii) this switch might be initiated by the adipocytes themselves.

  17. The expression of cancer stem cell markers in human colorectal carcinoma cells in a microenvironment dependent manner.

    PubMed

    Stankevicius, Vaidotas; Kunigenas, Linas; Stankunas, Edvinas; Kuodyte, Karolina; Strainiene, Egle; Cicenas, Jonas; Samalavicius, Narimantas E; Suziedelis, Kestutis

    2017-03-18

    Numerous lines of evidence support the hierarchical model of cancer development and tumor initiation. According to the theory, cancer stem cells play a crucial role in the formation of the tumor and should be targeted for more effective anticancer treatment. However, cancer stem cells quickly loose their characteristics when propagated as 2D cell culture, indicating that the 2D cell culture does not provide the appropriate settings to maintain an in vivo environment. In this study we have investigated the expression of self-renewal, cancer stem cell and epithelial to mesenchymal transition markers after the transfer of human colorectal carcinoma cell DLD1 and HT29 lines from 2D cell cultures to scaffold-attached laminin rich extracellular matrix and scaffold-free multicellular spheroid 3D culture models. Based on the up-regulated expression of multipotency, CSC and EMT markers, our data suggests that human colorectal carcinoma cells grown in 3D exhibit enhanced cancer stem cell characteristics. Therefore, in order to design more efficient targeted therapies, we suggest that 3D cell culture models should be employed in cancer stem cell research. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  18. Endothelial marker-expressing stromal cells are critical for kidney formation.

    PubMed

    Mukherjee, Elina; Maringer, Katherine; Papke, Emily; Bushnell, Daniel; Schaefer, Caitlin; Kramann, Rafael; Ho, Jacqueline; Humphreys, Benjamin D; Bates, Carlton; Sims-Lucas, Sunder

    2017-09-01

    Kidneys are highly vascularized and contain many distinct vascular beds. However, the origins of renal endothelial cells and roles of the developing endothelia in the formation of the kidney are unclear. We have shown that the Foxd1-positive renal stroma gives rise to endothelial marker-expressing progenitors that are incorporated within a subset of peritubular capillaries; however, the significance of these cells is unclear. The purpose of this study was to determine whether deletion of Flk1 in the Foxd1 stroma was important for renal development. To that end, we conditionally deleted Flk1 (critical for endothelial cell development) in the renal stroma by breeding-floxed Flk1 mice (Flk1(fl/fl) ) with Foxd1cre mice to generate Foxd1cre; Flk1(fl/fl) (Flk1(ST-/-) ) mice. We then performed FACsorting, histological, morphometric, and metabolic analyses of Flk1(ST-/-) vs. control mice. We confirmed decreased expression of endothelial markers in the renal stroma of Flk1(ST-/-) kidneys via flow sorting and immunostaining, and upon interrogation of embryonic and postnatal Flk1(ST-/-) mice, we found they had dilated peritubular capillaries. Three-dimensional reconstructions showed reduced ureteric branching and fewer nephrons in developing Flk1(ST-/-) kidneys vs. Juvenile Flk1(ST-/-) kidneys displayed renal papillary hypoplasia and a paucity of collecting ducts. Twenty-four-hour urine collections revealed that postnatal Flk1(ST-/-) mice had urinary-concentrating defects. Thus, while lineage-tracing revealed that the renal cortical stroma gave rise to a small subset of endothelial progenitors, these Flk1-expressing stromal cells are critical for patterning the peritubular capillaries. Also, loss of Flk1 in the renal stroma leads to nonautonomous-patterning defects in ureteric lineages. Copyright © 2017 the American Physiological Society.

  19. Tumour budding and the expression of cancer stem cell marker aldehyde dehydrogenase 1 in nasopharyngeal carcinoma.

    PubMed

    Luo, Wei-Ren; Gao, Fei; Li, Si-Yi; Yao, Kai-Tai

    2012-12-01

    To detect the prognostic significance of tumour budding and its expression of aldehyde dehydrogenase 1 (ALDH1) in nasopharyngeal carcinoma (NPC). Tumour budding was investigated in 105 patients with NPC by immunohistochemistry for pan-cytokeratin (AE1/AE3). The intensity of budding correlated strongly with T classification (P=0.008), lymphatic invasion (P<0.001), vascular invasion (P=0.029), lymph node metastasis (P < 0.001), and clinical stage (P=0.010). Univariate analysis revealed that patients with high budding grade had poorer survival than those with low grade (P=0.002). Multivariate analysis showed that tumour budding was an independent predictor of survival (P=0.001). Furthermore, budding cells showed high-level expression of the cancer stem cell (CSC) marker ALDH1. Budding cells with high-level ALDH1 expression contributed to several aggressive behaviours and poor survival (P=0.000).   We describe, for the first time, the presence of tumour budding and its correlation with aggressive tumour behaviour and poor patient survival in NPC. The degree of tumour budding could be a valuable predictive factor in NPC. In addition, we show, also for the first time, that budding cells in NPC might possess the invasive and metastatic properties of CSCs. © 2012 Blackwell Publishing Limited.

  20. Expression of a set of glial cell-specific markers in the Drosophila embryonic central nervous system.

    PubMed

    Ahn, Hui Jeong; Jeon, Sang-Hak; Kim, Sang Hee

    2014-06-01

    The types of glia in the central nervous system (CNS) of the Drosophila embryo include longitudinal glia (LG), cell body glia (CBG), and peripheral glia (PG). Transcription factors, such as glial cell missing and reverse polarity, are well-established general glial cell markers. Only a few glial cell-specific markers have been identified in the Drosophila embryonic CNS, thus far. In the present study, we employed the glial cell-specific markers for LG (vir-1/CG5453 and CG31235), CBG (fabp/CG6783 and CG11902), and PG (CG2310 and moody/CG4322), and comprehensively analyzed their expression patterns, during the embryonic CNS development. Our study validated the specificity of a set of glial markers, and further revealed their spatio-temporal expression patterns, which will aid in the understanding of the developmental lineage, and investigating their role in the development and homeostasis of the Drosophila CNS in vivo.

  1. Expression of surface markers on the blood cells during the delayed asthmatic response to allergen challenge

    PubMed Central

    2014-01-01

    Patients with bronchial asthma develop various types of asthmatic response to bronchial challenge with allergen, such as immediate/early asthmatic response (IAR), late asthmatic response (LAR) or delayed asthmatic response (DYAR), because of different immunologic mechanisms. The DYAR, occurring between 24 and 56 hours after the bronchial allergen challenge (p < 0.01), differs from IAR and LAR in clinical as well as immunologic features. This study investigates the expression of CD molecules (markers) on the surface of particular cell populations in the peripheral blood and their changes during the DYAR. In 17 patients developing the DYAR (p < 0.01), the bronchial challenge with allergen was repeated 2–6 weeks later. The repeated DYAR (p < 0.001) was combined with recording of CD molecule expression on various types of blood cells by means of flow cytometry up to 72 hours after the challenge. The results were expressed in percent of the mean relative fluorescence intensity. The DYAR was accompanied by (a) increased expression of CD11b, CD11b/18, CD16,CD32, CD35, CD62E, CD62L, CD64, and CD66b on neutrophils; CD203C on basophils; CD25 and CD62L on eosinophils; CD14, CD16, CD64, and CD86 on monocytes; CD3, CD4, CD8, CD11a, CD18, and CD69 on lymphocytes; CD16, CD56, CD57, and CD94 on natural killer (NK) cells; and CD31, CD41, CD61, CD62P, and CD63 on thrombocytes and (b) decreased expression of CD18 and CD62L on eosinophils, CD15 on neutrophils, and CD40 on lymphocytes. These results suggest involvement of cell-mediated hypersensitivity mechanism, on participation of Th1- lymphocytes, neutrophils, monocytes, NK cells, and thrombocytes in the DYAR. PMID:24988283

  2. Expression of the myeloid-associated marker CD33 is not an exclusive factor for leukemic plasmacytoid dendritic cells.

    PubMed

    Garnache-Ottou, Francine; Chaperot, Laurence; Biichle, Sabeha; Ferrand, Christophe; Remy-Martin, Jean-Paul; Deconinck, Eric; de Tailly, Patrick Darodes; Bulabois, Bénédicte; Poulet, Jacqueline; Kuhlein, Emilienne; Jacob, Marie-Christine; Salaun, Véronique; Arock, Michel; Drenou, Bernard; Schillinger, Françoise; Seilles, Estelle; Tiberghien, Pierre; Bensa, Jean-Claude; Plumas, Joel; Saas, Philippe

    2005-02-01

    A new entity of acute leukemia coexpressing CD4(+)CD56(+) markers without any other lineage-specific markers has been identified recently as arising from lymphoid-related plasmacytoid dendritic cells (pDCs). In our laboratory, cells from a patient with such CD4(+)CD56(+) lineage-negative leukemia were unexpectedly found to also express the myeloid marker CD33. To confirm the diagnosis of pDC leukemia despite the CD33 expression, we demonstrated that the leukemic cells indeed exhibited pDC phenotypic and functional properties. In 7 of 8 other patients with CD4(+)CD56(+) pDC malignancies, we were able to confirm that the tumor cells expressed CD33 although with variable expression levels. CD33 expression was shown by flow cytometry, reverse transcriptase-polymerase chain reaction, and immunoblot analysis. Furthermore, CD33 monoclonal antibody stimulation of purified CD4(+)CD56(+) leukemic cells led to cytokine secretion, thus confirming the presence of a functional CD33 on these leukemic cells. Moreover, we found that circulating pDCs in healthy individuals also weakly express CD33. Overall, our results demonstrate that the expression of CD33 on CD4(+)CD56(+) lineage-negative cells should not exclude the diagnosis of pDC leukemia and underline that pDC-specific markers should be used at diagnosis for CD4(+)CD56(+) malignancies.

  3. Increased expression of melanoma stem cell marker CD271 in metastatic melanoma to the brain

    PubMed Central

    Guo, Ruifeng; Fierro-Fine, Amelia; Goddard, Lindsey; Russell, Madison; Chen, Jie; Liu, Cheng Z; Fung, Kar-Ming; Hassell, Lewis A

    2014-01-01

    Human melanoma contains multipotent stem cells that express the neural crest stem cell marker CD271. CD271-expressing melanoma cells in murine xenografts give rise to metastatic tumor. However, a comprehensive clinical investigation of its role in different stages of melanomagenesis has not been well studied. We studied CD271 expression with immunohistochemistry in 11 cases of banal melanocytic nevus, 9 cases of primary cutaneous melanoma, 10 cases of primary mucosal melanoma, 5 cases of metastatic melanoma in regional lymph nodes, and 11 cases of metastatic melanoma in the brain. In addition, 9 cases of metastatic, high-grade adenocarcinomas from breast and lung to the brain were studied as controls. The staining was scored based on the number of positive cells and analyzed by student t-test. All banal melanocytic nevi showed negative to equivocal staining. Primary cutaneous melanomas showed variable patterns, mucosal melanomas were mostly negative, and metastases to lymph nodes ranged from negative to moderate positivity. In contrast, all 11 cases of metastatic melanoma to the brain showed moderate (4 cases) to strong positivity (7 cases). Metastases from lung and breast origin were used as controls and showed negative to weakly positive staining in all but one case. Statistically, CD271 has significantly increased expression in metastatic melanoma to the brain when compared to the other groups studied (P < 0.05). The findings suggest that CD271 expression is specifically increased in metastatic melanoma to the brain. Further prospective study for the role of CD271 in prediction of melanoma brain metastasis as well as prognosis assessment will be of great clinical significance. PMID:25674270

  4. Increased expression of melanoma stem cell marker CD271 in metastatic melanoma to the brain.

    PubMed

    Guo, Ruifeng; Fierro-Fine, Amelia; Goddard, Lindsey; Russell, Madison; Chen, Jie; Liu, Cheng Z; Fung, Kar-Ming; Hassell, Lewis A

    2014-01-01

    Human melanoma contains multipotent stem cells that express the neural crest stem cell marker CD271. CD271-expressing melanoma cells in murine xenografts give rise to metastatic tumor. However, a comprehensive clinical investigation of its role in different stages of melanomagenesis has not been well studied. We studied CD271 expression with immunohistochemistry in 11 cases of banal melanocytic nevus, 9 cases of primary cutaneous melanoma, 10 cases of primary mucosal melanoma, 5 cases of metastatic melanoma in regional lymph nodes, and 11 cases of metastatic melanoma in the brain. In addition, 9 cases of metastatic, high-grade adenocarcinomas from breast and lung to the brain were studied as controls. The staining was scored based on the number of positive cells and analyzed by student t-test. All banal melanocytic nevi showed negative to equivocal staining. Primary cutaneous melanomas showed variable patterns, mucosal melanomas were mostly negative, and metastases to lymph nodes ranged from negative to moderate positivity. In contrast, all 11 cases of metastatic melanoma to the brain showed moderate (4 cases) to strong positivity (7 cases). Metastases from lung and breast origin were used as controls and showed negative to weakly positive staining in all but one case. Statistically, CD271 has significantly increased expression in metastatic melanoma to the brain when compared to the other groups studied (P < 0.05). The findings suggest that CD271 expression is specifically increased in metastatic melanoma to the brain. Further prospective study for the role of CD271 in prediction of melanoma brain metastasis as well as prognosis assessment will be of great clinical significance.

  5. Schlafen-3 decreases cancer stem cell marker expression and autocrine/juxtacrine signaling in FOLFOX-resistant colon cancer cells

    PubMed Central

    Oh, Phil-Sun; Patel, Vaishali B.; Sanders, Matthew A.; Kanwar, Shailender S.; Yu, Yingjie; Nautiyal, Jyoti; Patel, Bhaumik B.

    2011-01-01

    We have previously demonstrated that expression of the novel gene schlafen-3 (Slfn-3) correlates with intestinal epithelial cell differentiation (Patel VB, Yu Y, Das JK, Patel BB, Majumdar AP. Biochem Biophys Res Commun 388: 752–756, 2009). The present investigation was undertaken to examine whether Slfn-3 plays a role in regulating differentiation of FOLFOX-resistant (5-fluorouracil + oxaliplatin) colon cancer cells that are highly enriched in cancer stem cells (CSCs). Transfection of Slfn-3 in FOLFOX-resistant colon cancer HCT-116 cells resulted in increase of alkaline phosphatase activity, a marker of intestinal differentiation. Additionally, Slfn-3 transfection resulted in reduction of mRNA and protein levels of the CSC markers CD44, CD133, CD166, and aldehyde dehydrogenase 1 in both FOLFOX-resistant HCT-116 and HT-29 cells. This was accompanied by decreased formation of tumorosphere/colonosphere (an in vitro model of tumor growth) in stem cell medium and inhibition of expression of the chemotherapeutic drug transporter protein ABCG2. Additionally, Slfn-3 transfection of FOLFOX-resistant HCT-116 and HT-29 cells reduced Hoechst 33342 dye exclusion. Finally, Slfn-3 transfection inhibited the expression of transforming growth factor-α in both FOLFOX-resistant colon cancer cells, but stimulated apoptosis in response to additional FOLFOX treatment. In summary, our data demonstrate that Slfn-3 expression inhibits multiple characteristics of CSC-enriched, FOLFOX-resistant colon cancer cells, including induction of differentiation and reduction in tumorosphere/colonosphere formation, drug transporter activity, and autocrine stimulation of proliferation. Thus Slfn-3 expression may render colon CSCs more susceptible to cancer chemotherapeutics. PMID:21596996

  6. Schlafen-3 decreases cancer stem cell marker expression and autocrine/juxtacrine signaling in FOLFOX-resistant colon cancer cells.

    PubMed

    Oh, Phil-Sun; Patel, Vaishali B; Sanders, Matthew A; Kanwar, Shailender S; Yu, Yingjie; Nautiyal, Jyoti; Patel, Bhaumik B; Majumdar, Adhip P N

    2011-08-01

    We have previously demonstrated that expression of the novel gene schlafen-3 (Slfn-3) correlates with intestinal epithelial cell differentiation (Patel VB, Yu Y, Das JK, Patel BB, Majumdar AP. Biochem Biophys Res Commun 388: 752-756, 2009). The present investigation was undertaken to examine whether Slfn-3 plays a role in regulating differentiation of FOLFOX-resistant (5-fluorouracil + oxaliplatin) colon cancer cells that are highly enriched in cancer stem cells (CSCs). Transfection of Slfn-3 in FOLFOX-resistant colon cancer HCT-116 cells resulted in increase of alkaline phosphatase activity, a marker of intestinal differentiation. Additionally, Slfn-3 transfection resulted in reduction of mRNA and protein levels of the CSC markers CD44, CD133, CD166, and aldehyde dehydrogenase 1 in both FOLFOX-resistant HCT-116 and HT-29 cells. This was accompanied by decreased formation of tumorosphere/colonosphere (an in vitro model of tumor growth) in stem cell medium and inhibition of expression of the chemotherapeutic drug transporter protein ABCG2. Additionally, Slfn-3 transfection of FOLFOX-resistant HCT-116 and HT-29 cells reduced Hoechst 33342 dye exclusion. Finally, Slfn-3 transfection inhibited the expression of transforming growth factor-α in both FOLFOX-resistant colon cancer cells, but stimulated apoptosis in response to additional FOLFOX treatment. In summary, our data demonstrate that Slfn-3 expression inhibits multiple characteristics of CSC-enriched, FOLFOX-resistant colon cancer cells, including induction of differentiation and reduction in tumorosphere/colonosphere formation, drug transporter activity, and autocrine stimulation of proliferation. Thus Slfn-3 expression may render colon CSCs more susceptible to cancer chemotherapeutics.

  7. Axonal Growth Arrests After an Increased Accumulation of Schwann Cells Expressing Senescence Markers and Stromal Cells in Acellular Nerve Allografts.

    PubMed

    Poppler, Louis H; Ee, Xueping; Schellhardt, Lauren; Hoben, Gwendolyn M; Pan, Deng; Hunter, Daniel A; Yan, Ying; Moore, Amy M; Snyder-Warwick, Alison K; Stewart, Sheila A; Mackinnon, Susan E; Wood, Matthew D

    2016-07-01

    Acellular nerve allografts (ANAs) and other nerve constructs do not reliably facilitate axonal regeneration across long defects (>3 cm). Causes for this deficiency are poorly understood. In this study, we determined what cells are present within ANAs before axonal growth arrest in nerve constructs and if these cells express markers of cellular stress and senescence. Using the Thy1-GFP rat and serial imaging, we identified the time and location of axonal growth arrest in long (6 cm) ANAs. Axonal growth halted within long ANAs by 4 weeks, while axons successfully regenerated across short (3 cm) ANAs. Cellular populations and markers of senescence were determined using immunohistochemistry, histology, and senescence-associated β-galactosidase staining. Both short and long ANAs were robustly repopulated with Schwann cells (SCs) and stromal cells by 2 weeks. Schwann cells (S100β(+)) represented the majority of cells repopulating both ANAs. Overall, both ANAs demonstrated similar cellular populations with the exception of increased stromal cells (fibronectin(+)/S100β(-)/CD68(-) cells) in long ANAs. Characterization of ANAs for markers of cellular senescence revealed that long ANAs accumulated much greater levels of senescence markers and a greater percentage of Schwann cells expressing the senescence marker p16 compared to short ANAs. To establish the impact of the long ANA environment on axonal regeneration, short ANAs (2 cm) that would normally support axonal regeneration were generated from long ANAs near the time of axonal growth arrest ("stressed" ANAs). These stressed ANAs contained mainly S100β(+)/p16(+) cells and markedly reduced axonal regeneration. In additional experiments, removal of the distal portion (4 cm) of long ANAs near the time of axonal growth arrest and replacement with long isografts (4 cm) rescued axonal regeneration across the defect. Neuronal culture derived from nerve following axonal growth arrest in long ANAs revealed no

  8. Axonal Growth Arrests After an Increased Accumulation of Schwann Cells Expressing Senescence Markers and Stromal Cells in Acellular Nerve Allografts

    PubMed Central

    Poppler, Louis H.; Ee, Xueping; Schellhardt, Lauren; Hoben, Gwendolyn M.; Pan, Deng; Hunter, Daniel A.; Yan, Ying; Moore, Amy M.; Snyder-Warwick, Alison K.; Stewart, Sheila A.; Mackinnon, Susan E.

    2016-01-01

    Acellular nerve allografts (ANAs) and other nerve constructs do not reliably facilitate axonal regeneration across long defects (>3 cm). Causes for this deficiency are poorly understood. In this study, we determined what cells are present within ANAs before axonal growth arrest in nerve constructs and if these cells express markers of cellular stress and senescence. Using the Thy1-GFP rat and serial imaging, we identified the time and location of axonal growth arrest in long (6 cm) ANAs. Axonal growth halted within long ANAs by 4 weeks, while axons successfully regenerated across short (3 cm) ANAs. Cellular populations and markers of senescence were determined using immunohistochemistry, histology, and senescence-associated β-galactosidase staining. Both short and long ANAs were robustly repopulated with Schwann cells (SCs) and stromal cells by 2 weeks. Schwann cells (S100β+) represented the majority of cells repopulating both ANAs. Overall, both ANAs demonstrated similar cellular populations with the exception of increased stromal cells (fibronectin+/S100β−/CD68− cells) in long ANAs. Characterization of ANAs for markers of cellular senescence revealed that long ANAs accumulated much greater levels of senescence markers and a greater percentage of Schwann cells expressing the senescence marker p16 compared to short ANAs. To establish the impact of the long ANA environment on axonal regeneration, short ANAs (2 cm) that would normally support axonal regeneration were generated from long ANAs near the time of axonal growth arrest (“stressed” ANAs). These stressed ANAs contained mainly S100β+/p16+ cells and markedly reduced axonal regeneration. In additional experiments, removal of the distal portion (4 cm) of long ANAs near the time of axonal growth arrest and replacement with long isografts (4 cm) rescued axonal regeneration across the defect. Neuronal culture derived from nerve following axonal growth arrest in long ANAs revealed no deficits

  9. Prognostic significance of stem cell-related marker expression and its correlation with histologic subtypes in lung adenocarcinoma

    PubMed Central

    Park, Eunhyang; Park, Soo Young; Sun, Ping-Li; Jin, Yan; Kim, Ji Eun; Jheon, Sanghoon; Kim, Kwhanmien; Lee, Choon Taek

    2016-01-01

    Cancer stem cells (CSCs) are a small subset of tumor cells that exhibit stem cell-like properties and contribute in treatment failure. To clarify the expression and prognostic significance of several CSC markers in non-small cell lung cancer, we retrospectively analyzed 368 patients with adenocarcinoma (n = 226) or squamous cell carcinoma (n = 142). We correlated the expression of six CSC markers – CD133, CD44, aldehyde dehydrogenase 1 (ALDH1), sex determining region Y-box 2 (SOX2), octamer binding transcription factor 4 (OCT4), and Nanog – with clinicopathologic and molecular variables and survival outcomes. In adenocarcinoma, CD133, ALDH1 and CD44 expression was associated with low pathologic stage and absence of lymphovascular invasion, while Nanog expression correlated with high histologic grade, lymphatic invasion and increased expression of Snail-1, a transcription factor associated with epithelial-mesenchymal transition. CSC marker expression was also associated with histologic subtypes in adenocarcinoma. Multivariate analysis showed that high Nanog expression was an independent factor associated with a poor prognosis in adenocarcinoma. CSC markers had no prognostic value in squamous cell carcinoma. These results suggest that Nanog is an independent negative prognostic factor that may be associated with epithelial-mesenchymal transition in lung adenocarcinoma. PMID:27285762

  10. Oct-4 expression in adult human differentiated cells challenges its role as a pure stem cell marker.

    PubMed

    Zangrossi, Stefano; Marabese, Mirko; Broggini, Massimo; Giordano, Rosaria; D'Erasmo, Marco; Montelatici, Elisa; Intini, Daniela; Neri, Antonino; Pesce, Maurizio; Rebulla, Paolo; Lazzari, Lorenza

    2007-07-01

    The Oct-4 transcription factor, a member of the POU family that is also known as Oct-3 and Oct3/4, is expressed in totipotent embryonic stem cells (ES) and germ cells, and it has a unique role in development and in the determination of pluripotency. ES may have their postnatal counterpart in the adult stem cells, recently described in various mammalian tissues, and Oct-4 expression in putative stem cells purified from adult tissues has been considered a real marker of stemness. In this context, normal mature adult cells would not be expected to show Oct-4 expression. On the contrary, we demonstrated, using reverse transcription-polymerase chain reaction (PCR) (total RNA, Poly A+), real-time PCR, immunoprecipitation, Western blotting, band shift, and immunofluorescence, that human peripheral blood mononuclear cells, genetically stable and mainly terminally differentiated cells with well defined functions and a limited lifespan, express Oct-4. These observations raise the question as to whether the role of Oct-4 as a marker of pluripotency should be challenged. Our findings suggest that the presence of Oct-4 is not sufficient to define a cell as pluripotent, and that additional measures should be used to avoid misleading results in the case of an embryonic-specific gene with a large number of pseudogenes that may contribute to false identification of Oct-4 in adult stem cells. These unexpected findings may provide new insights into the role of Oct-4 in fully differentiated cells. Disclosure of potential conflicts of interest is found at the end of this article.

  11. Stem cell marker CD271 is expressed by vasculogenic mimicry-forming uveal melanoma cells in three-dimensional cultures.

    PubMed

    Valyi-Nagy, Klara; Kormos, Bernadett; Ali, Mohamed; Shukla, Deepak; Valyi-Nagy, Tibor

    2012-01-01

    Cancer stem cells have increased resistance against a variety of anti-tumor treatment modalities. Vasculogenic mimicry (VM) patterns are present in numerous malignant tumor types, represent the formation of perfusion pathways by tumor cells, and their presence in tumors is associated with adverse outcome. Earlier we have shown that VM-forming tumor cells in three-dimensional (3D) uveal melanoma cultures have increased resistance against cytotoxic agents and oncolytic herpes simplex virus-mediated destruction. The purpose of the current study was to explore the possibility that this increased resistance of VM-forming tumor cells is due to a cancer stem cell phenotype. The expression of cancer stem cell marker cluster of differentiation 271 (CD271) was determined in traditional two-dimensional (2D) and 3D cultures of C918 uveal melanoma cells by fluorescent immunocytochemistry. We found that the VM-forming tumor cell subpopulation in 3D cultures expressed CD271. In contrast, cells grown in 2D cultures and tumor cell subpopulations not participating in VM formation in 3D cultures were negative for CD271. These findings suggest that VM-forming uveal melanoma cells acquire a cancer stem cell-like phenotype that may play a role in the increased therapy resistance of these cells.

  12. Stem cell marker CD271 is expressed by vasculogenic mimicry-forming uveal melanoma cells in three-dimensional cultures

    PubMed Central

    Valyi-Nagy, Klara; Kormos, Bernadett; Ali, Mohamed; Shukla, Deepak

    2012-01-01

    Purpose Cancer stem cells have increased resistance against a variety of anti-tumor treatment modalities. Vasculogenic mimicry (VM) patterns are present in numerous malignant tumor types, represent the formation of perfusion pathways by tumor cells, and their presence in tumors is associated with adverse outcome. Earlier we have shown that VM-forming tumor cells in three-dimensional (3D) uveal melanoma cultures have increased resistance against cytotoxic agents and oncolytic herpes simplex virus-mediated destruction. The purpose of the current study was to explore the possibility that this increased resistance of VM-forming tumor cells is due to a cancer stem cell phenotype. Methods The expression of cancer stem cell marker cluster of differentiation 271 (CD271) was determined in traditional two-dimensional (2D) and 3D cultures of C918 uveal melanoma cells by fluorescent immunocytochemistry. Results We found that the VM-forming tumor cell subpopulation in 3D cultures expressed CD271. In contrast, cells grown in 2D cultures and tumor cell subpopulations not participating in VM formation in 3D cultures were negative for CD271. Conclusions These findings suggest that VM-forming uveal melanoma cells acquire a cancer stem cell-like phenotype that may play a role in the increased therapy resistance of these cells. PMID:22419851

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

    PubMed

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

    2011-08-01

    To observe the expression and distribution of CD44, CD133, and ABCG2 in orbital adenoid cystic carcinoma (ACC) and investigate their correlations with pathological type and prognosis. Two steps method of immunohistochemical staining was employed in 33 cases of paraffin embedded surgical specimens of human orbital ACC, 5 cases of recurrence samples, 3 cases of an excised lacrimal gland caused by neither inflammation nor tumor diseases, and 6 cases of xenograft tumors in nude mice. A retrospective analysis was performed on the clinical material of these patients, which were collected from Jan. 1991 to Mar. 2009. The positive rate of CD44 was 54.5% (18/33), with 76.9% (10/13) in solid type and 40.0% (8/20) in adeno-tubiform type. There was no statistically significant difference between them (P = 0.072). In solid type the positive expression cells were often located at the marginal part of the cancer nest. In the adeno-tubiform type, positive cells were often located at the outer layer of the tubiform structure (myoepithelial cells). CD44 was also expressed in normal tissues. The positive rate of CD133 was 57.6% (19/33), with 76.9% (10/13) in solid type and 45.0% (9/20) in adeno-tubiform type. There was no significant difference between them (P = 0.087). CD133 antigen was expressed in either the cytoplasm or nucleus, or expressed in both the cytoplasm and nucleus. The positive rate of ABCG2 was 21.2% (7/33), with 30.77% (4/13) in solid type and 15.0% (3/20) in adeno-tubiform type. There was no significant difference between them (P = 0.393). Many positive cells surrounded the vessels in tumor tissues. There were no significant differences between different prognosis groups of these surface phenotypes. The correlative analysis results of three surface phenotypes showed that CD44(+) cells have positive correlation with CD133(+) cells (Spearman, r(s) = 0.416, P = 0.016). In six transplanted tumors of nude mice, the number of positive cases for CD44(+), CD133(+) and ABCG2

  14. Phenotypic analysis of cell surface markers and gene expression of human mesenchymal stem cells and chondrocytes during monolayer expansion.

    PubMed

    Cournil-Henrionnet, Christel; Huselstein, Céline; Wang, Yun; Galois, Laurent; Mainard, Didier; Decot, Véronique; Netter, Patrick; Stoltz, Jean-François; Muller, Sylvaine; Gillet, Pierre; Watrin-Pinzano, Astrid

    2008-01-01

    Both chondrocytes and mensenchymal stem cells (MSCs) are the most used cell sources for cartilage tissue engineering. However, monolayer expansion to obtain sufficient cells leads to a rapid chondrocyte dedifferentiation and a subsequent ancillary reduced ability of MSCs to differentiate into chondrocytes, thus limiting their application in cartilage repair. The aim of this study was to investigate the influence of the monolayer expansion on the immunophenotype and the gene expression profile of both cell types, and to find the appropriate compromise between monolayer expansion and the remaining chondrogenic characteristics. To this end, human chondrocytes, isolated enzymatically from femoral head slice, and human MSCs, derived from bone marrow, were maintained in monolayer culture up to passage 5. The respective expressions of cell surface markers (CD34, CD45, CD73, CD90, CD105, CD166) and several chondrogenic-related genes for each passage (P0-P5) of those cells were then analyzed using flow cytometry and quantitative real-time PCR, respectively. Flow cytometry analyses showed that, during the monolayer expansion, some qualitative and quantitative regulations occur for the expression of cell surface markers. A rapid increase in mRNA expression of type 1 collagen occurs whereas a significant decrease of type 2 collagen and Sox 9 was observed in chondrocytes through the successive passages. On the other hand, the expansion did not induced obvious change in MSCs gene expression. In conclusion, our results suggest that passage 1 might be the up-limit for chondrocytes in order to achieve their subsequent redifferentiation in 3D scaffold. Nevertheless, MSCs could be expanded in monolayer until passage 5 without loosing their undifferentiated phenotypes.

  15. Relationship of CD86 surface marker expression and cytotoxicity on dendritic cells exposed to chemical allergen

    SciTech Connect

    Hulette, Ben C.; Ryan, Cindy A.; Gildea, Lucy A.; Gerberick, G. Frank . E-mail: gerberick.gf@pg.com

    2005-12-01

    Human peripheral blood-derived dendritic cells (DC) respond to a variety of chemical allergens by up-regulating expression of the co-stimulatory molecule CD86. It has been postulated that this measure might provide the basis for an in vitro alternative approach for the identification of skin sensitizing chemicals. We recently reported that DC, exposed in culture to the highest non-cytotoxic concentrations of various chemical allergens, displayed marginal up-regulation of membrane CD86 expression; the interpretation being that such changes were insufficiently sensitive for the purposes of hazard identification. For the work presented here, immature DC were derived from human monocytes and treated with the chemical allergens 2,4-dinitrobenzenesulfonic acid (DNBS), nickel sulfate (NiSO{sub 4}), p-phenylenediamine (PPD), Bandrowski's base (BB), hydroquinone (HQ) and propyl gallate (PG) for 48 h at concentrations which induced both no to slight to moderate cytotoxicity. For comparison, DC were treated with the irritants sodium dodecyl sulfate (SDS), benzoic acid (BA), and benzalkonium chloride (BZC) at concentrations resulting in comparable levels of cytotoxicity. CD86 expression, as measured by flow cytometry, was consistently up-regulated (ranging from 162 to 386% control) on DC treated with concentrations of chemical allergens that induced approximately 10-15% cytotoxicity. The irritants BA and BZC did not induce up-regulation of CD86 expression when tested at concentrations that induced similar levels of cytotoxicity. SDS, however, up-regulated CD86 expression to 125-138% of control in 2/4 preparations when tested at concentrations which induced similar toxicity. Our results confirm that chemical allergens up-regulate CD86 expression on blood-derived DC and illustrate further that up-regulation of CD86 surface marker expression is more robust when DC are treated with concentrations of chemical allergen that induce slight to moderate cytotoxicity.

  16. Putative cancer stem cell marker expression in oral epithelial dysplasia and squamous cell carcinoma.

    PubMed

    Abdulmajeed, Ahmad A; Dalley, Andrew J; Farah, Camile S

    2013-11-01

    Multifactorial conditions underlie progression of potentially malignant oral lesions (PMOL) to oral squamous cell carcinoma (OSCC) and there is currently need for better prediction of malignant transformation. The hypothesised existence of cancer stem cells in dysplastic oral tissues provides the potential for more informed assessment of PMOL progression. Semi-quantitative immunohistochemical assessment of four putative cancer stem cell markers (CD24, CD44, CD271 and ALDH1) was conducted with a training cohort of 107 patient biopsies to establish clinically applicable score threshold values that were subsequently applied to a blind diagnosis in an independent validation cohort of 278 biopsies. Stain intensity scores for ALDH1, CD24 and CD44, but not CD271 were greater for OSCC than normal tissues. The intensity of ALDH1 and CD24 immunostaining correlated with increased oral epithelial disease severity, and CD24 was effective in distinguishing OSCC from non-malignant tissues, correctly diagnosing 71% of OSCC cases in the validation cohort. Importantly, CD24 immunostaining was effective in diagnosing the presence of dysplasia, correctly discriminating 69% of dysplasia tissues from normal tissues, although no distinction between mild and severe grades of dysplasia was achieved. The results highlight CD24 immunostain intensity as an effective marker of oral dysplasia and OSCC. In conclusion, CD24 immunostain intensity scoring may serve as a helpful technique to assist with the histological recognition of dysplasia in oral biopsies, but not for distinguishing between grades of dysplasia. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  17. Expression of basal cell marker revealed by RAM11 antibody during epithelial regeneration in rabbits.

    PubMed

    Lis, Grzegorz J; Jasek, Ewa; Litwin, Jan A; Gajda, Mariusz; Zarzecka, Joanna; Cichocki, Tadeusz

    2010-01-01

    RAM11 is a mouse monoclonal anti-rabbit macrophage antibody recognizing connective tissue and vascular macrophages. Our previous report showed that RAM11 reacted with basal cells of stratified squamous epithelia of rabbit skin, oral mucosa and esophagus. The aim of the present study was to follow the appearance of RAM11 immunoreactivity in basal cells of regenerating oral epithelium in rabbits. No RAM11 immunostaining was observed in the regenerating epithelium examined on days 1 and 3 of wound healing. A weak immunofluorescence first appeared on day 7 in single basal cells and 32% of RAM11- positive basal cells were observed on day 14. These findings indicate that expression of the antigen recognized by RAM11 antibody is a transient event in the differentiation of oral keratinocytes which not always occurs during epithelial repair, although it is a constant feature of epithelial turnover in mature epithelium. Therefore this antigen can be regarded as basal cell marker only in mature stratified squamous epithelia.

  18. Fibrin acts as biomimetic niche inducing both differentiation and stem cell marker expression of early human endothelial progenitor cells.

    PubMed

    Barsotti, M C; Magera, A; Armani, C; Chiellini, F; Felice, F; Dinucci, D; Piras, A M; Minnocci, A; Solaro, R; Soldani, G; Balbarini, A; Di Stefano, R

    2011-02-01

    Transplantation of endothelial progenitor cells (EPCs) is a promising approach for revascularization of tissue. We have used a natural and biocompatible biopolymer, fibrin, to induce cell population growth, differentiation and functional activity of EPCs. Peripheral blood mononuclear cells were cultured for 1 week to obtain early EPCs. Fibrin was characterized for stiffness and capability to sustain cell population expansion at different fibrinogen-thrombin ratios. Viability, differentiation and angiogenic properties of EPCs were evaluated and compared to those of EPCs grown on fibronectin. Fibrin had a nanometric fibrous structure forming a porous network. Fibrinogen concentration significantly influenced fibrin stiffness and cell growth: 9 mg/ml fibrinogen and 25 U/ml thrombin was the best ratio for enhanced cell viability. Moreover, cell viability was significantly higher on fibrin compared to being on fibronectin. Even though no significant difference was observed in expression of endothelial markers, culture on fibrin elicited marked induction of stem cell markers OCT 3/4 and NANOG. In vitro angiogenesis assay on Matrigel showed that EPCs grown on fibrin retain angiogenetic capability as EPCs grown on fibronectin, but significantly better release of cytokines involved in cell recruitment was produced by EPC grown on fibrin. Fibrin is a suitable matrix for EPC growth, differentiation and angiogenesis capability, suggesting that fibrin gel may be very useful for regenerative medicine. © 2010 Blackwell Publishing Ltd.

  19. Cloning and Expression of CD19, a Human B-Cell Marker in NIH-3T3 Cell Line

    PubMed Central

    Abbasi-Kenarsari, Hajar; Shafaghat, Farzaneh; Baradaran, Behzad; Movassaghpour, Ali Akbar; Shanehbandi, Dariush; Kazemi, Tohid

    2015-01-01

    Background CD19 is a pan B cell marker that is recognized as an attractive target for antibody-based therapy of B-cell disorders including autoimmune disease and hematological malignancies. The object of this study was to stably express the human CD19 antigen in the murine NIH-3T3 cell line aimed to be used as an immunogen in our future study. Methods Total RNA was extracted from Raji cells in which high expression of CD19 was confirmed by flow cytometry. Synthesized cDNA was used for CD19 gene amplification by conventional PCR method using Pfu DNA polymerase. PCR product was ligated to pGEM-T Easy vector and ligation mixture was transformed to DH5α competent bacteria. After blue/white selection, one positive white colony was subjected to plasmid extraction and direct sequencing. Then, CD19 cDNA was sub-cloned into pCMV6-Neo expression vector by double digestion using KpnI and HindIII enzymes. NIH-3T3 mouse fibroblast cell line was subsequently transfected by the construct using Jet-PEI transfection reagent. After 48 hours, surface expression of CD19 was confirmed by flow cytometry and stably transfected cells were selected by G418 antibiotic. Results Amplification of CD19 cDNA gave rise to 1701 bp amplicon confirmed by alignment to reference sequence in NCBI database. Flow cytometric analysis showed successful transient and stable expression of CD19 on NIH-3T3 cells (29 and 93%, respectively). Conclusion Stable cell surface expression of human CD19 antigen in a murine NIH-3T3 cell line may develop a proper immunogene which raises specific anti-CD19 antibody production in the mice immunized sera. PMID:25926951

  20. Early and Delayed Antiretroviral Therapy (ART) Result in Comparable Reductions in CD8+ T Cell Exhaustion Marker Expression.

    PubMed

    Rutishauser, Rachel; Hartogensis, Wendy; Deguit, Christian D; Krone, Melissa; Hoh, Rebecca; Hecht, Rick; Pilcher, Christopher D; Bacchetti, Peter; Deeks, Steven G; Hunt, Peter W; McCune, Joseph M

    2017-03-23

    In untreated HIV infection, CD8+ T cell exhaustion (i.e., decreased proliferative and effector capacity) is associated with high levels of expression of co-inhibitory receptors, including PD-1, TIGIT, CD160, and 2B4. This is evident for both HIV-specific and non-HIV-specific CD8+ T cells. Antiretroviral therapy (ART) initiated during chronic infection decreases but may not completely normalize the expression of such "exhaustion markers." Compared to initiation of ART later in the course of disease, initiation soon after infection reduces some parameters of chronic inflammation and adaptive immune dysfunction. However, it is not known if Early ART (e.g., initiated within the first six months after HIV infection) versus Delayed ART (e.g., initiated during chronic infection) preferentially reduces expression of exhaustion markers. We evaluated exhaustion marker expression on subsets of circulating effector and memory CD8+ T cells at longitudinal pre- and post-ART (two and five years on ART) time points from n=19 (Early ART) and n=23 (Delayed ART) individuals. Prior to ART, TIGIT and CD160 were expressed on a statistically significantly higher proportion of effector and transitional memory cells from individuals in the Delayed ART group: the timing of ART initiation, however, did not consistently affect the expression of the exhaustion markers once viral suppression was achieved. Understanding which factors do and do not regulate aspects of CD8+ T cell exhaustion, including the expression of exhaustion markers, is critical to inform the rational design of CD8+ T cell-based therapies to treat HIV, for which CD8+ T cell exhaustion remains an important barrier to efficacy.

  1. Spatial expression of CLAVATA3 in the shoot apical meristem suggests it is not a stem cell marker in soybean.

    PubMed

    Wong, Chui E; Singh, Mohan B; Bhalla, Prem L

    2013-12-01

    CLAVATA3 (CLV3), a stem cell marker in Arabidopsis thaliana, encodes a secreted peptide that maintains the stem cell population within the shoot apical meristem. This work investigated the CLV3 orthologue in a major legume crop, soybean (GmCLV3). Instead of being expressed in the three outermost layers of the meristem as in Arabidopsis, GmCLV3 was expressed deeper in the central zone beneath the fourth layer (L4) of the meristem, overlapping with the expression of soybean WUSCHEL. Subsequent investigation using an alternative stem cell marker (GmLOG1) revealed its expression within layers L2-L4, indicating that GmCLV3 is not a stem cell marker. Overexpression studies of GmCLV3 in Arabidopsis and complementation of clv3-2 mutant suggest similar functional capacity to that of Arabidopsis CLV3. The expression of soybean CLV1, which encodes a receptor for CLV3 in Arabidopsis, was not detectable in the central zone of the meristem via reverse-transcription PCR analysis of amplified RNA from laser-microdissected samples or in situ, implicating a diverged pathway in soybean. This study also reports the novel expression of GmLOG1 in initials of axillary meristem in the boundary region between the SAM and developing leaf primordia, before the expression of GmWUS or GmCLV3, indicating cytokinin as one of the earliest signals in initiating and specifying the stem cell population.

  2. Quantification of cells expressing markers of proliferation and apoptosis in chronic tonsilitis.

    PubMed

    Avramović, V; Petrović, V; Jović, M; Vlahović, P

    2015-10-01

    During chronic tonsillitis, the relationship between proliferation and apoptosis of lymphocytes in tonsillar follicles can be disturbed, which gives rise to attenuation of tonsil immunocompetence and diminishing its contribution in systemic immunity. In this study, we have quantified the cells expressing the markers of proliferation and apoptosis in the follicles of the palatine tonsil. Six tonsils from patients aged 10-29 years with hypertrophic tonsillitis and five tonsils from patients aged 18-22 years with recurrent tonsillitis were studied. The sections of paraffin blocks of tonsillar tissue were stained by the immunohistochemical LSAB/HRP method with the utilisation of antibodies for: Ki-67 antigen-cell marker of proliferation; Bcl-2 and survivin anti-apoptotic factors and Fas/CD95, caspase-3 and Bax pro-apoptotic factors. The size of lymphoid follicles, i.e. mean follicle area and number of lymphoid follicle immunopositive cells per mm2 of a slice area, i.e. numerical areal density were determined by the quantitative image analysis. The localisation of Ki-67, Bcl-2, survivin, Fas/CD95, caspase-3 and Bax- immunopositive cells inside the palatine tonsil was similar in both types of tonsillitis. The number of Ki-67 immunopositive cells was significantly (p < 0.01) larger in the tonsils with hypertrophic tonsillitis (14681.4 ± 1460.5) in comparison to those with recurrent tonsillitis (12491.4 ± 2321.6), although the number of survivin and caspase-3 immunopositive cells was significantly (p < 0.05) larger in recurrent tonsillitis (survivin, 406.9 ± 98.4; caspase-3, 350.4 ± 119.4) when compared to those with hypertrophic tonsillitis (survivin, 117.4 ± 14.5; caspase-3, 210 ± 24). Our results show that the rate of the proliferation and apoptosis of follicular lymphocytes is different in various types of tonsillitis. This suggests that the immunological potential of the palatine tonsil varies in patients with hypertrophic and recurrent tonsillitis, which in

  3. CD marker expression profiles of human embryonic stem cells and their neural derivatives, determined using flow-cytometric analysis, reveal a novel CD marker for exclusion of pluripotent stem cells.

    PubMed

    Sundberg, Maria; Jansson, Linda; Ketolainen, Johanna; Pihlajamäki, Harri; Suuronen, Riitta; Skottman, Heli; Inzunza, José; Hovatta, Outi; Narkilahti, Susanna

    2009-03-01

    Human embryonic stem cells (hESCs) are pluripotent cells that can differentiate into neural cell lineages. These neural populations are usually heterogeneous and can contain undifferentiated pluripotent cells that are capable of producing teratomas in cell grafts. The characterization of surface protein profiles of hESCs and their neural derivatives is important to determine the specific markers that can be used to exclude undifferentiated cells from neural populations. In this study, we analyzed the cluster of differentiation (CD) marker expression profiles of seven undifferentiated hESC lines using flow-cytometric analysis and compared their profiles to those of neural derivatives. Stem cell and progenitor marker CD133 and epithelial adhesion molecule marker CD326 were more highly expressed in undifferentiated hESCs, whereas neural marker CD56 (NCAM) and neural precursor marker (chemokine receptor) CD184 were more highly expressed in hESC-derived neural cells. CD326 expression levels were consistently higher in all nondifferentiated hESC lines than in neural cell derivatives. In addition, CD326-positive hESCs produced teratomas in SCID mouse testes, whereas CD362-negative neural populations did not. Thus, CD326 may be useful as a novel marker of undifferentiated hESCs to exclude undifferentiated hESCs from differentiated neural cell populations prior to transplantation.

  4. Different RNA and protein expression of surface markers in rabbit amniotic fluid-derived mesenchymal stem cells.

    PubMed

    Kovac, Michal; Vasicek, Jaromir; Kulikova, Barbora; Bauer, Miroslav; Curlej, Jozef; Balazi, Andrej; Chrenek, Peter

    2017-06-27

    Over the years, there has been much confusion in defining molecular markers of mesenchymal stem cells (MSCs) for other than human species due to a lack of species-specific antibodies. Therefore, the aim of our study was to define rabbit amniotic fluid-derived mesenchymal stem cells (rAF-MSCs) and to reflect upon the current identification of AF-MSCs by providing a summary of detected surface markers in different species. The expression of rAF-MSC surface markers was analyzed at the protein and mRNA level. Flow cytometry analyses showed that rAF-MSCs were positive for CD29 and CD44, low positive for CD90, but negative for CD73, CD105, and CD166. Interestingly, RT-PCR (reverse transcription-polymerase chain reaction) exposed a discprepancy between transcribed mRNA and protein expression, as the cells expressed mRNA of all MSC markers: CD29, CD44, CD73, CD90, CD105, and CD166. Our results also confirmed the mesenchymal nature of isolated cells by morphology, ultrastructure, and intracellular marker expression profile. In addition, the expression of few pluripotency markers was also detected. We also found that passaging did not affect apoptosis and viability. Similarly, changes in karyotype were not observed during passaging. In conclusion, the provided characteristics may be used as a comprehensive set of criteria to define and characterize rAF-MSCs, required for the identification of these cells in preclinical investigations. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 2017. © 2017 American Institute of Chemical Engineers.

  5. Clinical Use of Programmed Cell Death-1 and Its Ligand Expression as Discriminatory and Predictive Markers in Ovarian Cancer.

    PubMed

    Chatterjee, Jayanta; Dai, Wei; Aziz, Nor Haslinda Abd; Teo, Pei Yun; Wahba, John; Phelps, David L; Maine, Christian J; Whilding, Lynsey M; Dina, Roberto; Trevisan, Giorgia; Flower, Kirsty J; George, Andrew J T; Ghaem-Maghami, Sadaf

    2017-07-01

    Purpose: We aimed to establish whether programmed cell death-1 (PD-1) and programmed cell death ligand 1 (PD-L1) expression, in ovarian cancer tumor tissue and blood, could be used as biomarkers for discrimination of tumor histology and prognosis of ovarian cancer.Experimental Design: Immune cells were separated from blood, ascites, and tumor tissue obtained from women with suspected ovarian cancer and studied for the differential expression of possible immune biomarkers using flow cytometry. PD-L1 expression on tumor-associated inflammatory cells was assessed by immunohistochemistry and tissue microarray. Plasma soluble PD-L1 was measured using sandwich ELISA. The relationships among immune markers were explored using hierarchical cluster analyses.Results: Biomarkers from the discovery cohort that associated with PD-L1(+) cells were found. PD-L1(+) CD14(+) cells and PD-L1(+) CD11c(+) cells in the monocyte gate showed a distinct expression pattern when comparing benign tumors and epithelial ovarian cancers (EOCs)-confirmed in the validation cohort. Receiver operating characteristic curves showed PD-L1(+) and PD-L1(+) CD14(+) cells in the monocyte gate performed better than the well-established tumor marker CA-125 alone. Plasma soluble PD-L1 was elevated in patients with EOC compared with healthy women and patients with benign ovarian tumors. Low total PD-1(+) expression on lymphocytes was associated with improved survival.Conclusions: Differential expression of immunological markers relating to the PD-1/PD-L1 pathway in blood can be used as potential diagnostic and prognostic markers in EOC. These data have implications for the development and trial of anti-PD-1/PD-L1 therapy in ovarian cancer. Clin Cancer Res; 23(13); 3453-60. ©2016 AACR. ©2016 American Association for Cancer Research.

  6. Altered expression of a putative progenitor cell marker DCAMKL1 in the rat gastric mucosa in regeneration, metaplasia and dysplasia

    PubMed Central

    2010-01-01

    Background Doublecortin and calcium/calmodulin-dependent protein kinase-like-1 (DCAMKL1) is a candidate marker for progenitor cells in the gastrointestinal mucosa. Lineage cells in the gastric mucosa are derived from progenitor cells, but this process can be altered after injury. Therefore, we explored DCAMKL1 expression under pathological conditions. Methods An immunohistochemical analysis was performed in rat stomach with acute superficial injury, chronic ulcer, intestinal metaplasia and dysplasia. Results DCAMKL1 was exclusively expressed in immature quiescent cells in the isthmus of normal fundic glands, where putative progenitor cells are thought to reside. DCAMKL1-positive cells and proliferating cells shed into the lumen after superficial injury and re-appeared during the regenerative process, mainly in the superficial mucosa. In the marginal mucosa around the active ulcer, parietal and chief cells diminished, foveolar hyperplasia was evident, and trefoil factor family 2 (TFF2)/spasmolytic polypeptide-expressing metaplasia (SPEM) emerged at the gland base. DCAMKL1 cells re-emerged in the deep mucosa juxtaposed with SPEM and proliferating cells. In the healing ulcer, the TFF2 cell population expanded and seemed to redifferentiate to chief cells, while proliferating cells and DCAMKL1 cells appeared above and below the TFF2 cells to promote healing. SPEM appeared and PCNA cells increased in the intestinalized mucosa, and DCAMKL1 was expressed in the proximity of the PCNA cells in the deep mucosa. DCAMKL1, PCNA and TFF2 were expressed in different dysplastic cells lining dilated glands near SPEM. Conclusion The ultrastructural appearance of DCAMKL1-positive cells and the expression patterns of DCAMKL1 in normal and pathological states indicate that the cells belong to a progenitor cell population. DCAMKL1 expression is closely associated with TFF2/SPEM cells after injury. DCAMKL1 cells repopulate close to proliferating, hyperplastic, metaplastic and dysplastic

  7. Altered expression of a putative progenitor cell marker DCAMKL1 in the rat gastric mucosa in regeneration, metaplasia and dysplasia.

    PubMed

    Kikuchi, Miho; Nagata, Hiroshi; Watanabe, Norihito; Watanabe, Hiromitsu; Tatemichi, Masayuki; Hibi, Toshifumi

    2010-06-18

    Doublecortin and calcium/calmodulin-dependent protein kinase-like-1 (DCAMKL1) is a candidate marker for progenitor cells in the gastrointestinal mucosa. Lineage cells in the gastric mucosa are derived from progenitor cells, but this process can be altered after injury. Therefore, we explored DCAMKL1 expression under pathological conditions. An immunohistochemical analysis was performed in rat stomach with acute superficial injury, chronic ulcer, intestinal metaplasia and dysplasia. DCAMKL1 was exclusively expressed in immature quiescent cells in the isthmus of normal fundic glands, where putative progenitor cells are thought to reside. DCAMKL1-positive cells and proliferating cells shed into the lumen after superficial injury and re-appeared during the regenerative process, mainly in the superficial mucosa. In the marginal mucosa around the active ulcer, parietal and chief cells diminished, foveolar hyperplasia was evident, and trefoil factor family 2 (TFF2)/spasmolytic polypeptide-expressing metaplasia (SPEM) emerged at the gland base. DCAMKL1 cells re-emerged in the deep mucosa juxtaposed with SPEM and proliferating cells. In the healing ulcer, the TFF2 cell population expanded and seemed to redifferentiate to chief cells, while proliferating cells and DCAMKL1 cells appeared above and below the TFF2 cells to promote healing. SPEM appeared and PCNA cells increased in the intestinalized mucosa, and DCAMKL1 was expressed in the proximity of the PCNA cells in the deep mucosa. DCAMKL1, PCNA and TFF2 were expressed in different dysplastic cells lining dilated glands near SPEM. The ultrastructural appearance of DCAMKL1-positive cells and the expression patterns of DCAMKL1 in normal and pathological states indicate that the cells belong to a progenitor cell population. DCAMKL1 expression is closely associated with TFF2/SPEM cells after injury. DCAMKL1 cells repopulate close to proliferating, hyperplastic, metaplastic and dysplastic cells, and the progenitor zone shifts

  8. Expression of mesenchymal stem cell marker CD90 on dermal sheath cells of the anagen hair follicle in canine species

    PubMed Central

    Mercati, F.; Pascucci, L.; Ceccarelli, P.; Dall’Aglio, C.; Pedini, V.; Gargiulo, A.M.

    2009-01-01

    The dermal sheath (DS) of the hair follicle is comprised by fibroblast-like cells and extends along the follicular epithelium, from the bulb up to the infundibulum. From this structure, cells with stem characteristics were isolated: they have a mesenchymal origin and express CD90 protein, a typical marker of mesenchymal stem cells. It is not yet really clear in which region of hair follicle these cells are located but some experimental evidence suggests that dermal stem cells are localized prevalently in the lower part of the anagen hair follicle. As there are no data available regarding DS stem cells in dog species, we carried out a morphological analysis of the hair follicle DS and performed both an immunohistochemical and an immunocytochemical investigation to identify CD90+ cells. We immunohistochemically evidenced a clear and abundant positivity to CD90 protein in the DS cells located in the lower part of anagen hair follicle. The positive cells showed a typical fibroblast-like morphology. They were flat and elongated and inserted among bundles of collagen fibres.The whole structure formed a close and continuous sleeve around the anagen hair follicle. Our immunocytochemical study allowed us to localize CD90 protein at the cytoplasmic membrane level.

  9. Expression of mesenchymal stem cell marker CD90 on dermal sheath cells of the anagen hair follicle in canine species.

    PubMed

    Mercati, F; Pascucci, L; Ceccarelli, P; Dall'Aglio, C; Pedini, V; Gargiulo, A M

    2009-09-23

    The dermal sheath (DS) of the hair follicle is comprised by fibroblast-like cells and extends along the follicular epithelium, from the bulb up to the infundibulum. From this structure, cells with stem characteristics were isolated: they have a mesenchymal origin and express CD90 protein, a typical marker of mesenchymal stem cells. It is not yet really clear in which region of hair follicle these cells are located but some experimental evidence suggests that dermal stem cells are localized prevalently in the lower part of the anagen hair follicle. As there are no data available regarding DS stem cells in dog species, we carried out a morphological analysis of the hair follicle DS and performed both an immunohistochemical and an immunocytochemical investigation to identify CD90+ cells. We immunohistochemically evidenced a clear and abundant positivity to CD90 protein in the DS cells located in the lower part of anagen hair follicle. The positive cells showed a typical fibroblast-like morphology. They were flat and elongated and inserted among bundles of collagen fibres. The whole structure formed a close and continuous sleeve around the anagen hair follicle. Our immunocytochemical study allowed us to localize CD90 protein at the cytoplasmic membrane level.

  10. Craniopharyngiomas express embryonic stem cell markers (SOX2, OCT4, KLF4, and SOX9) as pituitary stem cells but do not coexpress RET/GFRA3 receptors.

    PubMed

    Garcia-Lavandeira, Montserrat; Saez, Carmen; Diaz-Rodriguez, Esther; Perez-Romero, Sihara; Senra, Ana; Dieguez, Carlos; Japon, Miguel A; Alvarez, Clara V

    2012-01-01

    Adult stem cells maintain some markers expressed by embryonic stem cells and express other specific markers depending on the organ where they reside. Recently, stem/progenitor cells in the rodent and human pituitary have been characterized as expressing GFRA2/RET, PROP1, and stem cell markers such as SOX2 and OCT4 (GPS cells). Our objective was to detect other specific markers of the pituitary stem cells and to investigate whether craniopharyngiomas (CRF), a tumor potentially derived from Rathke's pouch remnants, express similar markers as normal pituitary stem cells. We conducted mRNA and Western blot studies in pituitary extracts, and immunohistochemistry and immunofluorescence on sections from normal rat and human pituitaries and 20 CRF (18 adamantinomatous and two papillary). Normal pituitary GPS stem cells localized in the marginal zone (MZ) express three key embryonic stem cell markers, SOX2, OCT4, and KLF4, in addition to SOX9 and PROP1 and β-catenin overexpression. They express the RET receptor and its GFRA2 coreceptor but also express the coreceptor GFRA3 that could be detected in the MZ of paraffin pituitary sections. CRF maintain the expression of SOX2, OCT4, KLF4, SOX9, and β-catenin. However, RET and GFRA3 expression was altered in CRF. In 25% (five of 20), both RET and GFRA3 were detected but not colocalized in the same cells. The other 75% (15 of 20) lose the expression of RET, GFRA3, or both proteins simultaneously. Human pituitary adult stem/progenitor cells (GPS) located in the MZ are characterized by expression of embryonic stem cell markers SOX2, OCT4, and KLF4 plus the specific pituitary embryonic factor PROP1 and the RET system. Redundancy in RET coreceptor expression (GFRA2 and GFRA3) suggest an important systematic function in their physiological behavior. CRF share the stem cell markers suggesting a common origin with GPS. However, the lack of expression of the RET/GFRA system could be related to the cell mislocation and deregulated

  11. Differential expression of Yes-associated protein is correlated with expression of cell cycle markers and pathologic TNM staging in non-small-cell lung carcinoma.

    PubMed

    Kim, Jin Man; Kang, Dong Wook; Long, Liang Zhe; Huang, Song-Mei; Yeo, Min-Kyung; Yi, Eunhee S; Kim, Kyung-Hee

    2011-03-01

    Yes-associated protein, a downstream effector of the Hippo signaling pathway, has been linked to progression of non-small-cell lung carcinoma. The aim of this study was to investigate expression of Yes-associated protein in lung adenocarcinoma and squamous cell carcinoma. Associations of Yes-associated protein expression with clinicopathologic parameters, expression of cell cycle-specific markers, and epidermal growth factor receptor gene amplification were also analyzed. In a univariate analysis of the 66 adenocarcinomas, high nuclear expression of Yes-associated protein was significantly correlated with expression of cyclin A and mitogen-activated protein kinase. Multivariate analysis, including age and sex, showed that cyclin A expression was independently correlated with nuclear expression of Yes-associated protein in adenocarcinomas. Furthermore, high nuclear expression of Yes-associated protein was also a significant predictor of epidermal growth factor receptor gene amplification for adenocarcinoma. For the 102 squamous cell carcinomas, univariate analysis revealed that high cytoplasmic expression of Yes-associated protein was correlated with the low pathologic TNM staging (stage I) and histologic grading. Multivariate analysis, including age and sex, showed that cytoplasmic expression of Yes-associated protein was an independent predictor of low pathologic TNM staging. These results indicate that nuclear overexpression of Yes-associated protein contributes to pulmonary adenocarcinoma growth and that high cytoplasmic expression of Yes-associated protein is an independent predictor of low pathologic TNM staging and histologic grading. The differential effects of Yes-associated protein expression patterns in adenocarcinomas and squamous cell carcinomas suggest that Yes-associated protein may play important roles in different pathways in distinct tumor subtypes. These observations may, therefore, lead to new perspectives on therapeutic targeting of these tumor

  12. PAX-8 expression in renal tumours and distant sites: A useful marker of primary and metastatic renal cell carcinoma?

    PubMed Central

    Barr, Meaghan L; Jilaveanu, Lucia B; Camp, Robert L; Adeniran, Adebowale J; Kluger, Harriet M; Shuch, Brian

    2015-01-01

    Aims Immunohistochemical stains have greatly improved the diagnostic accuracy of renal cell carcinoma (RCC) for primary and distant tumours. We evaluate a marker that has recently been incorporated in clinical practice, PAX-8, in primary and metastatic RCCs. Methods Two distinct tissue microarrays were used, one consisting of over 334 renal tumours, 294 with adjacent normal kidney and the other with 40 matched nephrectomy and metastatic sites of RCC. PAX-8 expression was assessed by a method of quantitative immunofluorescence. Results PAX-8 was positive in 96% (146/152) of normal renal tissue and 83% (227/272) of renal tumours. PAX-8 staining was positive in clear cell, papillary and chromophobe tumours in 80% (165/207), 95% (39/41) and 100% (6/6) of samples, respectively. Overall, intensity of PAX-8 expression was significantly higher in RCC metastatic sites than in the primary site (p=0.0047), however, in matched sites there was no statistically significant difference in the proportion of positive versus negative specimens (p=0.274). Conclusions As the role of molecular markers expands in the diagnostic algorithm, this study confirms that PAX-8 expression is a useful diagnostic marker for RCC. PAX-8 expression was found in the primary tumour and distant sites. Compared with normal tissue and other histological types, clear cell RCC has lower PAX-8 expression and is less frequently positive, therefore, the lack of expression does not exclude a tumour of renal origin. PMID:25315900

  13. Association Between Expression of Cancer Stem Cell Markers and Poor Differentiation of Hepatocellular Carcinoma: A Meta-Analysis (PRISMA).

    PubMed

    Liu, Rui; Shen, Yuan; Nan, Kejun; Mi, Baibing; Wu, Tao; Guo, Jinyue; Li, Miaojing; Lv, Yi; Guo, Hui

    2015-08-01

    The role of cancer stem cell (CSC) markers in differentiation of hepatocellular carcinoma (HCC) remains uncertain. We conducted a meta-analysis to first investigate the association between expression of CSC markers (CD133, CD90, CD44, and EpCAM) and poor differentiation of HCC, and second, to determine if these CSC markers can be classified as biomarkers for patient classification and HCC differentiated therapy.The relevant literature was searched using PubMed, EMBASE, Elsevier, and Chinese Biological Medicine databases for association between CSC markers and HCC from January 1, 2000 to June 30, 2014. Data were synthesized using random-effect or fixed-effect models. The effect sizes were estimated by measuring odds ratios (OR) with 95% confidence interval (CI).The meta-analysis included 27 studies consisting of 2897 patients with HCC. The positive expression of CSC markers was associated with poor differentiation (OR = 2.37, 95% CI = 2.03-2.77, P < 0.00001). Similarly, the positive expression of CSC markers was only associated with HCC tissues compared with noncancerous liver tissues (OR = 9.26, 95% CI = 3.10-27.65, P < 0.0001). CD90 has a specificity of 91.9% for HCC and a sensitivity of 48.22% in predicting poor differentiation.The positive expression of CSC markers is associated with poor differentiation and aggressive phenotype of patients with HCC. The CD90 marker might be a promising target for patient with HCC classification and differentiation therapy.

  14. Gene expression markers in circulating tumor cells may predict bone metastasis and response to hormonal treatment in breast cancer.

    PubMed

    Wang, Haiying; Molina, Julian; Jiang, John; Ferber, Matthew; Pruthi, Sandhya; Jatkoe, Timothy; Derecho, Carlo; Rajpurohit, Yashoda; Zheng, Jian; Wang, Yixin

    2013-11-01

    Circulating tumor cells (CTCs) have recently attracted attention due to their potential as prognostic and predictive markers for the clinical management of metastatic breast cancer patients. The isolation of CTCs from patients may enable the molecular characterization of these cells, which may help establish a minimally invasive assay for the prediction of metastasis and further optimization of treatment. Molecular markers of proven clinical value may therefore be useful in predicting disease aggressiveness and response to treatment. In our earlier study, we identified a gene signature in breast cancer that appears to be significantly associated with bone metastasis. Among the genes that constitute this signature, trefoil factor 1 (TFF1) was identified as the most differentially expressed gene associated with bone metastasis. In this study, we investigated 25 candidate gene markers in the CTCs of metastatic breast cancer patients with different metastatic sites. The panel of the 25 markers was investigated in 80 baseline samples (first blood draw of CTCs) and 30 follow-up samples. In addition, 40 healthy blood donors (HBDs) were analyzed as controls. The assay was performed using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) with RNA extracted from CTCs captured by the CellSearch system. Our study indicated that 12 of the genes were uniquely expressed in CTCs and 10 were highly expressed in the CTCs obtained from patients compared to those obtained from HBDs. Among these genes, the expression of keratin 19 was highly correlated with the CTC count. The TFF1 expression in CTCs was a strong predictor of bone metastasis and the patients with a high expression of estrogen receptor β in CTCs exhibited a better response to hormonal treatment. Molecular characterization of these genes in CTCs may provide a better understanding of the mechanism underlying tumor metastasis and identify gene markers in CTCs for predicting disease progression and

  15. Gene expression markers in circulating tumor cells may predict bone metastasis and response to hormonal treatment in breast cancer

    PubMed Central

    WANG, HAIYING; MOLINA, JULIAN; JIANG, JOHN; FERBER, MATTHEW; PRUTHI, SANDHYA; JATKOE, TIMOTHY; DERECHO, CARLO; RAJPUROHIT, YASHODA; ZHENG, JIAN; WANG, YIXIN

    2013-01-01

    Circulating tumor cells (CTCs) have recently attracted attention due to their potential as prognostic and predictive markers for the clinical management of metastatic breast cancer patients. The isolation of CTCs from patients may enable the molecular characterization of these cells, which may help establish a minimally invasive assay for the prediction of metastasis and further optimization of treatment. Molecular markers of proven clinical value may therefore be useful in predicting disease aggressiveness and response to treatment. In our earlier study, we identified a gene signature in breast cancer that appears to be significantly associated with bone metastasis. Among the genes that constitute this signature, trefoil factor 1 (TFF1) was identified as the most differentially expressed gene associated with bone metastasis. In this study, we investigated 25 candidate gene markers in the CTCs of metastatic breast cancer patients with different metastatic sites. The panel of the 25 markers was investigated in 80 baseline samples (first blood draw of CTCs) and 30 follow-up samples. In addition, 40 healthy blood donors (HBDs) were analyzed as controls. The assay was performed using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) with RNA extracted from CTCs captured by the CellSearch system. Our study indicated that 12 of the genes were uniquely expressed in CTCs and 10 were highly expressed in the CTCs obtained from patients compared to those obtained from HBDs. Among these genes, the expression of keratin 19 was highly correlated with the CTC count. The TFF1 expression in CTCs was a strong predictor of bone metastasis and the patients with a high expression of estrogen receptor β in CTCs exhibited a better response to hormonal treatment. Molecular characterization of these genes in CTCs may provide a better understanding of the mechanism underlying tumor metastasis and identify gene markers in CTCs for predicting disease progression and

  16. Induced Expression of Cancer Stem Cell Markers ALDH1A3 and Sox-2 in Hierarchical Reconstitution of Apoptosis-resistant Human Breast Cancer Cells

    PubMed Central

    Kashii-Magaribuchi, Karin; Takeuchi, Rie; Haisa, Yuko; Sakamoto, Akemi; Itoh, Aimi; Izawa, Yuki; Isa, Miyuki; Fukuzawa, Mayu; Murakami, Motonobu; Takahashi, Rei

    2016-01-01

    We established an experimental system that can induce p53-dependent apoptosis by doxycycline treatment to analyze characteristics of the apoptosis-resistant cancer cell subpopulation in the human breast cancer cell line HCC1937. Expression patterns of the stem cell markers, ALDH1A3 and Sox-2, the luminal differentiation marker, GATA3 and the proliferation index marker, Ki-67 were analyzed using immunostaining and fluorescence-activated cell sorting (FACS). After doxycycline treatment, the number of viable cells was gradually decreased over seven days in a time-dependent manner due to p53-induced apoptosis; however, the number of smaller-sized ALDH1A3+ cells assessed by immunostaining increased sharply after 1 day of doxycycline treatment, suggesting their apoptosis-resistant nature. The expression of ALDH1A3 was also detected in 78% of small-sized Ki-67+ proliferating progenitor cells, followed by the transient expression of GATA3, which presumably indicated the ability to differentiate into luminal progenitor cells. Although 42.2–58.5% of residual cells were positive for both ALDH1A3 and GATA3, their expression patterns exhibited an inverse correlation. The expression pattern of another stem cell marker, Sox-2, was similar, but more drastically altered after p53 induction compared with ALDH1A3. These findings may aid in understanding the hierarchical responses of cancer stem cells to therapeutic stresses. PMID:27917009

  17. A population of human brain cells expressing phenotypic markers of more than one lineage can be induced in vitro to differentiate into mesenchymal cells

    SciTech Connect

    Rieske, Piotr; Augelli, Brian J.; Stawski, Robert; Gaughan, John; Azizi, S. Ausim; Krynska, Barbara

    2009-02-01

    Proliferating astrocytic cells from germinal, as well as mature areas of brain parenchyma, have the characteristics of neural stem/progenitor cells and are capable of generating both neurons and glia. We previously reported that primary fetal human brain cells, designated as Normal Human Astrocytes (NHA), expressed, in addition to GFAP, Vimentin and Nestin, low levels of {beta}III-Tubulin, an early neuronal marker, and differentiated into neurons and astrocytes in vitro. Here, we showed that primary NHA cells co-express low levels of mesenchymal markers Fibronectin and Collagen-1 in culture. These cells transitioned into mesenchymal-like cells when cultured in adherent conditions in serum containing media. The mesenchymal-like derivatives of these cells were characterized based on their morphological changes, high expression of Vimentin and extracellular matrix (ECM) proteins, Collagen-1 and Fibronectin, and decline of neural markers. When incubated in osteogenic and adipogenic induction media, the mesenchymal-like cells differentiated into osteoblasts and adipocytes. Furthermore, NHA cells express markers of neural crest cells, SOX-10 and p75. These data support the idea of ectoderm-derived mesenchymal lineages. These findings suggest that a population of primitive fetal brain cells with neural/neural crest/mesenchymal phenotype, resembles the remarkable phenotypic plasticity of neural crest cells, and differentiates into adipocytes and osteocytes under the influence of environmental factors.

  18. Expression and Prognostic Significance of a Panel of Tissue Hypoxia Markers in Head-and-Neck Squamous Cell Carcinomas

    SciTech Connect

    Le, Quynh-Thu Kong, Christina; Lavori, Phillip W.; O'Byrne, Ken; Erler, Janine T.; Huang Xin; Chen Yijun; Cao Hongbin; Tibshirani, Robert; Denko, Nic; Giaccia, Amato J.; Koong, Albert C.

    2007-09-01

    Purpose: To investigate the expression pattern of hypoxia-induced proteins identified as being involved in malignant progression of head-and-neck squamous cell carcinoma (HNSCC) and to determine their relationship to tumor pO{sub 2} and prognosis. Methods and Materials: We performed immunohistochemical staining of hypoxia-induced proteins (carbonic anhydrase IX [CA IX], BNIP3L, connective tissue growth factor, osteopontin, ephrin A1, hypoxia inducible gene-2, dihydrofolate reductase, galectin-1, I{kappa}B kinase {beta}, and lysyl oxidase) on tumor tissue arrays of 101 HNSCC patients with pretreatment pO{sub 2} measurements. Analysis of variance and Fisher's exact tests were used to evaluate the relationship between marker expression, tumor pO{sub 2}, and CA IX staining. Cox proportional hazard model and log-rank tests were used to determine the relationship between markers and prognosis. Results: Osteopontin expression correlated with tumor pO{sub 2} (Eppendorf measurements) (p = 0.04). However, there was a strong correlation between lysyl oxidase, ephrin A1, and galectin-1 and CA IX staining. These markers also predicted for cancer-specific survival and overall survival on univariate analysis. A hypoxia score of 0-5 was assigned to each patient, on the basis of the presence of strong staining for these markers, whereby a higher score signifies increased marker expression. On multivariate analysis, increasing hypoxia score was an independent prognostic factor for cancer-specific survival (p = 0.015) and was borderline significant for overall survival (p = 0.057) when adjusted for other independent predictors of outcomes (hemoglobin and age). Conclusions: We identified a panel of hypoxia-related tissue markers that correlates with treatment outcomes in HNSCC. Validation of these markers will be needed to determine their utility in identifying patients for hypoxia-targeted therapy.

  19. Increased expression of Beige/Brown adipose markers from host and breast cancer cells influence xenograft formation in mice

    PubMed Central

    Singh, Rajan; Parveen, Meher; Basgen, John M.; Fazel, Sayeda; Meshesha, Meron F.; Thames, Easter C.; Moore, Brandis; Martinez, Luis; Howard, Carolyn B.; Vergnes, Laurent; Reue, Karen; Pervin, Shehla

    2016-01-01

    The initiation and progression of breast cancer is a complex process that is influenced by heterogeneous cell populations within the tumor microenvironment (TME). Although adipocytes have been shown to promote breast cancer development, adipocyte characteristics involved in this process remain poorly understood. In this study, we demonstrate enrichment of beige/brown adipose markers, contributed from the host as well as tumor cells, in the xenografts from breast cancer cell lines. In addition to uncoupling protein-1 (UCP1) that is exclusively expressed in beige/brown adipocytes, gene expression for classical brown (MYF5, EVA1 and OPLAH), as well as beige (CD137/TNFRSF9 and TBX1) adipocyte markers, were also elevated in the xenografts. Enrichment of beige/brown characteristics in the xenografts was independent of the site of implantation of the breast tumor cells. Early stages of xenografts showed an expansion of a subset of mammary cancer stem cells (MCSCs) that expressed PRDM16, a master regulator of brown adipocyte differentiation. Depletion of UCP1+ or Myf5+ cells significantly reduced tumor development. There was increased COX-2 (MT-CO2) expression, which is known to stimulate formation of beige adipocytes in early xenografts and treatment with a COX-2 inhibitor (SC236) reduced tumor growth. By contrast, treatment with factors that induce brown adipocyte differentiation in vitro led to larger tumors in vivo. A panel of xenografts derived from established breast tumor cells as well as patient-tumor tissues were generated that expressed key brown adipose tissue (BAT)-related markers and contained cells that morphologically resembled brown adipocytes. Implications This is the first report demonstrating that beige /brown adipocyte characteristics could play an important role in breast tumor development and suggest a potential target for therapeutic drug design. PMID:26464213

  20. Characterization and Expression of Senescence Marker in Prolonged Passages of Rat Bone Marrow-Derived Mesenchymal Stem Cells

    PubMed Central

    Ridzuan, Noridzzaida; Al Abbar, Akram; Yip, Wai Kien; Maqbool, Maryam

    2016-01-01

    The present study is aimed at optimizing the in vitro culture protocol for generation of rat bone marrow- (BM-) derived mesenchymal stem cells (MSCs) and characterizing the culture-mediated cellular senescence. The initial phase of generation and characterization was conducted using the adherent cells from Sprague Dawley (SD) rat's BM via morphological analysis, growth kinetics, colony forming unit capacity, immunophenotyping, and mesodermal lineage differentiation. Mesenchymal stem cells were successfully generated and characterized as delineated by the expressions of CD90.1, CD44H, CD29, and CD71 and lack of CD11b/c and CD45 markers. Upon induction, rBM-MSCs differentiated into osteocytes and adipocytes and expressed osteocytes and adipocytes genes. However, a decline in cell growth was observed at passage 4 onwards and it was further deciphered through apoptosis, cell cycle, and senescence assays. Despite the enhanced cell viability at later passages (P4-5), the expression of senescence marker, β-galactosidase, was significantly increased at passage 5. Furthermore, the cell cycle analysis has confirmed the in vitro culture-mediated cellular senescence where cells were arrested at the G0/G1 phase of cell cycle. Although the currently optimized protocols had successfully yielded rBM-MSCs, the culture-mediated cellular senescence limits the growth of rBM-MSCs and its potential use in rat-based MSC research. PMID:27579045

  1. Activated peripheral lymphocytes with increased expression of cell adhesion molecules and cytotoxic markers are associated with dengue fever disease.

    PubMed

    Azeredo, Elzinandes L; Zagne, Sonia M O; Alvarenga, Allan R; Nogueira, Rita M R; Kubelka, Claire F; de Oliveira-Pinto, Luzia M

    2006-06-01

    The immune mechanisms involved in dengue fever and dengue hemorrhagic/dengue shock syndrome are not well understood. The ex vivo activation status of immune cells during the dengue disease in patients was examined. CD4 and CD8 T cells were reduced during the acute phase. Interestingly, CD8 T cells co-expressing activation marker HLA-DR, Q, P, and cytolytic granule protein-Tia-1 were significantly higher in dengue patients than in controls. Detection of adhesion molecules indicated that in dengue patients the majority of T cells (CD4 and CD8) express the activation/memory phenotype, characterized as CD44HIGH and lack the expression of the naïve cell marker, CD62L LOW. Also, the levels of T cells co-expressing ICAM-1 (CD54), VLA-4, and LFA-1 (CD11a) were significantly increased. CD8 T lymphocytes expressed predominantly low levels of anti-apoptotic molecule Bcl-2 in the acute phase, possibly leading to the exhibition of a phenotype of activated/effector cells. Circulating levels of IL-18, TGF-b1 and sICAM-1 were significantly elevated in dengue patients. Early activation events occur during acute dengue infection which might contribute to viral clearance. Differences in expression of adhesion molecules among CD4 and CD8 T cells might underlie the selective extravasation of these subsets from blood circulation into lymphoid organs and/or tissues. In addition, activated CD8 T cells would be more susceptible to apoptosis as shown by the alteration in Bcl-2 expression. Cytokines such as IL-18, TGF-b1, and sICAM-1 may be contributing by either stimulating or suppressing the adaptative immune response, during dengue infection, thereby perhaps establishing a relationship with disease severity.

  2. Expression analysis of human pterygium shows a predominance of conjunctival and limbal markers and genes associated with cell migration

    PubMed Central

    Aryankalayil-John, M.; Campos, M.M.; Fariss, R.N.; Rowsey, J.; Agarwalla, N.; Reid, T.W.; Dushku, N.; Cox, C.A.; Carper, D.; Wistow, G.

    2009-01-01

    Purpose Pterygium is a vision-impairing fibrovascular lesion that grows across the corneal surface and is associated with sunlight exposure. To increase our understanding of the cells types involved in pterygium, we have used expressed sequence tag analysis to examine the transcriptional repertoire of isolated pterygium and to identify marker genes for tissue origin and cell migration. Methods An unnormalized unamplified cDNA library was prepared from 15 pooled specimens of surgically removed pterygia as part of the NEIBank project. Gene expression patterns were compared with existing data for human cornea, limbus, and conjunctiva, and expression of selected genes was verified by immunofluorescence localization in normal eye ocular surface and in pterygium. Results Sequence analysis of 2,976 randomly selected clones produced over 1,800 unique clusters, potentially representing single genes. The most abundant complementary DNAs from pterygium include clusterin, keratins 13 (Krt13) and 4 (Krt4), S100A9/calgranulin B, and spermidine/spermine N1-acetyltransferase (SAT1). Markers for both conjunctiva (such as keratin 13/4 and AQP3) and corneal epithelium (such as keratin 12/3 and AQP5) were present. Immunofluorescence of Krt12 and 13 in the normal ocular surface showed specificity of Krt12 in cornea and Krt13 in conjunctival and limbal epithelia, with a fairly sharp boundary at the limbal–corneal border. In the pterygium there was a patchy distribution of both Krt12 and 13 up to a normal corneal epithelial region specific for Krt12. Immunoglobulins were also among the prominently expressed transcripts. Several of the genes expressed most abundantly in excised pterygium, particularly S100A9 and SAT1, have roles in cell migration. SAT1 exerts its effects through control of polyamine levels. IPENSpm, a polyamine analogue, showed a significant ability to reduce migration in primary cultures of pterygium. A number of genes highly expressed in cornea were not found in

  3. IGF2 expression is a marker for paraganglionic/SIF cell differentiation in neuroblastoma.

    PubMed Central

    Hedborg, F.; Ohlsson, R.; Sandstedt, B.; Grimelius, L.; Hoehner, J. C.; Pählman, S.

    1995-01-01

    Neuroblastoma is a childhood tumor of the sympathetic nervous system. Observations in the Beckwith-Wiedemann syndrome suggest that sympathetic embryonal cells with an abundant expression of the insulin-like growth factor 2 gene (IGF2) may be involved in the genesis of low-malignant infant neuroblastomas. We have therefore compared the cell type-specific IGF2 expression of the human sympathetic nervous system during early development with that of neuroblastoma. An abundant expression in normal sympathetic tissue was specific to extra-adrenal chromaffin cells, ie, paraganglia and small intensely fluorescent (SIF) cells, whereas sympathetic neuronal cells were IGF2-negative. A subpopulation of neuroblastomas expressed IGF2, which correlated with an early age at diagnosis, an extra-adrenal tumor origin, and severe hemodynamic signs of catecholamine secretion. Histologically IGF2-expressing tumors displayed a lobular growth pattern, and expression was restricted to the most mature and least proliferative cells. Typically, these cells were morphologically and histochemically similar to paraganglia/SIF cells and formed distinct ring-like zones in the center of the lobules around a core of apoptosis-like tumor cells. The similarities found between IGF2-expressing neuroblastoma cells and paraganglia/SIF cells in terms of histological features, anatomical origin, and age-dependent growth suggest a paraganglionic/SIF cell lineage of most infant tumors and also of extra-adrenal tumors diagnosed after infancy. Furthermore, since paraganglia/SIF cells undergo postnatal involution, the same cellular mechanism may be responsible for spontaneous regression in infant neuroblastoma. Images Figure 2 Figure 3 p839-a Figure 4 PMID:7717451

  4. Expression of Potential Cancer Stem Cell Marker ABCG2 is Associated with Malignant Behaviors of Hepatocellular Carcinoma

    PubMed Central

    Luo, Weihuan; Jiao, Hongbo; Jiang, Chunping

    2013-01-01

    Background. Despite improvement in treatment, the prognosis of hepatocellular carcinoma (HCC) remains disastrous. Cancer stem cells (CSCs) may be responsible for cancer malignant behaviors. ATP-binding cassette, subfamily G, member 2 (ABCG2) is widely expressed in both normal and cancer stem cells and may play an important role in cancer malignant behaviors. Methods. The expression of ABCG2 in HCC tissues and SMMC-7721 cells was examined, and the relevance of ABCG2 expression with clinical characteristics was analyzed. ABCG2+ and ABCG2− cells were sorted, and the potential of tumorigenicity was determined. Expression level of ABCG2 was manipulated by RNA interference and overexpression. Malignant behaviors including proliferation, drug resistance, migration, and invasion were studied in vitro. Results. Expression of ABCG2 was found in a minor group of cells in HCC tissues and cell lines. ABCG2 expression showed tendencies of association with unfavorable prognosis factors. ABCG2 positive cells showed a superior tumorigenicity. Upregulation of ABCG2 enhanced the capacity of proliferation, doxorubicin resistance, migration, and invasion potential, while downregulation of ABCG2 significantly decreased these malignant behaviors. Conclusion. Our results indicate that ABCG2 is a potential CSC marker for HCC. Its expression level has a close relationship with tumorigenicity, proliferation, drug resistance, and metastasis ability. PMID:24194752

  5. Expression of T-plastin, FoxP3 and other tumor-associated markers by leukemic T-cells of cutaneous T-cell lymphoma.

    PubMed

    Capriotti, Elisabetta; Vonderheid, Eric C; Thoburn, Christopher J; Wasik, Mariusz A; Bahler, David W; Hess, Allan D

    2008-06-01

    Peripheral blood cells from 28 patients with leukemic cutaneous T-cell lymphoma including 25 patients with Sezary syndrome were evaluated for expression of regulatory T-cell-associated markers (FoxP3, CD25, CTLA-4, neurophilin-1), T-cell activation markers (CD28 and its ligands B7.1 and B7.2) and NK cell-associated markers (NKG2D and its ligands Mic-A and Mic-B) using real-time quantitative polymerase chain reaction. T-plastin served as a positive genetic marker, and its expression correlated to blood tumor burden. More than 90% of samples had transcripts for CD28 and Mic-B, but less than 30% of samples expressed FoxP3, CTLA-4 and CD25. Expression of Mic-B by neoplastic cells could provide another mechanism to inhibit anti-tumor immune responses. FoxP3 expression correlated with a poor prognosis. Although the underlying mechanisms accounting for this correlation remain unclear, the expression of the Foxp3 and CTLA-4 regulatory elements indicates that a subset of leukemic cases displays a regulatory T-cell phenotype.

  6. Induction and expression of mutations at multiple drug-resistance marker loci in Chinese hamster ovary cells

    SciTech Connect

    Adair, G.M.; Carver, J.H.

    1983-01-01

    We observed quantitative and qualitative differences in the mutability and mutagen-specificity of various drug-resistance marker loci in Chinese hamster ovary (THO) cells, which suggest that mammalian gene loci may differ in their relative mutability by a given mutagenic agent. We have used the CHO-AT3-2 multiple-marker mutagenesis assay system to examine the dose-dependent induction and kinetics of expression of mutations at four well-characterized, drug-resistance marker loci, after treatment with chemical agents which produce various types of DNA damage. The CHO-AT3-2 subline allows simultaneous quantitation and direct comparison of induced mutation frequencies at the hgprt, oua (Na/sup +//K/sup +/ ATPase), aprt, and tk loci. The agents tested in this study included ethyl methanesulfonate, methyl methanesulfonate, mitomycin C, ICR-191, benzo(a)pyrene, and dimethylnitrosamine. The expression kinetics and optimal expression times for each drug-resistance marker were determined in dose-response experiments in which cells from mutagen-treated populations were plated at 1-2-day intervals over a period of 10 days following mutagenesis. Comparison of induced mutation frequencies for each drug-resistance marker after mutagen treatments yielding equivalent cell survivals (equitoxic doses resulting in relative cell survivals of 0.37) revealed locus-specific differences in the relative mutagenicities of the agents tested. These results indicate that the apparent mutagenicity of a particular agent at a single genetic locus may not necessarily be an accurate indicator of that agent's mutagenic potential for the genome as a whole.

  7. Strain magnitude-dependent calcific marker expression in valvular and vascular cells.

    PubMed

    Ferdous, Zannatul; Jo, Hanjoong; Nerem, Robert M

    2013-01-01

    Aortic valve disease and atherosclerosis tend to coexist in most patients with cardiovascular disease; however, the causes and mechanisms of disease development in heart valves are still not clearly understood. To understand the contributions of the magnitude of cyclic strain (5% hypotension, 10% physiological, and 15% hypertension) in calcification, we used a model system of tissue-engineered collagen gels containing human aortic smooth muscle cells and human aortic valvular interstitial cells, both isolated from noncalcific heart transplant tissue. The compacted collagen gels were cultured in osteogenic media for 3 weeks in a custom-designed bioreactor and all assessments were performed at the end of the culture period. The major finding of this study is that bone morphogenic protein (BMP)-2 and BMP-4 and transforming growth factor-β1 mRNA expression significantly changed in response to the magnitude of applied strain in valvular cells, while the lowest expression was observed for the representative physiological strain. On the other hand, mRNA expression in vascular cells did not vary in response to the magnitude of strain. Regarding BMP-2 and BMP-4 protein expression determined by immunostaining, trends were similar to mRNA expression in vascular and valvular cells, where only valvular cells showed a varied protein expression depending on the magnitude of the strain applied. Our results suggest that cellular differences exist between vascular and valvular cells in their response to altered levels of cyclic strain during calcification. Copyright © 2013 S. Karger AG, Basel.

  8. Regulatory T cells in B-cell-deficient and wild-type mice differ functionally and in expression of cell surface markers

    PubMed Central

    Ellis, Jason S; Braley-Mullen, Helen

    2015-01-01

    NOD.H-2h4 mice develop spontaneous autoimmune thyroiditis (SAT) with chronic inflammation of thyroids by T and B cells. B-cell deficient (B–/–) mice are resistant to SAT but develop SAT if regulatory T (Treg) cells are transiently depleted. We established a transfer model using splenocytes from CD28–/– B–/– mice (effector cells and antigen-presenting cells) cultured with or without sorted Treg cells from Foxp3-GFP wild-type (WT) or B–/– mice. After transfer to mice lacking T cells, mice given Treg cells from B–/– mice had significantly lower SAT severity scores than mice given Treg cells from WT mice, indicating that Treg cells in B–/– mice are more effective suppressors of SAT than Treg cells in WT mice. Treg cells from B–/– mice differ from WT Treg cells in expression of CD27, tumour necrosis factor receptor (TNFR) II p75, and glucocorticoid-induced TNFR-related protein (GITR). After transient depletion using anti-CD25 or diphtheria toxin, the repopulating Treg cells in B–/– mice lack suppressor function, and expression of CD27, GITR and p75 is like that of WT Treg cells. If B–/– Treg cells develop with B cells in bone marrow chimeras, their phenotype is like that of WT Treg cells. Addition of B cells to cultures of B–/– Treg and T effector cells abrogates their suppressive function and their phenotype is like that of WT Treg cells. These results establish for the first time that Treg cells in WT and B–/– mice differ both functionally and in expression of particular cell surface markers. Both properties are altered after transient depletion and repopulation of B–/– Treg cells, and by the presence of B cells during Treg cell development or during interaction with effector T cells. PMID:25318356

  9. Expression of early developmental markers predicts the efficiency of embryonic stem cell differentiation into midbrain dopaminergic neurons.

    PubMed

    Salti, Ahmad; Nat, Roxana; Neto, Sonya; Puschban, Zoe; Wenning, Gregor; Dechant, Georg

    2013-02-01

    Dopaminergic neurons derived from pluripotent stem cells are among the best investigated products of in vitro stem cell differentiation owing to their potential use for neurorestorative therapy of Parkinson's disease. However, the classical differentiation protocols for both mouse and human pluripotent stem cells generate a limited percentage of dopaminergic neurons and yield a considerable cellular heterogeneity comprising numerous scarcely characterized cell populations. To improve pluripotent stem cell differentiation protocols for midbrain dopaminergic neurons, we established extensive and strictly quantitative gene expression profiles, including markers for pluripotent cells, neural progenitors, non-neural cells, pan-neuronal and glial cells, neurotransmitter phenotypes, midbrain and nonmidbrain populations, floor plate and basal plate populations, as well as for Hedgehog, Fgf, and Wnt signaling pathways. The profiles were applied to discrete stages of in vitro differentiation of mouse embryonic stem cells toward the dopaminergic lineage and after transplantation into the striatum of 6-hydroxy-dopamine-lesioned rats. The comparison of gene expression in vitro with stages in the developing ventral midbrain between embryonic day 11.5 and 13.5 ex vivo revealed dynamic changes in the expression of transcription factors and signaling molecules. Based on these profiles, we propose quantitative gene expression milestones that predict the efficiency of dopaminergic differentiation achieved at the end point of the protocol, already at earlier stages of differentiation.

  10. PAX-8 expression in renal tumours and distant sites: a useful marker of primary and metastatic renal cell carcinoma?

    PubMed

    Barr, Meaghan L; Jilaveanu, Lucia B; Camp, Robert L; Adeniran, Adebowale J; Kluger, Harriet M; Shuch, Brian

    2015-01-01

    Immunohistochemical stains have greatly improved the diagnostic accuracy of renal cell carcinoma (RCC) for primary and distant tumours. We evaluate a marker that has recently been incorporated in clinical practice, PAX-8, in primary and metastatic RCCs. Two distinct tissue microarrays were used, one consisting of over 334 renal tumours, 294 with adjacent normal kidney and the other with 40 matched nephrectomy and metastatic sites of RCC. PAX-8 expression was assessed by a method of quantitative immunofluorescence. PAX-8 was positive in 96% (146/152) of normal renal tissue and 83% (227/272) of renal tumours. PAX-8 staining was positive in clear cell, papillary and chromophobe tumours in 80% (165/207), 95% (39/41) and 100% (6/6) of samples, respectively. Overall, intensity of PAX-8 expression was significantly higher in RCC metastatic sites than in the primary site (p=0.0047), however, in matched sites there was no statistically significant difference in the proportion of positive versus negative specimens (p=0.274). As the role of molecular markers expands in the diagnostic algorithm, this study confirms that PAX-8 expression is a useful diagnostic marker for RCC. PAX-8 expression was found in the primary tumour and distant sites. Compared with normal tissue and other histological types, clear cell RCC has lower PAX-8 expression and is less frequently positive, therefore, the lack of expression does not exclude a tumour of renal origin. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

  11. Expression kinetics of hepatic progenitor markers in cellular models of human liver development recapitulating hepatocyte and biliary cell fate commitment.

    PubMed

    Chaudhari, Pooja; Tian, Lipeng; Deshmukh, Abhijeet; Jang, Yoon-Young

    2016-09-01

    Due to the limitations of research using human embryos and the lack of a biological model of human liver development, the roles of the various markers associated with liver stem or progenitor cell potential in humans are largely speculative, and based on studies utilizing animal models and certain patient tissues. Human pluripotent stem cell-based in vitro multistage hepatic differentiation systems may serve as good surrogate models for mimicking normal human liver development, pathogenesis and injury/regeneration studies. Here, we describe the implications of various liver stem or progenitor cell markers and their bipotency (i.e. hepatocytic- and biliary-epithelial cell differentiation), based on the pluripotent stem cell-derived model of human liver development. Future studies using the human cellular model(s) of liver and biliary development will provide more human relevant biological and/or pathological roles of distinct markers expressed in heterogeneous liver stem/progenitor cell populations. © 2016 by the Society for Experimental Biology and Medicine.

  12. Expression of the stem cell marker, SOX2, in ameloblastoma and dental epithelium.

    PubMed

    Juuri, Emma; Isaksson, Sanna; Jussila, Maria; Heikinheimo, Kristiina; Thesleff, Irma

    2013-12-01

    Ameloblastomas are locally invasive odontogenic tumors that exhibit a high rate of recurrence and often associate with the third molars. They are suggested to originate from dental epithelium because the tumor cells resemble epithelial cells of developing teeth. Expression of the transcription factor SOX2 has been previously localized in epithelial stem and progenitor cells in developing teeth as well as in various tumors. Here, we show that SOX2 is expressed in the epithelial cells of follicular and plexiform ameloblastomas. SOX2 was localized in the dental lamina of developing human primary molars. It was also expressed in the fragmented dental lamina associated with the third molars and in the epithelium budding from its posterior aspect in mice. However, no SOX2 expression was detected in either Hertwig's epithelial root sheath directing the formation of roots or in the epithelial cell rests of Malassez covering the completed roots. SOX2 was associated with supernumerary tooth formation in odontoma-like tumors induced by Wnt signal activation in mice. We propose that SOX2 functions in maintaining the progenitor state of epithelium in ameloblastomas and that ameloblastomas may originate from SOX2-expressing dental lamina epithelium. © 2013 Eur J Oral Sci.

  13. Expression of Putative Stem Cell Marker, Hepatocyte Nuclear Factor 4 Alpha, in Mammary Gland of Water Buffalo.

    PubMed

    Choudhary, Ratan K; Choudhary, Shanti; Kaur, Harmanjot; Pathak, Devendra

    2016-01-01

    Buffaloes account for more than 56% of total milk production in India. Cyclic remodeling of mammary glands of human, mice, cow, sheep, and goat is determined by mammary stem cells. It is logical to assume that buffalo mammary gland will have mammary stem/progenitor cells. Thus far, no report exists on identification of buffalo mammary stem cells. Hepatocyte nuclear factor 4 alpha (HNF4A) is a candidate marker for hepatic progenitor cells and has recently been suggested as a marker of bovine mammary stem/progenitor cells. We hypothesized that ( 1 ) HNF4A identifies putative buffalo mammary stem/progenitor cells and ( 2 ) the number of HNF4A-positive cells increases during mastitis. Sixteen buffalo mammary samples were collected from a local slaughterhouse. Hematoxylin and eosin staining were performed on 5-micron thick sections and on the basis of gross examination and histomorphology of the mammary glands, physiological stages of the animals were estimated as non-lactating (n = 4), mastitis (n = 9), and prepubertal (n = 3). In total, 24048 cells were counted (5-10 microscopic fields/animal; n = 16 animals) of which, 40% cells were mammary epithelial cells (MEC) and 60% cells were the stromal cells. The percentage of MEC in non-lactating animals was higher compared to mastitic animals (47.3% vs. 37.3%), which was likely due to loss of MEC in mastitis. HNF4A staining was observed in nuclei of MEC of ducts, alveoli, and stromal cells. Basal location and low frequency of HNF4A-positive MEC (ranges from 0.4-4.5%) were consistent with stem cell characteristics. Preliminary study showed coexpression of HNF4A with MSI1 (a mammary stem cell marker in sheep), suggesting HNF4A was likely to be a putative mammary stem/progenitor cell marker in buffalo. HNF4A-positive MEC (basal and luminal; light and dark stained) tended to be higher in non-lactating than the mastitic animals (8.73 ± 1.71% vs. 4.29 ± 1.19%; P = 0.07). The first hypothesis that HNF4A identify

  14. Inhibition of Phosphatidylinositol 3-Kinase/Akt Signaling Suppresses Tumor Cell Proliferation and Neuroendocrine Marker Expression in GI Carcinoid Tumors

    PubMed Central

    Pitt, Susan C.; Chen, Herbert; Kunnimalaiyaan, Muthusamy

    2010-01-01

    Background Over-activation of PI3K/Akt signaling facilitates tumor proliferation in several cancers. We have shown that various signal transduction pathways promote tumorigenesis in carcinoid tumors, which exhibit endogenously high levels of active, phosphorylated Akt. Therefore, we hypothesized that inhibition of the PI3K/Akt pathway would suppress carcinoid tumor cell growth and neuroendocrine (NE) marker production. Methods Human carcinoid BON cells were treated in vitro with LY294002, a PI3 kinase inhibitor, or transfected with Akt1 siRNA. Tumor cell proliferation was measured by MTT for six days. The effect of LY294002 or Akt1 siRNA treatment was assessed by western analysis. We examined the levels of phosphorylated Akt, total Akt, Akt1, and the NE markers human achaete-scute homolog1 (ASCL1) and chromogranin A (CgA). Results Treatment of BON cells with LY294002 reduced tumor cell proliferation (76%) in a dose-dependent manner. Growth also decreased in Akt1 siRNA transfected cells (29%). Levels of active, phosphorylated Akt and the NE tumor markers, ASCL1 and CgA, were diminished with both LY294002 and Akt1 siRNA treatments proportional to the degree of Akt inhibition. Total Akt, Akt2, and Akt3 levels were unaffected by these experiments. Conclusions These data indicate that PI3K/Akt signaling performs a critical role in human carcinoid tumor cell survival and NE hormone generation. Furthermore, the development of novel therapeutics targeting Akt1 or components of the PI3K/Akt pathway may enhance the management of carcinoid disease. Synopsis Carcinoid tumor cells were treated with a PI3K inhibitor, LY294002, and Akt1 siRNA to delineate the role of PI3K/Akt signaling in carcinoids. The effects of treatment on cellular proliferation and neuroendocrine marker expression were observed. PMID:19588205

  15. Changes in the expression of stem cell markers in oral lichen planus and hyperkeratotic lesions.

    PubMed

    Köse, Osman; Lalli, Anand; Kutulola, Adegun O; Odell, Edward W; Waseem, Ahmad

    2007-06-01

    Despite the pivotal role of stem cells in homeostasis of oral epithelia the location of this cell population within the tissue is uncertain. How disease influences these cells in vivo also remains to be elucidated. In this study we have used six molecular markers to identify stem cells in normal and diseased buccal mucosa. Samples of normal oral mucosa (NOM), hyperkeratosis (OHK) and oral lichen planus (OLP) were immunostained for alpha6 and beta1 integrins, keratin 15 (K15), melanoma-associated chondroitin sulphate proteoglycan (MCSP), NG2 the rat homologue of human MCSP and notch 1. K15, NG2 and beta1 staining was continuous in the basal layer of NOM whilst alpha6 and MCSP were limited to basal cells at the tips of connective tissue papillae. K15 was downregulated in OLP whereas alpha6, beta1 and MCSP were upregulated in both OLP and OHK. NG2 remained unchanged and notch 1 was absent in all samples. Therefore, the stem cell phenotype in OLP and OHK maybe altered in response to pathological signaling. Classification of these changes is essential to understand the role of adult stem cells in the pathogenesis of oral diseases characterised by abnormal keratinocyte proliferation and differentiation.

  16. Expression of stem cell markers on mononuclear cells derived from heparinized cadaveric organ donors before and after disconnection from the respirator.

    PubMed

    Machaliński, B; Paczkowska, E; Hałasa, M; Pabisiak, K; Walczak, M; Sieńko, J; Kozik, W; Ostrowski, M; Syrenicz, A; Sulikowski, T; Machalińska, A

    2006-01-01

    We have attempted to evaluate the level of the earliest human hematopoietic cell marker expression (CD34, CD117, CD133, CD184) on cells obtained from heparinized cadaveric organ donors before and after disconnection from the respirator. Moreover, we compared various cell populations: (1) coexpressing CD34/CD117; (2) CD34/CD133; (3) highly enriched hematopoietic stem cells (CD34+CXCR4+CD45+); and (4) highly enriched tissue-committed stem cells (CD34+CXCR4+CD45-). Finally, we analyzed whether the level of hematopoietic stem cell marker expression depended on the age of the donor. The expression of the membrane receptors (CD34, CD45, CD117, CD133, CD184) was studied by flow cytometry. We observed that the proportion of mononuclear cells expressing these markers slightly decreased in bone marrow harvested after disconnection from the respirator compared with the samples obtained before disconnection. Moreover, the proportion of cells expressing CD117 antigen depended on age of the donor.

  17. Genetic expression of adipose derived stem cell and smooth muscle cell markers to monitor differentiation potential following low intensity laser irradiation

    NASA Astrophysics Data System (ADS)

    Abrahamse, Heidi

    2014-02-01

    Mesenchymal stem cells (MSCs) have the capacity to differentiate into a variety of cell types that could potentially be used in tissue engineering and regenerative medicine. Low intensity laser irradiation (LILI) has been shown to induce a significant increase in cell viability and proliferation. Growth factors such as retinoic acid (RA) and transforming growth factor β1 (TGF-β1) play important roles in the differentiation of cells. The aim of this study was to investigate whether LILI in combination with growth factors could induce the differentiation of adipose derived stem cells (ADSCs) cocultured with smooth muscle cells (SMCs). The study used primary and continuous ADSC cell lines and a SMC line (SKUT-1) as control. Cells were co-cultured directly at a ratio of 1:1 using established methods, with and without growth factors and then exposed to LILI at 5 J/cm2 using a 636 nm diode laser. The cellular morphology, viability and proliferation of the co-cultures were assessed over a period of one week. The study also monitored the expression of cell specific markers over the same period of time. Genetic expression of the markers for both adipose derived stem cells (β1 Integrin and Thymidine 1) and smooth muscle cells (Heavy Myosin Chain) was monitored using flow cytometry. Cell viability and proliferation increased significantly in the co-cultured groups that were exposed to laser alone, as well as in combination with growth factors. Furthermore, there was a significant decrease in the expression of stem cell markers in the ADSCs over time. The results indicate that LILI in combination with growth factors not only increases the viability and proliferation of co-cultured cells but also decreases the expression of ADSC stem cell markers. This could indicate the possible differentiation of ADSCs into SMCs.

  18. Physiological tonicity improves human chondrogenic marker expression through nuclear factor of activated T-cells 5 in vitro.

    PubMed

    van der Windt, Anna E; Haak, Esther; Das, Ruud H J; Kops, Nicole; Welting, Tim J M; Caron, Marjolein M J; van Til, Niek P; Verhaar, Jan A N; Weinans, Harrie; Jahr, Holger

    2010-01-01

    Chondrocytes experience a hypertonic environment compared with plasma (280 mOsm) due to the high fixed negative charge density of cartilage. Standard isolation of chondrocytes removes their hypertonic matrix, exposing them to nonphysiological conditions. During in vitro expansion, chondrocytes quickly lose their specialized phenotype, making them inappropriate for cell-based regenerative strategies. We aimed to elucidate the effects of tonicity during isolation and in vitro expansion on chondrocyte phenotype. Human articular chondrocytes were isolated and subsequently expanded at control tonicity (280 mOsm) or at moderately elevated, physiological tonicity (380 mOsm). The effects of physiological tonicity on chondrocyte proliferation and chondrogenic marker expression were evaluated. The role of Tonicity-responsive Enhancer Binding Protein in response to physiological tonicity was investigated using nuclear factor of activated T-cells 5 (NFAT5) RNA interference. Moderately elevated, physiological tonicity (380 mOsm) did not affect chondrocyte proliferation, while higher tonicities inhibited proliferation and diminished cell viability. Physiological tonicity improved expression of chondrogenic markers and NFAT5 and its target genes, while suppressing dedifferentiation marker collagen type I and improving type II/type I expression ratios >100-fold. Effects of physiological tonicity were similar in osteoarthritic and normal (nonosteoarthritic) chondrocytes, indicating a disease-independent mechanism. NFAT5 RNA interference abolished tonicity-mediated effects and revealed that NFAT5 positively regulates collagen type II expression, while suppressing type I. Physiological tonicity provides a simple, yet effective, means to improve phenotypical characteristics during cytokine-free isolation and in vitro expansion of human articular chondrocytes. Our findings will lead to the development of improved cell-based repair strategies for chondral lesions and provides important

  19. Physiological tonicity improves human chondrogenic marker expression through nuclear factor of activated T-cells 5 in vitro

    PubMed Central

    2010-01-01

    Introduction Chondrocytes experience a hypertonic environment compared with plasma (280 mOsm) due to the high fixed negative charge density of cartilage. Standard isolation of chondrocytes removes their hypertonic matrix, exposing them to nonphysiological conditions. During in vitro expansion, chondrocytes quickly lose their specialized phenotype, making them inappropriate for cell-based regenerative strategies. We aimed to elucidate the effects of tonicity during isolation and in vitro expansion on chondrocyte phenotype. Methods Human articular chondrocytes were isolated and subsequently expanded at control tonicity (280 mOsm) or at moderately elevated, physiological tonicity (380 mOsm). The effects of physiological tonicity on chondrocyte proliferation and chondrogenic marker expression were evaluated. The role of Tonicity-responsive Enhancer Binding Protein in response to physiological tonicity was investigated using nuclear factor of activated T-cells 5 (NFAT5) RNA interference. Results Moderately elevated, physiological tonicity (380 mOsm) did not affect chondrocyte proliferation, while higher tonicities inhibited proliferation and diminished cell viability. Physiological tonicity improved expression of chondrogenic markers and NFAT5 and its target genes, while suppressing dedifferentiation marker collagen type I and improving type II/type I expression ratios >100-fold. Effects of physiological tonicity were similar in osteoarthritic and normal (nonosteoarthritic) chondrocytes, indicating a disease-independent mechanism. NFAT5 RNA interference abolished tonicity-mediated effects and revealed that NFAT5 positively regulates collagen type II expression, while suppressing type I. Conclusions Physiological tonicity provides a simple, yet effective, means to improve phenotypical characteristics during cytokine-free isolation and in vitro expansion of human articular chondrocytes. Our findings will lead to the development of improved cell-based repair strategies for

  20. Cancer stem cell, cytokeratins and epithelial to mesenchymal transition markers expression in oral squamous cell carcinoma derived from ortothopic xenoimplantation of CD44(high) cells.

    PubMed

    de Andrade, Nathália Paiva; Rodrigues, Maria Fernanda Setúbal Destro; Rodini, Camila Oliveira; Nunes, Fabio Daumas

    2017-03-01

    Oral squamous cell carcinoma (OSCC) is the most prevalent neoplasia of oral cavity worldwide and prognosis remains unchanged in decades. Recently, different authors reported that head and neck squamous cell carcinomas have a subpopulation of tumor initiating cells that apparently correspond to cancer stem cells (CSC) and are also responsible for tumor growth and metastasis. The purpose of the present study was to investigate the microscopic and phenotypic characteristics of OSCC tumors induced after orthotopic xenoimplantation of SCC9(WT) cell line and CSC-enriched subpopulation isolated from SCC9 cell line based on high expression of the putative CSC marker CD44. Different numbers of FACS-sorted SCC9 CD44(high) and CD44(low) cells as well as SCC9(WT) (wild type) were transplanted into the tongue of BALB/C nude (NOD/SCID) mice to evaluate their tumorigenic potential. Sixty days post-induction, tumors were morphologically characterized and immunostained for CSC markers (CD44, Nanog and Bmi-1), epithelial-mesenchymal transition (Snail, Slug) and epithelial differentiating cell markers (cytokeratins 4, 13, 15, 17 and 19), as well as E-cadherin and β-catenin. The data presented here shows that SCC9 CD44(high) cells have higher ability to form tumors than SCC9 CD44(low) cells, even when significantly lower numbers of SCC9 CD44(high) cells were transplanted. Immunoassessment of tumors derived from SCC9 CD44(high) cells revealed high expression of cytokeratin CK19, β-catenin, E-cadherin and CD44, and negative or low expression of CK17, CK4, CK15, CK13, Nanog, Bmi-1, Snail and Slug. While tumors derived from SCC9(WT) showed high expression of CK17, CK19, CD44, Nanog, Bmi-1, Snail and Slug, and negative or low expression of CK4, CK15, CK13, β-catenin and E-cadherin. Thus, SCC9 CD44(high) cells were highly tumorigenic, capable of originating heterogeneous tumors and these tumors have a immunohistochemical profile different from those formed by the wild type cell line.

  1. A Novel Gemini Vitamin D Analog Represses the Expression of a Stem Cell Marker CD44 in Breast Cancer

    PubMed Central

    So, Jae Young; Lee, Hong Jin; Smolarek, Amanda K.; Paul, Shiby; Wang, Chung-Xiou; Maehr, Hubert; Uskokovic, Milan; Zheng, Xi; Conney, Allan H.; Cai, Li; Liu, Fang

    2011-01-01

    CD44 is a multifunctional transmembrane protein involved in cell proliferation, angiogenesis, invasion, and metastasis. CD44 is identified as a cancer stem cell marker, and the CD44-positive breast cancer cells are enriched in residual breast cancer cell populations after conventional therapies, suggesting that CD44 may be an important target for cancer prevention and therapy. Therefore, we investigated for the inhibitory effect of a novel Gemini vitamin D analog, 1α,25-dihydroxy-20R-21(3-hydroxy-3-deuteromethyl-4,4,4-trideuterobutyl)-23-yne-26,27-hexafluoro-cholecalciferol (BXL0124), on mammary tumor growth and CD44 expression in MCF10DCIS.com human breast cancer in vitro and in vivo. MCF10DCIS.com cells were injected into mammary fat pads in immunodeficient mice, and BXL0124 was then administered intraperitoneally (0.1 μg/kg body weight) or orally (0.03 or 0.1 μg/kg body weight) 6 days a week for 5 weeks. At necropsy, mammary tumors and blood were collected for evaluating tumor growth, CD44 expression, and serum calcium level. BXL0124 suppressed mammary tumor growth and markedly decreased the expression of CD44 protein in MCF10DCIS xenograft tumors without causing hypercalcemic toxicity. BXL0124 also inhibited the expression of CD44 protein and mRNA as well as the transcriptional activity of the CD44 promoter in cultured MCF10DCIS.com cells. The repression of CD44 expression induced by BXL0124 was blocked by siRNA vitamin D receptor (VDR), indicating that the regulation of CD44 expression by BXL0124 is a VDR-dependent event. The novel Gemini vitamin D analog, BXL0124, represses CD44 expression in MCF10DCIS.com cells in vitro and in xenograft tumors, suggesting an inhibitory role of a Gemini vitamin D derivative on breast cancer stem cells. PMID:21115634

  2. Oleic acid promotes the expression of neural markers in differentiated human endometrial stem cells.

    PubMed

    Kojour, Maryam Ali Mohammadie; Ebrahimi-Barough, Somayeh; Kouchesfehani, Homa Mohseni; Jalali, Hanieh; Ebrahim, Mohammah Hosein Karbalaie

    2017-01-01

    Variety of neurodegenerative diseases in humans are caused by loss of cells along with loss of function and disability. Cell replacement therapy is a potential strategy to cure neurodegenerative diseases. Mesenchymal stem cells are pluripotent non-hematopoietic cells that can be isolated from numerous tissues. Human endometrial-derived stem cell (hEnSC) are the abundant and easy available source with no immunological response, for cell replacement therapy. In the nervous system, where fatty acids are found in huge amounts, they participate in its development and maintenance throughout life. Oleic acid is a kind of the saturated fatty acids which plays crucial role in brain development. Oleic acid released by astrocytes is used by neurons for the synthesis of phospholipids and is specifically incorporated into growth cones. Human endometrial-derived stem cells in the third passage were divided into 3 groups including: control, sham (cultured in full differentiation medium without oleic acid) and experimental group (cultured in full differentiation medium with oleic acid) to differentiate over a 18-day period. Data from Real-Time PCR showed that mRNA levels of NF and β-TUBULIN were increased significantly (p<0.05) in oleic acid treated cells in comparison to control and sham groups. Immunocytochemistry analysis of Chat and NF expression also showed the same results. The present study clearly demonstrates that oleic acid promotes neural differentiation of hEnSC through regulation of gene expression.

  3. Cell-surface Vimentin: A mislocalized protein for isolating csVimentin(+) CD133(-) novel stem-like hepatocellular carcinoma cells expressing EMT markers.

    PubMed

    Mitra, Abhisek; Satelli, Arun; Xia, Xueqing; Cutrera, Jeffrey; Mishra, Lopa; Li, Shulin

    2015-07-15

    Recent advances in cancer stem cell biology have shown that cancer stem-like cells with epithelial-mesenchymal transition (EMT) phenotypes are more aggressive and cause relapse; however, absence of a specific marker to isolate these EMT stem-like cells hampers research in this direction. Cell surface markers have been identified for isolating cancer stem-like cells, but none has been identified for isolating cancer stem-like cells with EMT phenotype. Recently, we discovered that Vimentin, an intracellular EMT tumor cell marker, is present on the surface of colon metastatic tumor nodules in the liver. In our study, we examined the potential of targeting cell surface Vimentin (CSV) to isolate stem-like cancer cells with EMT phenotype, by using a specific CSV-binding antibody, 84-1. Using this antibody, we purified the CSV-positive, CD133-negative (csVim(+) CD133(-) ) cell population from primary liver tumor cell suspensions and characterized for stem cell properties. The results of sphere assays and staining for the stem cell markers Sox2 and Oct4A demonstrated that csVim(+) CD133(-) cells have stem-like properties similar to csVim(-) CD133(+) population. Our investigation further revealed that the csVim(+) CD133(-) cells had EMT phenotypes, as evidenced by the presence of Twist and Slug in the nucleus, the absence of EpCAM on the cell surface and basal level of expression of epithelial marker E-cadherin. The csVimentin-negative CD133-positive stem cells do not have any EMT phenotypes. csVim(+) CD133(-) cells exhibited more aggressively metastatic in livers than csVim(-) CD133(+) cells. Our findings indicate that csVim(+) CD133(-) cells are promising targets for treatment and prevention of metastatic hepatocellular carcinoma.

  4. Ovarian Surface Epithelium in Patients with Severe Ovarian Infertility: A Potential Source of Cells Expressing Markers of Pluripotent/Multipotent Stem Cells

    PubMed Central

    Virant-Klun, Irma; Skutella, Thomas; Stimpfel, Martin; Sinkovec, Jasna

    2011-01-01

    The aim of this study was to confirm the presence of stem cells in the ovarian surface epithelium of patients with premature ovarian failure and no mature follicles and oocytes. In these patients, small round cells of unknown origin expressing SOX-2 marker of pluripotency were observed among the epithelial cells just after the ovarian surface epithelium scraping. These cells were an integral part of the ovarian surface epithelium. When the scraped cells were cultured in a medium with added follicular fluid to provide some ovarian niche, primitive oocyte-like cells and typical round-shaped cell clusters positively stained on alkaline phosphatase, and markers of pluripotency, such as SOX-2 and SSEA-4, were developed. These markers were expressed early and also later in the culture. Single oocyte-like cells expressed genes OCT4A, SOX-2, NANOG, NANOS, STELLA, CD9, LIN28, KLF4, GDF3, and MYC, characteristic for pluripotent stem cells. The results of this study confirmed the presence of putative stem cells in the ovarian surface epithelium of these patients and provided some basis to create a stem cell line in the future. PMID:22187524

  5. Changes of neural markers expression during late neurogenic differentiation of human adipose-derived stem cells

    PubMed Central

    Razavi, Shahnaz; Khosravizadeh, Zahra; Bahramian, Hamid; Kazemi, Mohammad

    2015-01-01

    Background: Different studies have been done to obtain sufficient number of neural cells for treatment of neurodegenerative diseases, spinal cord, and traumatic brain injury because neural stem cells are limited in central nerves system. Recently, several studies have shown that adipose-derived stem cells (ADSCs) are the appropriate source of multipotent stem cells. Furthermore, these cells are found in large quantities. The aim of this study was an assessment of proliferation and potential of neurogenic differentiation of ADSCs with passing time. Materials and Methods: Neurosphere formation was used for neural induction in isolated human ADSCs (hADSCs). The rate of proliferation was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and potential of neural differentiation of induced hADSCs was evaluated by immunocytochemical and real-time reverse transcription polymerase chain reaction analysis after 10 and 14 days post-induction. Results: The rate of proliferation of induced hADSCs increased after 14 days while the expression of nestin, glial fibrillary acidic protein, and microtubule-associated protein 2 was decreased with passing time during neurogenic differentiation. Conclusion: These findings showed that the proliferation of induced cells increased with passing time, but in early neurogenic differentiation of hADSCs, neural expression was higher than late of differentiation. Thus, using of induced cells in early differentiation may be suggested for in vivo application. PMID:26605238

  6. Adipose tissue-derived stromal cells (ADSC) express oligodendrocyte and myelin markers, but they do not function as oligodendrocytes.

    PubMed

    Vellosillo, Lara; Muñoz, Maria Paz; Paíno, Carlos Luis

    2017-06-15

    Mesenchymal cells cultured from the vasculo-stromal fraction of adipose tissue (ADSC) show adult stem cell characteristics and several groups have claimed generating neural cells from them. However, we have observed that many markers commonly used for the identification of neural cells are spontaneously expressed by ADSC in culture. In the present study, we have examined the expression of characteristic oligodendrocyte molecules in cultured ADSC, aiming to test if myelinating cells could be generated from accessible non-neural adult tissues. In basal growth conditions, rat ADSC spontaneously expressed CNPase, MBP, MOG, protein zero, GAP43, Sox10, and Olig2, as shown by immunocytrochemistry and western blot. A small population of cultured ADSC expressed membrane galactocerebroside (O1 antibody), but no cell stained with O4 antibody. RT-PCR analyses showed the expression of CNPase, MBP, DM20, and low levels of Olig2, Sox10, and Sox2 mRNA by rat ADSC. When rat ADSC were treated with combinations of factors commonly used in neural-inducing media (retinoic acid, dbcAMP, EGF, basic FGF, NT3, and/or PDGF), the number of O1-positive cells changed, but in no case, mRNA expression of Sox10 and Olig2 transcription factors approached CNS oligodendrocyte levels. In co-culture with rat dorsal root ganglion neurons, no sign of axonal myelination by rat ADSC was observed. These studies show that the expression of oligodendrocyte traits by cultured ADSC is not a proof of functional competence as oligodendroglia and suggest that in culture conditions, ADSC acquire intermediate, uncommitted phenotypes.

  7. Cell cycle S phase markers are expressed in cerebral neuron nuclei of cats infected by the Feline Panleukopenia Virus.

    PubMed

    Poncelet, Luc; Garigliany, Mutien; Ando, Kunie; Franssen, Mathieu; Desmecht, Daniel; Brion, Jean-Pierre

    2016-12-16

    The cell cycle-associated neuronal death hypothesis, which has been proposed as a common mechanism for most neurodegenerative diseases, is notably supported by evidencing cell cycle effectors in neurons. However, in naturally occurring nervous system diseases, these markers are not expressed in neuron nuclei but in cytoplasmic compartments. In other respects, the Feline Panleukopenia Virus (FPV) is able to complete its cycle in mature brain neurons in the feline species. As a parvovirus, the FPV is strictly dependent on its host cell reaching the cell cycle S phase to start its multiplication. In this retrospective study on the whole brain of 12 cats with naturally-occurring, FPV-associated cerebellar atrophy, VP2 capsid protein expression was detected by immunostaining not only in some brain neuronal nuclei but also in neuronal cytoplasm in 2 cats, suggesting that viral mRNA translation was still occurring. In these cats, double immunostainings demonstrated the expression of cell cycle S phase markers cyclin A, cdk2 and PCNA in neuronal nuclei. Parvoviruses are able to maintain their host cells in S phase by triggering the DNA damage response. S139 phospho H2A1, a key player in the cell cycle arrest, was detected in some neuronal nuclei, supporting that infected neurons were also blocked into the S phase. PCR studies did not support a co-infection with an adeno or herpes virus. ERK1/2 nuclear accumulation was observed in some neurons suggesting that the ERK signaling pathway might be involved as a mechanism driving these neurons far into the cell cycle.

  8. Rheumatoid arthritis and pigmented villonodular synovitis: comparative analysis of cell polyploidy, cell cycle phases and expression of macrophage and fibroblast markers in proliferating synovial cells.

    PubMed

    Berger, I; Weckauf, H; Helmchen, B; Ehemann, V; Penzel, R; Fink, B; Bernd, L; Autschbach, F

    2005-05-01

    Rheumatoid arthritis (RA) and pigmented villonodular synovitis (PVNS) are aggressive diseases with progressive joint destruction. The present study aims to define cell cycle phases, polyploidy and the immunophenotype of proliferating synovial cells in both diseases. Synovial tissues from patients with proliferative-active RA, localized and diffuse PVNS were analysed by DNA flow cytometry, immunohistochemistry and double immunofluorescence with confocal laser scan microscopy. Expression of macrophage markers (CD68/CD163), fibroblast markers (h4Ph/CD55) and Ki67 antigen was examined. Synovial cells positive for either macrophage or fibroblast markers as well as double-labelled cells were found in both RA and PVNS. In RA, CD68/CD163+ synoviocytes were preferentially located in the vicinity of the synovial lining layer, while they were more randomly distributed in PVNS. Of cases with diffuse PVNS, 20% showed an aneuploid cell pattern. All samples of localized PVNS and RA were diploid. Proliferative activity was significantly higher in aneuploid PVNS. In spite of their histologically homogeneous appearance, proliferating synovial cells display a heterogeneous immunophenotype in both RA and PVNS, indicating functional properties of both macrophages and fibroblasts. Aneuploidy seems to be a special feature of diffuse PVNS.

  9. Clinical and prognostic significance of aberrant T-cell marker expression in 225 cases of de novo diffuse large B-cell lymphoma and 276 cases of other B-cell lymphomas.

    PubMed

    Tsuyama, Naoko; Ennishi, Daisuke; Yokoyama, Masahiro; Baba, Satoko; Asaka, Reimi; Mishima, Yuko; Terui, Yasuhito; Hatake, Kiyohiko; Takeuchi, Kengo

    2017-03-23

    Expression of T-cell markers, generally investigated for immunophenotyping of T-cell lymphomas, is also observed in several types of B-cell lymphomas, including diffuse large B-cell lymphoma (DLBCL). We previously reported that CD5 expression in DLBCL is an inferior prognostic factor in the era of rituximab. However, data regarding the frequencies, histological relevance, and prognostic importance of T-cell markers other than CD5 are currently unavailable. In the present study, we comprehensively evaluated the expression of T-cell markers (CD2, CD3, CD4, CD5, CD7, and CD8) in 501 B-cell lymphomas, including 225 DLBCLs, by flow cytometry and subsequent immunohistochemistry. T-cell markers other than CD5, such as CD2, CD4, CD7, and CD8, were expressed in 27 (5%) patients, and notably, all of these cases were classified as large B-cell lymphoma subtypes: 25 DLBCLs and 2 intravascular large B-cell lymphomas. CD5 and other T-cell markers were expressed in 15% (31/225) and 10% (25/225) of DLBCL cases, respectively. Five of them co-expressed CD5 and other T-cell markers. Retrospectively analyzing the prognostic relevance of T-cell markers in 169 patients with primary DLBCL treated with rituximab-based chemotherapy, we showed that only CD5 was a strong predictor of poor survival. This study provides information about the occurrence of T-cell markers other than CD5 in B-cell lymphomas, their frequent histological subtypes, and their prognostic significance in DLBCL. CD5 was reconfirmed as a negative prognostic marker in DLBCL patients receiving rituximab-inclusive chemotherapy, whereas T-cell markers other than CD5 were found to have no impact on clinicopathological and survival analyses.

  10. Paneth cell marker expression in intestinal villi and colon crypts characterizes dietary induced risk for mouse sporadic intestinal cancer.

    PubMed

    Wang, Donghai; Peregrina, Karina; Dhima, Elena; Lin, Elaine Y; Mariadason, John M; Augenlicht, Leonard H

    2011-06-21

    Nutritional and genetic risk factors for intestinal tumors are additive on mouse tumor phenotype, establishing that diet and genetic factors impact risk by distinct combinatorial mechanisms. In a mouse model of dietary-induced sporadic small and large intestinal cancer in WT mice in which tumor etiology, lag, incidence, and frequency reflect >90% of intestinal cancer in Western societies, dietary-induced risk altered gene expression profiles predominantly in villus cells of the histologically normal mucosa, in contrast to targeting of crypt cells by inheritance of an Apc(1638N) allele or homozygous inactivation of p21(Waf1/cip1), and profiles induced by each risk factor were distinct at the gene or functional group level. The dietary-induced changes in villus cells encompassed ectopic expression of Paneth cell markers (a lineage normally confined to the bottom of small intestinal crypts), elevated expression of the Wnt receptor Fzd5 and of EphB2 (genes necessary for Paneth cell differentiation and localization to the crypt bottom), and increased Wnt signaling in villus cells. Ectopic elevation of these markers was also present in the colon crypts, which are also sites of sporadic tumors in the nutritional model. Elevating dietary vitamin D(3) and calcium, which prevents tumor development, abrogated these changes in the villus and colon cells. Thus, common intestinal cancer driven by diet involves mechanisms of tumor development distinct from those mechanisms that cause tumors induced by the rare inheritance of a mutant adenomatous polyposis coli (Apc) allele. This is fundamental for understanding how common sporadic tumors arise and in evaluating relative risk in the population.

  11. Suprabasal expression of Ki-67 as a marker for the severity of oral epithelial dysplasia and oral squamous cell carcinoma.

    PubMed

    Dwivedi, Nidhi; Chandra, Shaleen; Kashyap, Bina; Raj, Vineet; Agarwal, Akhil

    2013-01-01

    Transition of the normal oral epithelium to dysplasia and to malignancy is featured by increased cell proliferation. To evaluate the hypothesis of distributional disturbances in proliferating and stem cells in oral epithelial dysplasia and oral squamous cell carcinoma (OSCC). To evaluate layer wise expression of Ki-67 in oral epithelial dysplasia and in OSCC. Thirty histologically confirmed cases of oral epithelial dysplasia, fifteen cases of OSCC and five cases of normal buccal mucosa were immunohistochemically examined and nuclear expression of Ki-67 was counted according to basal, parabasal, and suprabasal layers in epithelial dysplasia and number of positive cells per 100 cells in OSCC as labeling index (LI). Suprabasal expression of Ki-67 increased according to the severity of epithelial dysplasia and the difference was statistically significant (P < 0.001). The mean Ki-67LI was 12.78 for low risk lesions, 28.68 for high risk lesions, 39.45 for OSCC and 13.6 for normal buccal mucosa. The results of the present study demonstrate the use of proliferative marker Ki-67 in assessing the severity of epithelial dysplasia. Suprabasal expression of Ki-67 provides an objective criteria for determining the severity of epithelial dysplasia and histological grading of OSCC.

  12. [Regulation of moxibustion for expression of gastric mucosa cell-related marker protein in rats with acute gastric ulcer].

    PubMed

    Yang, Zong-Bao; Wang, Chen-Guang; Gong, An; Xie, Yu-feng; Liu, Qiong; Yang, Qing

    2013-11-01

    To explore relevant material basis of moxibustion for recovering gastric mucosal lesion. METHODL Forty-five SD rats were randomly divided into a normal goup, a model group, an acupoint group and a control group, 15 rats in the model group and 10 rats in the rest three groups. Except the normal group, binding and cold stress method were used to establish gastric mucosa injury model. The suspended moxibustion was applied in the acupoint group and control group at acupoints of the stomach meridian ("Liangmen" (ST 21) and "Zusanli" (ST36) and control acupoints (Laterally 1cm next to the "Liangmen" (ST 21) and Zusanli" (ST36), once a day, consectutively for 12 days. After 12 days, morphology of gastric mucosal was observed under optical microscope; protein fingerprints of gastric mucosa cell in rats were detected by protein fingerprint technology, weak cation chip and weak anion chip. Also mass to charge ratio of differential proteins in groups were compared and analyzed. Compared with the model group, index of gastric mucosal lesion in the acupoint group was reduced and its morphology was obviously improved (P<0.05). Campared with control group, index and morphology of gastric mucosal lesion were significantly improved in the acupoint group (P<0.05). According to test of weak cation chip, there was four marker proteins that had expression differences, indicating moxibustion at acupoints of stomach meridian could inrease expression of three marker protein whose molecular weight was 1354Da, 5692Da and 8432Da (all P<0.05) while reduce expression of marker protein with molecular weight of 3287Da (_<0.05). According to test of weak anion chip, moxibustion at acupoints of stomach meridian could increase expression of three marker proteins whose molecular weight was 2412 Da, 3026Da and 6475 Da (allP<0.05). Moxibustion at acupoints of the stomach meridian could regulate differential expression of gastric mucosa cell-related marker protein in rats with acute gastric ulcer and

  13. Reduced expression of CXCR4, a novel renal cancer stem cell marker, is associated with high-grade renal cell carcinoma.

    PubMed

    Rasti, Arezoo; Abolhasani, Maryam; Zanjani, Leili Saeednejad; Asgari, Mojgan; Mehrazma, Mitra; Madjd, Zahra

    2017-01-01

    Cancer stem cells (CSCs) represent a population with tumour-initiating, self-renewal, and differentiation potential. This study aimed to evaluate the expression patterns and clinical significance of chemokine receptor type 4 (CXCR4) as a novel CSC marker in renal cell carcinoma (RCC). The expression of CXCR4 was examined in 173 well-defined renal tumour tissues, including 106 (61.5 %) clear cell renal cell carcinomas (ccRCCs), 35 (20 %) papillary renal cell carcinomas (pRCCs), and 32 (18.5 %) chromophobe renal cell carcinomas (ChRCCs), by immunohistochemistry on a tissue microarray. The association between expression of this marker and clinicopathologic parameters was then analysed. There was a significant difference in the expression levels of CXCR4 in the ccRCC samples compared to the ChRCC and pRCC samples (P < 0.001). Increased expression of CXCR4 was significantly correlated with higher-grade tumours (P < 0.001) and worse stage (P = 0.001). A significant association was also found between expression of CXCR4 and microvascular invasion (P = 0.018). Among RCC subtypes, comparison of the differences between CXCR4 expression in low- and high-grade tumours demonstrated that pRCC tumours had a significantly higher expression of CXCR4 (P < 0.001) than ccRCC tumours (P = 0.01). Significantly higher expression levels of CXCR4 were found in pRCC and ccRCC samples. Increased CXCR4 expression was associated with more aggressive tumour behaviour in RCC patients, especially in pRCC and ccRCC subtypes due to their more metastatic behaviour. These findings suggest that CXCR4 can be considered as a novel diagnostic and therapeutic marker for targeted therapy of renal carcinoma.

  14. Increased expression of the regulatory T cell-associated marker CTLA-4 in bovine leukemia virus infection.

    PubMed

    Suzuki, Saori; Konnai, Satoru; Okagawa, Tomohiro; Ikebuchi, Ryoyo; Nishimori, Asami; Kohara, Junko; Mingala, Claro N; Murata, Shiro; Ohashi, Kazuhiko

    2015-02-15

    Regulatory T cells (Tregs) play a critical role in the maintenance of the host's immune system. Tregs, particularly CD4(+)CD25(+)Foxp3(+) T cells, have been reported to be involved in the immune evasion mechanism of tumors and several pathogens that cause chronic infections. Recent studies showed that a Treg-associated marker, cytotoxic T-lymphocyte antigen 4 (CTLA-4), is closely associated with the progression of several diseases. We recently reported that the proportion of Foxp3(+)CD4(+) cells was positively correlated with the number of lymphocytes, virus titer, and virus load but inversely correlated with IFN-γ expression in cattle infected with bovine leukemia virus (BLV), which causes chronic infection and lymphoma in its host. Here the kinetics of CTLA-4(+) cells were analyzed in BLV-infected cattle. CTLA-4 mRNA was predominantly expressed in CD4(+) T cells in BLV-infected cattle, and the expression was positively correlated with Foxp3 mRNA expression. To test for differences in the protein expression level of CTLA-4, we measured the proportion of CTLA-4-expressing cells by flow cytometry. In cattle with persistent lymphocytosis (PL), mean fluorescence intensities (MFIs) of CTLA-4 on CD4(+) and CD25(+) T cells were significantly increased compared with that in control and aleukemic (AL) cattle. The percentage of CTLA-4(+) cells in the CD4(+) T cell subpopulation was positively correlated with TGF-β mRNA expression, suggesting that CD4(+)CTLA-4(+) T cells have a potentially immunosuppressive function in BLV infection. In the limited number of cattle that were tested, the anti-CTLA-4 antibody enhanced the expression of CD69, IL-2, and IFN-γ mRNA in anti-programmed death ligand 1 (PD-L1) antibody-treated peripheral blood mononuclear cells from BLV-infected cattle. Together with previous findings, the present results indicate that Tregs may be involved in the inhibition of T cell function during BLV infection. Copyright © 2014 Elsevier B.V. All rights

  15. Expressed sequence tag analysis of adult human optic nerve for NEIBank: Identification of cell type and tissue markers

    PubMed Central

    Bernstein, Steven L; Guo, Yan; Peterson, Katherine; Wistow, Graeme

    2009-01-01

    Background The optic nerve is a pure white matter central nervous system (CNS) tract with an isolated blood supply, and is widely used in physiological studies of white matter response to various insults. We examined the gene expression profile of human optic nerve (ON) and, through the NEIBANK online resource, to provide a resource of sequenced verified cDNA clones. An un-normalized cDNA library was constructed from pooled human ON tissues and was used in expressed sequence tag (EST) analysis. Location of an abundant oligodendrocyte marker was examined by immunofluorescence. Quantitative real time polymerase chain reaction (qRT-PCR) and Western analysis were used to compare levels of expression for key calcium channel protein genes and protein product in primate and rodent ON. Results Our analyses revealed a profile similar in many respects to other white matter related tissues, but significantly different from previously available ON cDNA libraries. The previous libraries were found to include specific markers for other eye tissues, suggesting contamination. Immune/inflammatory markers were abundant in the new ON library. The oligodendrocyte marker QKI was abundant at the EST level. Immunofluorescence revealed that this protein is a useful oligodendrocyte cell-type marker in rodent and primate ONs. L-type calcium channel EST abundance was found to be particularly low. A qRT-PCR-based comparative mammalian species analysis reveals that L-type calcium channel expression levels are significantly lower in primate than in rodent ON, which may help account for the class-specific difference in responsiveness to calcium channel blocking agents. Several known eye disease genes are abundantly expressed in ON. Many genes associated with normal axonal function, mRNAs associated with axonal transport, inflammation and neuroprotection are observed. Conclusion We conclude that the new cDNA library is a faithful representation of human ON and EST data provide an initial overview

  16. Horse bone marrow mesenchymal stem cells express embryo stem cell markers and show the ability for tenogenic differentiation by in vitro exposure to BMP-12

    PubMed Central

    Violini, Stefania; Ramelli, Paola; Pisani, Laura F; Gorni, Chiara; Mariani, Paola

    2009-01-01

    Background Mesenchymal stem cells (MSCs) have been recently investigated for their potential use in regenerative medicine. MSCs, in particular, have great potential, as in various reports they have shown pluripotency for differentiating into many different cell types. However, the ability of MSCs to differentiate into tendon cells in vitro has not been fully investigated. Results In this study, we show that equine bone marrow mesenchymal stem cells (BM-MSCs), defined by their expression of markers such as Oct4, Sox-2 and Nanog, have the capability to differentiate in tenocytes. These differentiated cells express tendon-related markers including tenomodulin and decorin. Moreover we show that the same BM-MSCs can differentiate in osteocytes, as confirmed by alkaline phosphatase and von Kossa staining. Conclusion As MSCs represent an attractive tool for tendon tissue repair strategies, our data suggest that bone marrow should be considered the preferred MSC source for therapeutic approaches. PMID:19383177

  17. Progenitor/Stem Cell Markers in Brain Adjacent to Glioblastoma: GD3 Ganglioside and NG2 Proteoglycan Expression.

    PubMed

    Lama, Gina; Mangiola, Annunziato; Proietti, Gabriella; Colabianchi, Anna; Angelucci, Cristiana; D' Alessio, Alessio; De Bonis, Pasquale; Geloso, Maria Concetta; Lauriola, Libero; Binda, Elena; Biamonte, Filippo; Giuffrida, Maria Grazia; Vescovi, Angelo; Sica, Gigliola

    2016-02-01

    Characterization of tissue surrounding glioblastoma (GBM) is a focus for translational research because tumor recurrence invariably occurs in this area. We investigated the expression of the progenitor/stem cell markers GD3 ganglioside and NG2 proteoglycan in GBM, peritumor tissue (brain adjacent to tumor, BAT) and cancer stem-like cells (CSCs) isolated from GBM (GCSCs) and BAT (PCSCs). GD3 and NG2 immunohistochemistry was performed in paired GBM and BAT specimens from 40 patients. Double-immunofluorescence was carried out to characterize NG2-positive cells of vessel walls. GD3 and NG2 expression was investigated in GCSCs and PCSCs whose tumorigenicity was also evaluated in Scid/bg mice. GD3 and NG2 expression was higher in tumor tissue than in BAT. NG2 decreased as the distance from tumor margin increased, regardless of the tumor cell presence, whereas GD3 correlated with neoplastic infiltration. In BAT, NG2 was coexpressed with a-smooth muscle actin (a-SMA) in pericytes and with nestin in the endothelium. Higher levels of NG2 mRNA and protein were found in GCSCs while GD3 synthase was expressed at similar levels in the 2 CSC populations. PCSCs had lower tumorigenicity than GCSCs. These data suggest the possible involvement of GD3 and NG2 in pre/pro-tumorigenic events occurring in the complex microenvironment of the tissue surrounding GBM.

  18. Co-expression of autophagic markers following photodynamic therapy in SW620 human colon adenocarcinoma cells

    PubMed Central

    Ziółkowska, Barbara; Woźniak, Marta; Ziółkowski, Piotr

    2016-01-01

    Photodynamic therapy (PDT) is a minimally invasive cancer treatment. It involves the combination of a photosensitizer and light of a specific wavelength to generate singlet oxygen and other reactive oxygen species that lead to tumor cell death. Autophagy is one of the pathways that tumor cells undergo during photodamage and it is common in photodynamic therapy. The aim of this study was to examine the effect of in vitro PDT on the expression of autophagy-related proteins, autophagy related 7 (Atg7), light chain 3 (LC3) and Beclin-1. Human SW620 colon carcinoma cells were treated with 5-aminolevulinic acid (ALA)-based PDT at a dose of 3 mM. The irradiation was performed using 4.5 J/cm2 total light and a fluence rate of 60 mW/cm2. Autophagy was evaluated by immunocytochemistry using specific antibodies to Atg7, Beclin-1 and LC3. The evaluation was repeated at several time points (0, 4, 8 and 24 h) following irradiation. The induction of autophagy was observed directly following the 5-ALA-mediated PDT procedure with the strongest expression of autophagy-related proteins at 4 and 8 h after irradiation as demonstrated using immunocytochemistry. It was characterized by significantly increased expression of Beclin-1, Atg7 and LC3. To the best of our knowledge this is the first study to analyze Beclin-1, Atg7 and LC3 expression in a PDT-related experiment. This study enhances the understanding of the role of autophagy in PDT, which may contribute to better and more effective tumor responses to this therapy. PMID:27485939

  19. Expression Patterns of Cancer Stem Cell Markers During Specific Celecoxib Therapy in Multistep Rat Colon Carcinogenesis Bioassays.

    PubMed

    Salim, Elsayed I; Hegazi, Mona M; Kang, Jin Seok; Helmy, Hager M

    2016-01-01

    The purpose of this study was to investigate the role of colon cancer stem cells (CSCs) during chemicallyinduced rat multi-step colon carcinogenesis with or without the treatment with a specific cyclooxygenase-2 inhibitor drug (celecoxib). Two experiments were performed, the first, a short term 12 week colon carcinogenesis bioassay in which only surrogate markers for colon cancer, aberrant crypt foci (ACF) lesions, were formed. The other experiment was a medium term colon cancer rat assay in which tumors had developed after 32 weeks. Treatment with celecoxib lowered the numbers of ACF, as well as the tumor volumes and multiplicities after 32 weeks. Immunohistochemical proliferating cell nuclear antigen (PCNA) labeling indexes LI (%) were downregulated after treatment by celecoxib. Also different cell surface antigens known to associate with CSCs such as the epithelial cell adhesion molecule (EpCAM), CD44 and CD133 were compared between the two experiments and showed differential expression patterns depending on the stage of carcinogenesis and treatment with celecoxib. Flow cytometric analysis demonstrated that the numbers of CD133 cells were increased in the colonic epithelium after 12 weeks while those of CD44 but not CD133 cells were increased after 32 weeks. Moreover, aldehyde dehydrogenase-1 activity levels in the colonic epithelium (a known CSC marker) detected by ELISA assay were found down-regulated after 12 weeks, but were up-regulated after 32 weeks. The data have also shown that the protective effect of celecoxib on these specific markers and populations of CSCs and on other molecular processes such as apoptosis targeted by this drug may vary depending on the genetic and phenotypic stages of carcinogenesis. Therefore, uncovering these distinction roles of CSCs during different phases of carcinogenesis and during specific treatment could be useful for targeted therapy.

  20. Characterization of protein marker expression, tumorigenicity, and doxorubicin chemoresistance in two new canine mammary tumor cell lines.

    PubMed

    Hsiao, Yen-Ling; Hsieh, Tai-Zu; Liou, Chian-Jiun; Cheng, Yeong-Hsiang; Lin, Chung-Tien; Chang, Chi-Yao; Lai, Yu-Shen

    2014-09-30

    Canine mammary tumors (CMTs) are the most common type of cancer found in female dogs. Establishment and evaluation of tumor cell lines can facilitate investigations of the biological mechanisms of cancer. Different cell models are used to investigate genetic, epigenetic, and cellular pathways, cancer progression, and cancer therapeutics. Establishment of new cell models will greatly facilitate research in this field. In the present study, we established and characterized two new CMT cell lines derived from a single CMT. We established two cell lines from a single malignant CMT specimen: DTK-E and DTK-SME. Morphologically, the DTK-E cells were large, flat, and epithelial-like, whereas DTK-SME cells were round and epithelial-like. Doubling times were 24 h for DTK-E and 18 h for DTK-SME. On western blots, both cell lines expressed cytokeratin AE1, vimentin, cytokeratin 7 (CK7), and heat shock protein 27 (HSP27). Moreover, investigation of chemoresistance revealed that DTK-SME was more resistant to doxorubicin-induced apoptosis than DTK-E was. After xenotransplantation, both DTK-E and DTK-SME tumors appeared within 14 days, but the average size of DTK-SME tumors was greater than that of DTK-E tumors after 56 days. We established two new cell lines from a single CMT, which exhibit significant diversity in cell morphology, protein marker expression, tumorigenicity, and chemoresistance. The results of this study revealed that the DTK-SME cell line was more resistant to doxorubicin-induced apoptosis and exhibited higher tumorigenicity in vivo than the DTK-E cell line. We anticipate that the two novel CMT cell lines established in this study will be useful for investigating the tumorigenesis of mammary carcinomas and for screening anticancer drugs.

  1. Eurycomanone suppresses expression of lung cancer cell tumor markers, prohibitin, annexin 1 and endoplasmic reticulum protein 28.

    PubMed

    Wong, Pooi-Fong; Cheong, Wei-Fun; Shu, Meng-Hooi; Teh, Chin-Hoe; Chan, Kit-Lam; AbuBakar, Sazaly

    2012-01-15

    Bioactive compounds from the medicinal plant, Eurycoma longifolia Jack have been shown to promote anti-proliferative effects on various cancer cell lines. Here we examined the effects of purified eurycomanone, a quassinoid found in Eurycoma longifolia Jack extract, on the expression of selected genes of the A549 lung cancer cells. Eurycomanone inhibited A549 lung cancer cell proliferation in a dose-dependent manner at concentrations ranging from 5 to 20 μg/ml. The concentration that inhibited 50% of cell growth (GI(50)) was 5.1 μg/ml. The anti-proliferative effects were not fully reversible following the removal of eurycomanone, in which 30% of cell inhibition still remained (p<0.0001, T-test). At 8 μg/ml (GI(70)), eurycomanone suppressed anchorage-independent growth of A549 cells by >25% (p<0.05, T-test, n=8) as determined using soft agar colony formation assay. Cisplatin, a chemotherapy drug used for the treatment of non small cell lung cancer on the other hand, inhibited A549 cells proliferation at concentrations ranging from 0.2 μg/ml to 15 μg/ml with a GI(50) of 0.58 μg/ml. The treatment with eurycomanone reduced the abundance expression of the lung cancer markers, heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1, p53 tumor suppressor protein and other cancer-associated genes including prohibitin (PHB), annexin 1 (ANX1) and endoplasmic reticulum protein 28 (ERp28) but not the house keeping genes. The mRNA expressions of all genes with the exception of PHB were significantly downregulated, 72 h after treatment (p<0.05, T-test, n=9). These findings suggest that eurycomanone at viable therapeutic concentrations of 5-20 μg/ml exhibited significant anti-proliferative and anti-clonogenic cell growth effects on A549 lung cancer cells. The treatment also resulted in suppression of the lung cancer cell tumor markers and several known cancer cell growth-associated genes. Copyright © 2011 Elsevier GmbH. All rights reserved.

  2. Inflammatory Marker Testing Identifies CD74 Expression in Melanoma Tumor Cells, and Its Expression Associates with Favorable Survival for Stage III Melanoma.

    PubMed

    Ekmekcioglu, Suhendan; Davies, Michael A; Tanese, Keiji; Roszik, Jason; Shin-Sim, Myung; Bassett, Roland L; Milton, Denái R; Woodman, Scott E; Prieto, Victor G; Gershenwald, Jeffrey E; Morton, Donald L; Hoon, Dave S; Grimm, Elizabeth A

    2016-06-15

    Inflammatory marker expression in stage III melanoma tumors was evaluated for association with outcome, using two independent cohorts of stage III melanoma patients' tumor tissues. Fifteen markers of interest were selected for analysis, and their expression in melanoma tissues was determined by immunohistochemistry. Proteins associating with either overall survival (OS) or recurrence-free survival (RFS) in the retrospective discovery tissue microarray (TMA; n = 158) were subsequently evaluated in an independent validation TMA (n = 114). Cox proportional hazards regression models were used to assess the association between survival parameters and covariates, the Kaplan-Meier method to estimate the distribution of survival, and the log-rank test to compare distributions. Expression of CD74 on melanoma cells was unique, and in the discovery TMA, it associated with favorable patient outcome (OS: HR, 0.53; P = 0.01 and RFS: HR, 0.56; P = 0.01). The validation data set confirmed the CD74 prognostic significance and revealed that the absence of macrophage migration inhibitory factor (MIF) and inducible nitric oxide synthase (iNOS) was also associated with poor survival parameters. Consistent with the protein observation, tumor CD74 mRNA expression also correlated positively (P = 0.003) with OS in the melanoma TCGA data set. Our data validate CD74 as a useful prognostic tumor cell protein marker associated with favorable RFS and OS in stage III melanoma. Low or negative expression of MIF in both TMAs and of iNOS in the validation set also provided useful prognostic data. A disease-specific investigation of CD74's functional significance is warranted, and other markers appear intriguing to pursue. Clin Cancer Res; 22(12); 3016-24. ©2016 AACR. ©2016 American Association for Cancer Research.

  3. Inflammatory Marker Testing Identifies CD74 Expression in Melanoma Tumor Cells, and its Expression Associates with Favorable Survival for Stage III Melanoma

    PubMed Central

    Ekmekcioglu, Suhendan; Davies, Michael A.; Tanese, Keiji; Roszik, Jason; Shin-Sim, Myung; Bassett, Roland L.; Milton, Denái R.; Woodman, Scott E.; Prieto, Victor G.; Gershenwald, Jeffrey E.; Morton, Donald L.; Hoon, Dave S.; Grimm, Elizabeth A.

    2016-01-01

    Purpose Inflammatory marker expression in stage III melanoma tumors was evaluated for association with outcome, using two independent cohorts of stage III melanoma patients’ tumor tissues. Experimental Design Fifteen markers of interest were selected for analysis, and their expression in melanoma tissues was determined by immunohistochemistry. Proteins associating with either overall survival (OS) or relapse-free survival (RFS) in the retrospective discovery tissue microarray (TMA) (n = 158) were subsequently evaluated in an independent validation TMA (n = 114). Cox proportional hazards regression models were used to assess the association between survival parameters and covariates, the Kaplan-Meier method to estimate the distribution of survival, and the log-rank test to compare distributions. Results Expression of CD74 on melanoma cells was unique, and in the discovery TMA it associated with favorable patient outcome (OS: HR, 0.53, P = 0.01 and RFS: HR, 0.56; P = 0.01). The validation dataset confirmed the CD74 prognostic significance and revealed that the absence of MIF and iNOS was also associated with poor survival parameters. Consistent with the protein observation, tumor CD74 mRNA expression also correlated positively (p = 0.003) with OS in the melanoma TCGA dataset. Conclusions Our data validate CD74 as a useful prognostic tumor cell protein marker associated with favorable RFS and OS in stage III melanoma. Low or negative expression of MIF in both TMAs, and of iNOS in the validation set also provided useful prognostic data. A disease-specific investigation of CD74’s functional significance is warranted, and other markers appear intriguing to pursue. PMID:26783288

  4. Expression of Carcinoembryonic Cell Adhesion Molecule 6 and Alveolar Epithelial Cell Markers in Lungs of Human Infants with Chronic Lung Disease.

    PubMed

    Gonzales, Linda W; Gonzalez, Robert; Barrette, Anne Marie; Wang, Ping; Dobbs, Leland; Ballard, Philip L

    2015-12-01

    The membrane protein carcinoembryonic antigen cell adhesion molecule (CEACAM6) is expressed in the epithelium of various tissues, participating in innate immune defense, cell proliferation and differentiation, with overexpression in gastrointestinal tract, pancreatic and lung tumors. It is developmentally and hormonally regulated in fetal human lung, with an apparent increased production in preterm infants with respiratory failure. To further examine the expression and cell localization of CEACAM6, we performed immunohistochemical and biochemical studies in lung specimens from infants with and without chronic lung disease. CEACAM6 protein and mRNA were increased ~4-fold in lungs from infants with chronic lung disease as compared with controls. By immunostaining, CEACAM6 expression was markedly increased in the lung parenchyma of infants and children with a variety of chronic lung disorders, localizing to hyperplastic epithelial cells with a ~7-fold elevated proliferative rate by PCNA staining. Some of these cells also co-expressed membrane markers of both type I and type II cells, which is not observed in normal postnatal lung, suggesting they are transitional epithelial cells. We suggest that CEACAM6 is both a marker of lung epithelial progenitor cells and a contributor to the proliferative response after injury due to its anti-apoptotic and cell adhesive properties. © The Author(s) 2015.

  5. Proliferation and mRNA expression of absorptive villous cell markers and mineral transporters in prolactin-exposed IEC-6 intestinal crypt cells.

    PubMed

    Teerapornpuntakit, Jarinthorn; Wongdee, Kannikar; Thongbunchoo, Jirawan; Krishnamra, Nateetip; Charoenphandhu, Narattaphol

    2012-06-01

    During pregnancy and lactation, prolactin (PRL) enhances intestinal absorption of calcium and other minerals for fetal development and milk production. Although an enhanced absorptive efficiency is believed to mainly result from the upregulation of mineral transporters in the absorptive villous cells, some other possibilities, such as PRL-enhanced crypt cell proliferation and differentiation to increase the absorptive area, have never been ruled out. Here, we investigated cell proliferation and mRNA expression of mineral absorption-related genes in the PRL-exposed IEC-6 crypt cells. As expected, the cell proliferation was not altered by PRL. Inasmuch as the mRNA expressions of villous cell markers, including dipeptidylpeptidase-4, lactase and glucose transporter-5, were not increased, PRL was not likely to enhance crypt cell differentiation into the absorptive villous cells. In contrast to the previous findings in villous cells, PRL was found to downregulate the expression of calbindin-D(9k), claudin-3 and occludin in IEC-6 crypt cells, while having no effect on transient receptor potential vanilloid family channels-5/6, plasma membrane Ca(2+)-ATPase (PMCA)-1b and Na(+)/Ca(2+) exchanger-1 expression. In conclusion, IEC-6 crypt cells did not respond to PRL by increasing proliferation or differentiation into villous cells. The present results thus supported the previous hypothesis that PRL enhanced mineral absorption predominantly by increasing transporter expression and activity in the absorptive villous cells. Copyright © 2012 John Wiley & Sons, Ltd.

  6. Expression of CD90 and P75NTR stem cell markers in ameloblastomas: a possible role in their biological behavior.

    PubMed

    Silva, Fernanda Paula Yamamoto; Dias, Andrielle; Coelho, Carolinne Almeida; Guerra, Eliete Neves; Marques, Ana Elizia Mascarenhas; Decurcio, Daniel de Almeida; Mantesso, Andrea; Cury, Sérgio Elias Vieira; Silva, Brunno Santos de Freitas

    2016-10-10

    Multicystic and unicystic ameloblastomas are benign odontogenic tumors that present distinct biological behavior. The investigation of stem cells has become an important branch of tumor biology, with several studies addressing the possible role of these cells in tumor growth, angiogenesis, progression, infiltration and invasiveness. This study evaluated the immunohistochemical expression of CD90(Thy-1) and P75NTR stem cell markers in multicystic and unicystic ameloblastomas. Seventeen (17) samples of ameloblastomas (multicystic, n = 10; unicystic, n = 7) were submitted to immunohistochemical reactions and graded semi-quantitatively. The Kolmogorov-Smirnov test was used to verify possible differences in CD90 and P75NTR expressions between multicystic and unicystic ameloblastomas (p < 0.05). CD90 immunostaining was observed in all multicystic ameloblastoma specimens (n = 10), in the cytoplasm of the fibroblasts and vascular endothelial cells of the tumor stroma, near the neoplastic odontogenic epithelia. The staining of stromal CD90 was significantly higher in multicystic than in unicystic ameloblastomas (p = 0.003). Nuclear P75NTR immunostaining was observed in all ameloblastoma specimens. A significant difference was seen in the epithelial staining of P75NTR between multicystic and unicystic types (p = 0.007). The increased expression of CD90 and P75NTR found in multicystic ameloblastomas suggests a behavioral biological difference between multicystic and unicystic ameloblastomas, as well as a difference in ameloblastoma development.

  7. αvβ6 Expression in myoepithelial cells: a novel marker for predicting DCIS progression with therapeutic potential.

    PubMed

    Allen, Michael D; Marshall, John F; Jones, J Louise

    2014-11-01

    The tumor microenvironment dynamically regulates the progression of cancer. In the breast, a unique component of the microenvironment is the myoepithelial cell. Normal myoepithelial cells act as "natural tumor suppressors"; however, more recent evidence suggests that these cells develop phenotypic changes, which may contribute to loss of tumor suppressor activity. We have shown that myoepithelial cells in a subset of preinvasive ductal carcinoma in situ (DCIS) upregulate expression of the integrin αvβ6, switching on tumor promoter activity through activation of TGFβ and MMP9. This makes the tumor microenvironment more permissive to invasion, seen both in vitro and in vivo. In human tissue samples, increased myoepithelial αvβ6 expression correlated with increased risk of disease progression and recurrence. Current estimates suggest that as many as 50% of DCIS cases will never progress in the patient's lifetime, but there are no markers to predict the outcome of individual cases. The identification of αvβ6 in a subset of DCIS presents a unique way to stratify patients with DCIS into those who may or may not progress to more serious disease. As αvβ6 is not expressed on most normal adult tissues, this finding may also provide novel targets for therapy in this high-risk group. ©2014 American Association for Cancer Research.

  8. A population of serumdeprivation-induced bone marrow stem cells (SD-BMSC) expresses marker typical for embryonic and neural stem cells

    SciTech Connect

    Sauerzweig, Steven Munsch, Thomas; Lessmann, Volkmar; Reymann, Klaus G.; Braun, Holger

    2009-01-01

    The bone marrow represents an easy accessible source of adult stem cells suitable for various cell based therapies. Several studies in recent years suggested the existence of pluripotent stem cells within bone marrow stem cells (BMSC) expressing marker proteins of both embryonic and tissue committed stem cells. These subpopulations were referred to as MAPC, MIAMI and VSEL-cells. Here we describe SD-BMSC (serumdeprivation-induced BMSC) which are induced as a distinct subpopulation after complete serumdeprivation. SD-BMSC are generated from small-sized nestin-positive BMSC (S-BMSC) organized as round-shaped cells in the top layer of BMSC-cultures. The generation of SD-BMSC is caused by a selective proliferation of S-BMSC and accompanied by changes in both morphology and gene expression. SD-BMSC up-regulate not only markers typical for neural stem cells like nestin and GFAP, but also proteins characteristic for embryonic cells like Oct4 and SOX2. We hypothesize, that SD-BMSC like MAPC, MIAMI and VSEL-cells represent derivatives from a single pluripotent stem cell fraction within BMSC exhibiting characteristics of embryonic and tissue committed stem cells. The complete removal of serum might offer a simple way to specifically enrich this fraction of pluripotent embryonic like stem cells in BMSC cultures.

  9. The stem cell/cancer stem cell marker ALDH1A3 regulates the expression of the survival factor tissue transglutaminase, in mesenchymal glioma stem cells

    PubMed Central

    Sullivan, Kelly E.; Rojas, Kathy; Cerione, Richard A.; Nakano, Ichiro; Wilson, Kristin F.

    2017-01-01

    Tissue transglutaminase (tTG), a dual-function enzyme with GTP-binding and acyltransferase activities, has been implicated in the survival and chemotherapy resistance of aggressive cancer cells and cancer stem cells, including glioma stem cells (GSCs). Using a model system comprising two distinct subtypes of GSCs referred to as proneural (PN) and mesenchymal (MES), we find that the phenotypically aggressive and radiation therapy-resistant MES GSCs exclusively express tTG relative to PN GSCs. As such, the self-renewal, proliferation, and survival of these cells was sensitive to treatment with tTG inhibitors, with a benefit being observed when combined with the standard of care for high grade gliomas (i.e. radiation or temozolomide). Efforts to understand the molecular drivers of tTG expression in MES GSCs revealed an unexpected link between tTG and a common marker for stem cells and cancer stem cells, Aldehyde dehydrogenase 1A3 (ALDH1A3). ALDH1A3, as well as other members of the ALDH1 subfamily, can function in cells as a retinaldehyde dehydrogenase to generate retinoic acid (RA) from retinal. We show that the enzymatic activity of ALDH1A3 and its product, RA, are necessary for the observed expression of tTG in MES GSCs. Additionally, the ectopic expression of ALDH1A3 in PN GSCs is sufficient to induce the expression of tTG in these cells, further demonstrating a causal link between ALDH1A3 and tTG. Together, these findings ascribe a novel function for ALDH1A3 in an aggressive GSC phenotype via the up-regulation of tTG, and suggest the potential for a similar role by ALDH1 family members across cancer types. PMID:28423611

  10. The stem cell/cancer stem cell marker ALDH1A3 regulates the expression of the survival factor tissue transglutaminase, in mesenchymal glioma stem cells.

    PubMed

    Sullivan, Kelly E; Rojas, Kathy; Cerione, Richard A; Nakano, Ichiro; Wilson, Kristin F

    2017-04-04

    Tissue transglutaminase (tTG), a dual-function enzyme with GTP-binding and acyltransferase activities, has been implicated in the survival and chemotherapy resistance of aggressive cancer cells and cancer stem cells, including glioma stem cells (GSCs). Using a model system comprising two distinct subtypes of GSCs referred to as proneural (PN) and mesenchymal (MES), we find that the phenotypically aggressive and radiation therapy-resistant MES GSCs exclusively express tTG relative to PN GSCs. As such, the self-renewal, proliferation, and survival of these cells was sensitive to treatment with tTG inhibitors, with a benefit being observed when combined with the standard of care for high grade gliomas (i.e. radiation or temozolomide). Efforts to understand the molecular drivers of tTG expression in MES GSCs revealed an unexpected link between tTG and a common marker for stem cells and cancer stem cells, Aldehyde dehydrogenase 1A3 (ALDH1A3). ALDH1A3, as well as other members of the ALDH1 subfamily, can function in cells as a retinaldehyde dehydrogenase to generate retinoic acid (RA) from retinal. We show that the enzymatic activity of ALDH1A3 and its product, RA, are necessary for the observed expression of tTG in MES GSCs. Additionally, the ectopic expression of ALDH1A3 in PN GSCs is sufficient to induce the expression of tTG in these cells, further demonstrating a causal link between ALDH1A3 and tTG. Together, these findings ascribe a novel function for ALDH1A3 in an aggressive GSC phenotype via the up-regulation of tTG, and suggest the potential for a similar role by ALDH1 family members across cancer types.

  11. Slc27a2 expression in peripheral blood mononuclear cells as a molecular marker for overweight development.

    PubMed

    Caimari, A; Oliver, P; Rodenburg, W; Keijer, J; Palou, A

    2010-05-01

    Peripheral blood mononuclear cells (PBMC) can be collected easily and repeatedly. Their potential use to reflect the individual's biological status is increasingly explored. Obesity is becoming the most common health problem of the 21st century, being dietary intake an important determinant of this pathology and numerous chronic health conditions. The aim of this study is to identify PBMC genes involved in energy homeostasis, which could be good markers of overweight development. Using whole-genome microarray analysis, we evaluated the gene expression in PBMC of normoweight and diet-induced obese (cafeteria-fed) Wistar rats. Microarray analysis showed 566 genes differentially expressed between normoweight and cafeteria-fed rats. Of these, 35 genes were particularly involved in energy homeostasis. The gene with the biggest fold change was the 'solute carrier family 27 (fatty acid transporter), member 2' (slc27a2), which is implicated in lipid biosynthesis and fatty acid degradation. Scl27a2 was 33-fold overexpressed in cafeteria-fed rats compared with normoweight rats. This result was confirmed by quantitative PCR, although the overexpression was smaller (sixfold). Moreover, the increase in slc27a2 expression in PBMC of cafeteria-fed rats from 2 to 6 months of age paralleled the increase in body weight. The progressive overexpression of slc27a2 in PBMC of cafeteria-fed rats as the body weight increases suggests this gene as an early marker of overweight development related to the intake of a hyperlipidic diet.

  12. ST6Gal-I protein expression is upregulated in human epithelial tumors and correlates with stem cell markers in normal tissues and colon cancer cell lines.

    PubMed

    Swindall, Amanda F; Londoño-Joshi, Angelina I; Schultz, Matthew J; Fineberg, Naomi; Buchsbaum, Donald J; Bellis, Susan L

    2013-04-01

    The ST6Gal-I sialyltransferase adds an α2-6-linked sialic acid to the N-glycans of certain receptors. ST6Gal-I mRNA has been reported to be upregulated in human cancer, but a prior lack of antibodies has limited immunochemical analysis of the ST6Gal-I protein. Here, we show upregulated ST6Gal-I protein in several epithelial cancers, including many colon carcinomas. In normal colon, ST6Gal-I localized selectively to the base of crypts, where stem/progenitor cells are found, and the tissue staining patterns were similar to the established stem cell marker ALDH1. Similarly, ST6Gal-I expression was restricted to basal epidermal layers in skin, another stem/progenitor cell compartment. ST6Gal-I was highly expressed in induced pluripotent stem (iPS) cells, with no detectable expression in the fibroblasts from which iPS cells were derived. On the basis of these observations, we investigated further an association of ST6Gal-I with cancer stem cells (CSC). Selection of irinotecan resistance in colon carcinoma cells led to a greater proportion of CSCs compared with parental cells, as measured by the CSC markers CD133 and ALDH1 activity (Aldefluor). These chemoresistant cells exhibited a corresponding upregulation of ST6Gal-I expression. Conversely, short hairpin RNA (shRNA)-mediated attenuation of ST6Gal-I in colon carcinoma cells with elevated endogenous expression decreased the number of CD133/ALDH1-positive cells present in the cell population. Collectively, our results suggest that ST6Gal-I promotes tumorigenesis and may serve as a regulator of the stem cell phenotype in both normal and cancer cell populations.

  13. ST6Gal-I protein expression is upregulated in human epithelial tumors and correlates with stem cell markers in normal tissues and colon cancer cell lines

    PubMed Central

    Swindall, Amanda F.; Londoño-Joshi, Angelina I.; Schultz, Matthew J.; Fineberg, Naomi; Buchsbaum, Donald J.; Bellis, Susan L.

    2014-01-01

    The ST6Gal-I sialyltransferase adds an α2–6-linked sialic acid to the N-glycans of certain receptors. ST6Gal-I mRNA has been reported to be upregulated in human cancer, but a prior lack of antibodies has limited immunochemical analysis of the ST6Gal-I protein. Here we show upregulated ST6Gal-I protein in several epithelial cancers, including many colon carcinomas. In normal colon, ST6Gal-I localized selectively to the base of crypts, where stem/progenitor cells are found, and the tissue staining patterns were similar to the established stem cell marker ALDH1. Similarly, ST6Gal-I expression was restricted to basal epidermal layers in skin, another stem/progenitor cell compartment. ST6Gal-I was highly expressed in induced pluripotent stem (iPS) cells, with no detectable expression in the fibroblasts from which iPS cells were derived. On the basis of these observations we investigated further an association of ST6Gal-I with cancer stem cells (CSCs). Selection of irinotecan resistance in colon carcinoma cells led to a greater proportion of CSCs compared with parental cells, as measured by the CSC markers CD133 and ALDH1 activity (Aldefluor). These chemoresistant cells exhibited a corresponding upregulation of ST6Gal-I expression. Conversely, shRNA-mediated attenuation of ST6Gal-I in colon carcinoma cells with elevated endogenous expression decreased the number of CD133/ALDH1-positive cells present in the cell population. Collectively, our results suggest that ST6Gal-I promotes tumorigenesis and may serve as a regulator of the stem cell phenotype in both normal and cancer cell populations. PMID:23358684

  14. Hic-5 Regulates Actin Cytoskeletal Reorganization and Expression of Fibrogenic Markers and Myocilin in Trabecular Meshwork Cells

    PubMed Central

    Pattabiraman, Padmanabhan Paranji; Rao, Ponugoti Vasantha

    2015-01-01

    Purpose To explore the role of inducible focal adhesion (FA) protein Hic-5 in actin cytoskeletal reorganization, FA formation, fibrogenic activity, and expression of myocilin in trabecular meshwork (TM) cells. Methods Using primary cultures of human TM (HTM) cells, the effects of various external factors on Hic-5 protein levels, as well as the effects of recombinant Hic-5 and Hic-5 small interfering RNA (siRNA) on actin cytoskeleton, FAs, myocilin, α-smooth muscle actin (αSMA), and collagen-1 were determined by immunofluorescence and immunoblot analyses. Results Hic-5 distributes discretely to the FAs in HTM cells and throughout the TM and Schlemm's canal of the human aqueous humor (AH) outflow pathway. Transforming growth factor-β2 (TGF-β2), endothelin-1, lysophosphatidic acid, hydrogen peroxide, and RhoA significantly increased Hic-5 protein levels in HTM cells in association with reorganization of actin cytoskeleton and FAs. While recombinant Hic-5 induced actin stress fibers, FAs, αv integrin redistribution to the FAs, increased levels of αSMA, collagen-1, and myocilin, Hic-5 siRNA suppressed most of these responses in HTM cells. Hic-5 siRNA also suppressed TGF-β2-induced fibrogenic activity and dexamethasone-induced myocilin expression in HTM cells. Conclusions Taken together, these results reveal that Hic-5, whose levels were increased by various external factors implicated in elevated intraocular pressure, induces actin cytoskeletal reorganization, FAs, expression of fibrogenic markers, and myocilin in HTM cells. These characteristics of Hic-5 in TM cells indicate its importance in regulation of AH outflow through the TM in both normal and glaucomatous eyes. PMID:26313302

  15. Expression of p75(NGFR), a Proliferative and Basal Cell Marker, in the Buccal Mucosa Epithelium during Re-epithelialization.

    PubMed

    Ishii, Akihiro; Muramatsu, Takashi; Lee, Jong-Min; Higa, Kazunari; Shinozaki, Naoshi; Jung, Han-Sung; Shibahara, Takahiko

    2014-08-29

    We investigated the expression of p75(NGFR), a proliferative and basal cell marker, in the mouse buccal mucosa epithelium during wound healing in order to elucidate the role of epithelial stem cells. Epithelial defects were generated in the epithelium of the buccal mucosa of 6-week-old mice using CO2 laser irradiation. BrdU was immediately administered to mice following laser irradiation. They were then sacrificed after 1, 3, 7, and 14 days. Paraffin sections were prepared and the irradiated areas were analyzed using immunohistochemistry with anti-p75(NGFR), BrdU, PCNA, and CK14 antibodies. During re-epithelialization, PCNA (-)/p75(NGFR) (+) cells extended to the wound, which then closed, whereas PCNA (+)/p75(NGFR) (+) cells were not observed at the edge of the wound. In addition, p75(NGFR) (-)/CK14 (+), which reflected the presence of post-mitotic differentiating cells, was observed in the supra-basal layers of the extended epithelium. BrdU (+)/p75(NGFR) (+), which reflected the presence of epithelial stem cells, was detected sparsely in buccal basal epithelial cells after healing, and disappeared after 7 days. These results suggest that p75(NGFR) (+) keratinocytes are localized in the basal layer, which contains oral epithelial stem cells, and retain the ability to proliferate in order to regenerate the buccal mucosal epithelium.

  16. Identification of gene expression-based prognostic markers in the hematopoietic stem cells of patients with myelodysplastic syndromes.

    PubMed

    Pellagatti, Andrea; Benner, Axel; Mills, Ken I; Cazzola, Mario; Giagounidis, Aristoteles; Perry, Janet; Malcovati, Luca; Della Porta, Matteo G; Jädersten, Martin; Verma, Amit; McDonald, Emma-Jane; Killick, Sally; Hellström-Lindberg, Eva; Bullinger, Lars; Wainscoat, James S; Boultwood, Jacqueline

    2013-10-01

    The diagnosis of patients with myelodysplastic syndromes (MDS) is largely dependent on morphologic examination of bone marrow aspirates. Several criteria that form the basis of the classifications and scoring systems most commonly used in clinical practice are affected by operator-dependent variation. To identify standardized molecular markers that would allow prediction of prognosis, we have used gene expression profiling (GEP) data on CD34+ cells from patients with MDS to determine the relationship between gene expression levels and prognosis. GEP data on CD34+ cells from 125 patients with MDS with a minimum 12-month follow-up since date of bone marrow sample collection were included in this study. Supervised principal components and lasso penalized Cox proportional hazards regression (Coxnet) were used for the analysis. We identified several genes, the expression of which was significantly associated with survival of patients with MDS, including LEF1, CDH1, WT1, and MN1. The Coxnet predictor, based on expression data on 20 genes, outperformed other predictors, including one that additionally used clinical information. Our Coxnet gene signature based on CD34+ cells significantly identified a separation of patients with good or bad prognosis in an independent GEP data set based on unsorted bone marrow mononuclear cells, demonstrating that our signature is robust and may be applicable to bone marrow cells without the need to isolate CD34+ cells. We present a new, valuable GEP-based signature for assessing prognosis in MDS. GEP-based signatures correlating with clinical outcome may significantly contribute to a refined risk classification of MDS.

  17. Isolation of neural progenitor cells from the human adult subventricular zone based on expression of the cell surface marker CD271.

    PubMed

    van Strien, Miriam E; Sluijs, Jacqueline A; Reynolds, Brent A; Steindler, Dennis A; Aronica, Eleonora; Hol, Elly M

    2014-04-01

    Neural progenitor cells (NPCs) in the subventricular zone (SVZ) hold promise for future therapy for neurodegenerative disorders, because the stimulation of adult neurogenesis could potentially restore the function of degenerating neurons and glia. To obtain more knowledge on these NPCs, we developed a method to specifically isolate NPCs from postmortem adult human brains based on the expression of the specific human adult neural stem/progenitor cell marker glial fibrillary acidic protein δ (GFAPδ). An extensive immunophenotyping analysis for cell surface markers resulted in the observation that CD271 was limited to the SVZ-derived GFAPδ-positive cells. CD271(+) cells developed into neurospheres and could be differentiated into astrocytes, neurons, and oligodendrocytes. We are the first to show that a pure population of NPCs can be isolated from the adult human SVZ, which is highly instrumental for developing future therapies based on stimulating endogenous SVZ neurogenesis.

  18. Isolation of Neural Progenitor Cells From the Human Adult Subventricular Zone Based on Expression of the Cell Surface Marker CD271

    PubMed Central

    Sluijs, Jacqueline A.; Reynolds, Brent A.; Steindler, Dennis A.; Aronica, Eleonora

    2014-01-01

    Neural progenitor cells (NPCs) in the subventricular zone (SVZ) hold promise for future therapy for neurodegenerative disorders, because the stimulation of adult neurogenesis could potentially restore the function of degenerating neurons and glia. To obtain more knowledge on these NPCs, we developed a method to specifically isolate NPCs from postmortem adult human brains based on the expression of the specific human adult neural stem/progenitor cell marker glial fibrillary acidic protein δ (GFAPδ). An extensive immunophenotyping analysis for cell surface markers resulted in the observation that CD271 was limited to the SVZ-derived GFAPδ-positive cells. CD271+ cells developed into neurospheres and could be differentiated into astrocytes, neurons, and oligodendrocytes. We are the first to show that a pure population of NPCs can be isolated from the adult human SVZ, which is highly instrumental for developing future therapies based on stimulating endogenous SVZ neurogenesis. PMID:24604282

  19. A PCR-based forward genetics screening, using expression domain-specific markers, identifies mutants in endosperm transfer cell development

    PubMed Central

    Muñiz, Luis M.; Gómez, Elisa; Guyon, Virginie; López, Maribel; Khbaya, Bouchaib; Sellam, Olivier; Peréz, Pascual; Hueros, Gregorio

    2014-01-01

    Mutant collections are an invaluable source of material on which forward genetic approaches allow the identification of genes affecting a wide variety of biological processes. However, some particular developmental stages and morphological structures may resist analysis due to their physical inaccessibility or to deleterious effects associated to their modification. Furthermore, lethal mutations acting early in development may escape detection. We have approached the characterization of 101 maize seed mutants, selected from a collection of 27,500 visually screened Mu-insertion lines, using a molecular marker approach based on a set of genes previously ascribed to different tissue compartments within the early developing kernel. A streamlined combination of qRT-PCR assays has allowed us to preliminary pinpoint the affected compartment, establish developmental comparisons to WT siblings and select mutant lines with alterations in the different compartments. Furthermore, clusters of markers co-affected by the underlying mutation were identified. We have analyzed more extensively a set of lines presenting significant variation in transfer cell-associated expression markers, and have performed morphological observations, and immunolocalization experiments to confirm the results, validating this approach as an efficient mutant description tool. PMID:24808899

  20. Reduced expression of autophagy markers correlates with high-risk human papillomavirus infection in human cervical squamous cell carcinoma

    PubMed Central

    WANG, HUA-YI; YANG, GUI-FANG; HUANG, YAN-HUA; HUANG, QI-WEN; GAO, JUN; ZHAO, XIAN-DA; HUANG, LI-MING; CHEN, HONG-LEI

    2014-01-01

    Infection by an oncogenic human papillomavirus (HPV), in particular HPV16 and 18, is a high risk factor for developing cervical cancer; however, viral infection alone is not sufficient for cancer progression. Autophagy is hypothesized to be an important process during carcinogenesis. The aim of the present study was to investigate the association between autophagy and high-risk HPV (hrHPV) infection in human cervical squamous cell carcinomas (SCCs), and to analyze the clinical significance of this association. Quantum dot (QD)-based immunofluorescence histochemistry was used to detect the expression of autophagy markers, Beclin-1 and microtubule-associated proteins 1A/1B light chain 3B (LC3B) proteins, in 104 cases of cervical cancer (including 80 SCCs and 24 adenocarcinomas) and 20 normal cervical tissues. hrHPV (HPV16/18) infection was detected by QDs based fluorescence in situ hybridization in cervical cancers. The results revealed that the expression levels of Beclin-1 and LC3B were significantly lower in cervical cancer cells when compared with those of normal cervical squamous epithelial cells, and were found to negatively correlate with hrHPV infection. The expression levels of Beclin-1 and LC3B were not associated with age, tumor grade, tumor stage, tumor node metastasis stage or lymph node metastasis. However, a positive correlation was identified between Beclin-1 and LC3B protein expression. In addition, the absence of autophagy in combination with hrHPV infection may accelerate the progression of cervical SCC. In conclusion, decreased expression of Beclin-1 and LC3B may be important in cervical carcinogenesis. The hrHPV-host cell interaction may inhibit autophagy, which may aid virus duplication and infection, as well as cervical cancer development. PMID:25202355

  1. Influence of inositol hexaphosphate on the expression of selected proliferation markers in IL-1β-stimulated intestinal epithelial cells.

    PubMed

    Kapral, Małgorzata; Sośnicki, Stanisław; Wawszczyk, Joanna; Węglarz, Ludmiła

    2014-01-01

    The aim of the present study was to examine the influence of IP6, a naturally occurring phytochem- ical, on the expression of genes coding for proliferation markers, i.e., cyclin D1 (CCND1) and histone H3 in IL-1β-stimulated intestinal cancer cell line Caco-2. Quantification of genes expression was carried out using real time RT-QPCR technique in Caco-2 cells after treatment with IL-1β, 1 and 2.5 mM of IP6 for 3, 6 and 12 h. In separate cultures, cells were incubated with IL-1β for the indicated times. The untreated Caco-2 cells were used as the control. In a time course experiment, stimulation of cells with IL-1β only resulted in an overex- pression of both CCND1 and histone H3 mRNAs as compared with control. IP6 had no influence on IL-1β-stimulated CCND1 expression for 3 and 6 h. After 12 h, statistically significant decrease in CCND1 mRNA was observed in cells exposed to IL-1β and IP6 (1 and 2.5 mM) in relation to cells treated with IL-1β only. The levels of H3 mRNA in IL-1β-stimulated cells and cells treated with IL-1β and IP6 revealed no statistically significant differences after 3 h. IP6 at 1 and 2.5 mM enhanced IL1β-stimulated transcription of H3 gene after 6 h. Subsequently (12 h), the combination of IP6 and IL-1β decreased H3 mRNA level compared to IL1β-treated cells. In conclusion, pro-inflammatory cytokine IL-1β up-regulates CCND1 and histone H3 mRNAs expression in Caco-2 cells. These results suggest that the ability of IP6 to inhibit colon cancer cells proliferation may be mediated through downregulation of genes encoding cyclin D1 and histone H3 at the mRNA level.

  2. The putative human stem cell marker, Rex-1 (Zfp42): structural classification and expression in normal human epithelial and carcinoma cell cultures.

    PubMed

    Mongan, Nigel P; Martin, Kisha M; Gudas, Lorraine J

    2006-12-01

    Human Rex-1 (hRex-1) (also referred to as zinc-finger protein-42, Zfp42) encodes a zinc finger protein expression of which is believed to be characteristic of pluripotent stem cells. We have applied bioinformatics to classify the relationship of human, rat, and mouse REX1 proteins in the C2H2 family of zinc finger proteins and demonstrate that REX1 is a member of the YY1 sub-family of transcription factors, which includes the Drosophila pleiohomeotic (Pho) protein. We have generated a molecular model of the human REX1 zinc finger domains based on the crystal structure of the YY1 transcription factor. To date, expression of hRex-1 and its extensively studied mouse homolog mRex-1, has been reported only in embryonic and adult stem cells and in differentiated spermatocytes. In this study, reverse transcription-PCR and Western analysis were employed to assay for hRex-1 expression in cultured normal human epithelial cells and human carcinoma cell lines. Expression of hRex-1 mRNA was detected in normal human epidermal keratinocytes, normal prostate epithelial cells (PrEC), bronchial, and small airway lung epithelial cells. Other stem cell markers, such as Oct 4, DAB2, and cMyc were also detected in normal human epidermal keratinocyte cultures. Expression of hRex-1 was also detected in some human tumor cell lines including MDA-MB-468 mammary carcinoma, SCC-15 head and neck squamous cell carcinoma, and N-TERA2 human teratocarcinoma cells. Western analyses confirmed expression of the human REX1 (ZFP42) protein in MDA-MB-468 cells and normal human keratinocytes. This research has identified model human cell culture systems, in addition to embryonic stem (ES) cells, in which Rex-1 is expressed, and this should enable the characterization of REX1 functions in normal adult epithelial cells and tumorigenic stem cells.

  3. CD44v6-competent tumor exosomes promote motility, invasion and cancer-initiating cell marker expression in pancreatic and colorectal cancer cells

    PubMed Central

    Wang, Zhe; von Au, Anja; Schnölzer, Martina; Hackert, Thilo; Zöller, Margot

    2016-01-01

    Cancer-initiating cells (CIC) account for metastatic spread, which may rely mostly on CIC exosomes (TEX) that affect host cells and can transfer CIC features into Non-CIC. The CIC marker CD44 variant isoform v6 (CD44v6) being known for metastasis-promotion, we elaborated in cells its contribution to migration and invasion and in TEX the tranfer of migratory and invasive capacity to Non-CIC, using a CD44v6 knockdown (CD44v6kd) as Non-CIC model. A CD44v6kd in human pancreatic and colorectal cancer (PaCa, CoCa) lines led to loss of CIC characteristics including downregulation of additional CIC markers, particularly Tspan8. This aggravated the loss of CD44v6-promoted motility and invasion. Loss of motility relies on the distorted cooperation of CD44v6 and Tspan8 with associated integrins and loss of invasiveness on reduced protease expression. These deficits, transferred into TEX, severely altered the CD44v6kd-TEX composition. As a consequence, unlike the CIC-TEX, CD44v6kd TEX were not taken up by CD44v6kd cells and CIC. The uptake of CIC-TEX was accompanied by partial correction of CIC marker and protease expression in CD44v6kd cells, which regained migratory, invasive and metastatic competence. CIC-TEX also fostered angiogenesis and expansion of myeloid cells, likely due to a direct impact of CIC-TEX on the host, which could be supported by reprogrammed CD44v6kd cells. Taken together, the striking loss of tumor progression by a CD44v6kd relies on the capacity of CD44v6 to cooperate with associating integrins and proteases and its promotion of additional CIC marker expression. The defects by a CD44v6kd are efficiently corrected upon CIC-TEX uptake. PMID:27419629

  4. CD44v6-competent tumor exosomes promote motility, invasion and cancer-initiating cell marker expression in pancreatic and colorectal cancer cells.

    PubMed

    Wang, Zhe; von Au, Anja; Schnölzer, Martina; Hackert, Thilo; Zöller, Margot

    2016-08-23

    Cancer-initiating cells (CIC) account for metastatic spread, which may rely mostly on CIC exosomes (TEX) that affect host cells and can transfer CIC features into Non-CIC. The CIC marker CD44 variant isoform v6 (CD44v6) being known for metastasis-promotion, we elaborated in cells its contribution to migration and invasion and in TEX the tranfer of migratory and invasive capacity to Non-CIC, using a CD44v6 knockdown (CD44v6kd) as Non-CIC model.A CD44v6kd in human pancreatic and colorectal cancer (PaCa, CoCa) lines led to loss of CIC characteristics including downregulation of additional CIC markers, particularly Tspan8. This aggravated the loss of CD44v6-promoted motility and invasion. Loss of motility relies on the distorted cooperation of CD44v6 and Tspan8 with associated integrins and loss of invasiveness on reduced protease expression. These deficits, transferred into TEX, severely altered the CD44v6kd-TEX composition. As a consequence, unlike the CIC-TEX, CD44v6kd TEX were not taken up by CD44v6kd cells and CIC. The uptake of CIC-TEX was accompanied by partial correction of CIC marker and protease expression in CD44v6kd cells, which regained migratory, invasive and metastatic competence. CIC-TEX also fostered angiogenesis and expansion of myeloid cells, likely due to a direct impact of CIC-TEX on the host, which could be supported by reprogrammed CD44v6kd cells.Taken together, the striking loss of tumor progression by a CD44v6kd relies on the capacity of CD44v6 to cooperate with associating integrins and proteases and its promotion of additional CIC marker expression. The defects by a CD44v6kd are efficiently corrected upon CIC-TEX uptake.

  5. Effect of Cryopreserved Amniotic Membrane Orientation on the Expression of Limbal Mesenchymal and Epithelial Stem Cell Markers in Prolonged Limbal Explant Cultures

    PubMed Central

    Lužnik, Zala; Hawlina, Marko; Maličev, Elvira; Bertolin, Marina; Kopitar, Andreja Nataša; Ihan, Alojz; Ferrari, Stefano; Schollmayer, Petra

    2016-01-01

    Purpose To evaluate the effect of prolonged limbal explants cultured without any scaffolds or on amniotic membrane (AM) on the viability, proliferation and differentiation potential of putative phenotypically defined cultured limbal mesenchymal (LMSC) and epithelial stem cells (LESC). Methods Limbal explants were cultivated on cryopreserved intact AM or plastic plates using medium supplemented with only human serum. AM was positioned with either the epithelial or stromal side up. The outgrowing cells were immunophenotyped for the co-expression of mesenchymal stem cell markers (CD73/CD90/CD105 positive and CD45 negative), proliferation and putative progenitor markers (CXCR4, CD117), epithelial markers and antigen presenting cell markers (CD80, CD83, CD86) by flow cytometry. Immunohistochemistry on limbal cultures cultivated on AM was carried out with antibodies against pan-cytokeratin, p63, Ki67. Results Morphological and immunostaining analyses revealed two distinct stem cell population types, which could be identified over prolonged culturing time periods. Expression of LMSC markers and CXCR4 was significantly higher (p < 0.05) in cultures cultivated without AM. However, no statistically significant difference was observed in CD117 expression. The cells cultivated on AM retained an epithelial cell structure, which was further confirmed by histology examination. Histology revealed limbal epithelial growth and p63, Ki67 positive cells on both sides of AM. Conclusion Limbal cells cultivated on AM exhibited a lower expression profile of LMSC and CXCR4 markers as limbal cells cultivated on plastic culture plates. However, CD117 expression was similar. Histology confirmed limbal epithelial cell growth on both sides of AM, with no morphological differences, or positivity of cells for p63 and Ki67. PMID:27723792

  6. Acute damage by naphthalene triggers expression of the neuroendocrine marker PGP9.5 in airway epithelial cells

    PubMed Central

    Poulsen, Thomas T.; Naizhen, Xu; Poulsen, Hans S.; Linnoila, R. Ilona

    2008-01-01

    Protein Gene Product 9.5 (PGP9.5) is highly expressed in nervous tissue. Recently PGP9.5 expression has been found to be upregulated in the pulmonary epithelium of smokers and in non-small cell lung cancer, suggesting that it also plays a role in carcinogen-inflicted lung epithelial injury and carcinogenesis. We investigated the expression of PGP9.5 in mice in response to two prominent carcinogens found in tobacco smoke: Naphthalene and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). By immunostaining, we found that PGP9.5 protein was highly expressed throughout the airway epithelium in the days immediately following a single injection of naphthalene. In contrast, PGP9.5 was exclusively confined to neurons and neuroendocrine cells in the control and NNK-exposed lungs. Furthermore, we investigated the expression of PGP9.5 mRNA in the lungs by quantitative RT-PCR (qPCR). PGP9.5 mRNA expression was highly upregulated in the days immediately following naphthalene injection and gradually returning to that of control mice 5 days after naphthalene injection. In contrast, exposure to NNK did not result in a significant increase in PGP9.5 mRNA 10 weeks after exposure. No increased expression of two other neuroendocrine markers was found in the non-neuroendocrine epithelial cells after naphthalene exposure. In contrast, immunostaining for the cell cycle regulator p27Kip1, which has previously been associated with PGP9.5 in lung cancer cells, revealed transient downregulation of p27Kip1 in naphthalene exposed airways compared to controls, indicating that the rise in PGP9.5 in the airway epithelium is related to downregulation of p27Kip1. This study is the first to specifically identify the carcinogen naphthalene as an inducer of PGP9.5 expression in non-neuroendocrine epithelium after acute lung injury and further strengthens the accumulating evidence of PGP9.5 as a central player in lung epithelial damage and early carcinogenesis. PMID:18687389

  7. Human non-small cell lung cancer expresses putative cancer stem cell markers and exhibits the transcriptomic profile of multipotent cells.

    PubMed

    Zakaria, Norashikin; Yusoff, Narazah Mohd; Zakaria, Zubaidah; Lim, Moon Nian; Baharuddin, Puteri J Noor; Fakiruddin, Kamal Shaik; Yahaya, Badrul

    2015-02-25

    Despite significant advances in staging and therapies, lung cancer remains a major cause of cancer-related lethality due to its high incidence and recurrence. Clearly, a novel approach is required to develop new therapies to treat this devastating disease. Recent evidence indicates that tumours contain a small population of cells known as cancer stem cells (CSCs) that are responsible for tumour maintenance, spreading and resistant to chemotherapy. The genetic composition of CSCs so far is not fully understood, but manipulation of the specific genes that maintain their integrity would be beneficial for developing strategies to combat cancer. Therefore, the goal of this study isto identify the transcriptomic composition and biological functions of CSCs from non-small cell lung cancer (NSCLC). We isolated putative lung CSCs from lung adenocarcinoma cells (A549 and H2170) and normal stem cells from normal bronchial epithelial cells (PHBEC) on the basis of positive expression of stem cell surface markers (CD166, CD44, and EpCAM) using fluorescence-activated cell sorting. The isolated cells were then characterised for their self-renewal characteristics, differentiation capabilities, expression of stem cell transcription factor and in vivo tumouregenicity. The transcriptomic profiles of putative lung CSCs then were obtained using microarray analysis. Significantly regulated genes (p < 0.05, fold change (FC) > 2.0) in putative CSCs were identified and further analysed for their biological functions using the Database for Annotation, Visualization, and Integrated Discovery (DAVID). The putative lung CSCs phenotypes of CD166(+)/CD44(+) and CD166(+)/EpCAM(+) showed multipotent characteristics of stem cells, including the ability to differentiate into adipogenic and osteogenic cells, self-renewal, and expression of stem cell transcription factors such as Sox2 and Oct3/4. Moreover, the cells also shows the in vivo tumouregenicity characteristic when transplanted into

  8. Cytoskeletal proteins and stem cell markers gene expression in human bone marrow mesenchymal stromal cells after different periods of simulated microgravity

    NASA Astrophysics Data System (ADS)

    Gershovich, P. M.; Gershovich, J. G.; Zhambalova, A. P.; Romanov, Yu. A.; Buravkova, L. B.

    2012-01-01

    Mesenchymal stem (stromal) cells (MSCs) are present in a variety of tissues during prenatal and postnatal human development. In adult organism, they are prevalent in bone marrow and supposed to be involved in space-flight induced osteopenia. We studied expression of various genes in human bone marrow MSCs after different terms of simulated microgravity (SMG) provided by Random Positioning Machine. Simulated microgravity induced transient changes in expression level of genes associated with actin cytoskeleton, especially after 48 h of SMG. However, after 120 h exposure in SMG partial restoration of gene expression levels (relative to the control) was found. Similar results were obtained with bmMSCs subjected to 24 h readaptation in static state after 24 h in SMG. Analysis of 84 genes related to identification, growth and differentiation of stem cells revealed that expression of nine genes was changed slightly after 48 h in SMG. More pronounced changes in gene expression of "stem cells markers" were observed after 120 h of simulated microgravity. Among 84 investigated genes, 30 were up-regulated and 24 were down-regulated. Finally, MSCs osteogenesis induced by long-term (10-20 days) simulation of microgravity was accompanied by down-regulation of gene expression of the main osteogenic differentiation markers ( ALPL, OMD) and master transcription osteogenic factor of MSCs ( Runx2). Thus, our study demonstrated that changes in expression level of some genes associated with actin cytoskeleton and stem cell markers are supposed to be one of the mechanisms, which contribute to precursor's cellular adaptation to the microgravity conditions. These results can clarify genomic mechanisms through which SMG reduces osteogenic differentiation of bmMSCs.

  9. Dental pulp stem cells express tendon markers under mechanical loading and are a potential cell source for tissue engineering of tendon-like tissue

    PubMed Central

    Chen, Yu-Ying; He, Sheng-Teng; Yan, Fu-Hua; Zhou, Peng-Fei; Luo, Kai; Zhang, Yan-Ding; Xiao, Yin; Lin, Min-Kui

    2016-01-01

    Postnatal mesenchymal stem cells have the capacity to differentiate into multiple cell lineages. This study explored the possibility of dental pulp stem cells (DPSCs) for potential application in tendon tissue engineering. The expression of tendon-related markers such as scleraxis, tenascin-C, tenomodulin, eye absent homologue 2, collagens I and VI was detected in dental pulp tissue. Interestingly, under mechanical stimulation, these tendon-related markers were significantly enhanced when DPSCs were seeded in aligned polyglycolic acid (PGA) fibre scaffolds. Furthermore, mature tendon-like tissue was formed after transplantation of DPSC-PGA constructs under mechanical loading conditions in a mouse model. This study demonstrates that DPSCs could be a potential stem cell source for tissue engineering of tendon-like tissue. PMID:27811845

  10. Dental pulp stem cells express tendon markers under mechanical loading and are a potential cell source for tissue engineering of tendon-like tissue.

    PubMed

    Chen, Yu-Ying; He, Sheng-Teng; Yan, Fu-Hua; Zhou, Peng-Fei; Luo, Kai; Zhang, Yan-Ding; Xiao, Yin; Lin, Min-Kui

    2016-12-16

    Postnatal mesenchymal stem cells have the capacity to differentiate into multiple cell lineages. This study explored the possibility of dental pulp stem cells (DPSCs) for potential application in tendon tissue engineering. The expression of tendon-related markers such as scleraxis, tenascin-C, tenomodulin, eye absent homologue 2, collagens I and VI was detected in dental pulp tissue. Interestingly, under mechanical stimulation, these tendon-related markers were significantly enhanced when DPSCs were seeded in aligned polyglycolic acid (PGA) fibre scaffolds. Furthermore, mature tendon-like tissue was formed after transplantation of DPSC-PGA constructs under mechanical loading conditions in a mouse model. This study demonstrates that DPSCs could be a potential stem cell source for tissue engineering of tendon-like tissue.

  11. Vitamin D compounds reduce mammosphere formation and decrease expression of putative stem cell markers in breast cancer.

    PubMed

    Wahler, Joseph; So, Jae Young; Cheng, Larry C; Maehr, Hubert; Uskokovic, Milan; Suh, Nanjoo

    2015-04-01

    Breast cancer stem cells (BCSCs) are a subset of tumor cells that are believed to be the cells responsible for the establishment and maintenance of tumors. Moreover, BCSCs are suggested to be the main cause of progression to metastasis and recurrence of cancer because of their tumor-initiating abilities and resistance to conventional therapies. Ductal carcinoma in situ (DCIS) is an early precursor in breast carcinogenesis which progresses to invasive ductal carcinoma (IDC). We have previously reported that a vitamin D compound, BXL0124, inhibits the progression of DCIS to IDC. In the present study we sought to determine whether this effect was mediated through an influence on BCSCs. In MCF10DCIS cells treated with vitamin D compounds (1α25(OH)2D3 or BXL0124), the breast cancer stem cell-like population, identified by the CD44(+)/CD24(-/low) and CD49f(+)/CD24(-/low) subpopulations, was reduced. To determine the effects of vitamin D compounds on cancer stem cell activity, the MCF10DCIS mammosphere cell culture system, which enriches for mammary progenitor cells and putative BCSCs, was utilized. Untreated MCF10DCIS mammospheres showed a disorganized and irregular shape. When MCF10DCIS cells were treated with 1α25(OH)2D3 or BXL0124, the mammospheres that formed exhibited a more organized, symmetrical and circular shape, similar to the appearance of spheres formed by the non-malignant, normal mammary epithelial cell line, MCF10A. The mammosphere forming efficiency (MFE) was significantly decreased upon treatment with 1α25(OH)2D3 or BXL0124, indicating that these compounds have an inhibitory effect on mammosphere development. Treatment with 1α25(OH)2D3 or BXL0124 repressed markers associated with the stem cell-like phenotype, such as CD44, CD49f, c-Notch1, and pNFκB. Furthermore, 1α25(OH)2D3 and BXL0124 reduced the expression of pluripotency markers, OCT4 and KLF-4 in mammospheres. This study suggests that vitamin D compounds repress the breast cancer stem cell

  12. Circulating tumor cells expressing cancer stem cell marker CD44 as a diagnostic biomarker in patients with gastric cancer

    PubMed Central

    Watanabe, Toru; Okumura, Tomoyuki; Hirano, Katsuhisa; Yamaguchi, Tetsuji; Sekine, Shinichi; Nagata, Takuya; Tsukada, Kazuhiro

    2017-01-01

    Epithelial cell adhesion molecule (EpCAM) is a marker for circulating tumor cells (CTCs) in various types of cancer, while cluster of differentiation 44 (CD44) is a marker for gastric cancer (GC) stem cells. To evaluate the clinical significance of CD44+ CTCs in patients with GC in the present study, the number of EpCAM+CD44+ and EpCAM+CD44− cells were detected in the peripheral blood of 26 GC patients and 12 healthy volunteers using flow cytometry. The number (mean ± standard deviation) of EpCAM+CD44+ cells in the GC patients and healthy volunteers was 69.9±52.0 and 0.91±2.10, respectively (P=0.0001), while that of EpCAM+CD44− cells was 59.1±88.0 and 9.83±9.91, respectively (P=0.0313). The sensitivity and specificity of EpCAM+CD44+ cell detection for the identification of GC patients were 92.3 and 100%, respectively. By contrast, the values of EpCAM+CD44− cell detection were 76.9 and 83.3%, respectively. The number of EpCAM+CD44+ cells in the GC patients was correlated with the disease stage (P=0.0423), the depth of the tumor (P=0.0314) and venous invasion (P=0.0184) in the resected tumor specimens, while the number of EpCAM+CD44− cells did not correlate with any clinicopathological factors. The number of EpCAM+CD44+ cells significantly decreased following surgical resection of the tumor or induction of systemic chemotherapy. Additionally, atypical cells with a high nuclear to cytoplasmic ratio were morphologically detected in the sorted EpCAM+CD44+ cells. These results suggested that CD44+ CTCs, but not CD44− CTCs, reflect the malignant status of the primary tumor in patients with GC, providing a candidate biomarker for diagnosis and treatment response. PMID:28123556

  13. MET expression and copy number status in clear-cell renal cell carcinoma: prognostic value and potential predictive marker

    PubMed Central

    Macher-Goeppinger, Stephan; Keith, Martina; Endris, Volker; Penzel, Roland; Tagscherer, Katrin E.; Pahernik, Sascha; Hohenfellner, Markus; Gardner, Humphrey; Grüllich, Carsten; Schirmacher, Peter; Roth, Wilfried

    2017-01-01

    Multiple targeted therapy for advanced clear-cell renal cell carcinoma (RCC) has substantially improved patient outcome, but complete remission is uncommon and many tumors eventually develop resistance. Mechanistic, preclinical, and early clinical data highlight c-Met / hepatocyte growth factor receptor as a promising target for RCC therapeutic agents. We have examined MET expression, frequency of MET gene copy gains and MET gene mutation in a large, hospital-based series of renal cell carcinomas with long-term follow-up information. Out of a total of 572 clear-cell RCC, only 17% were negative for MET expression whereas 32% showed high protein levels. High MET expression and MET copy number gains were associated with an aggressive phenotype and an unfavorable patient outcome. Elevated protein levels in absence of gene amplification were not attributed to mutations, based on results of targeted next-generation sequencing. Our data reveal that clear-cell RCC with MET upregulation show an aggressive behavior and MET copy number increase is evident in a substantial percentage of patients with high-grade carcinomas and metastatic disease. Diagnostic assessment of MET expression and amplification may be of predictive value to guide targeted therapy against MET signaling in patients with clear-cell RCC. PMID:27894094

  14. Expression pattern of pluripotent markers in different embryonic developmental stages of buffalo (Bubalus bubalis) embryos and putative embryonic stem cells generated by parthenogenetic activation.

    PubMed

    Singh, Karn P; Kaushik, Ramakant; Garg, Veena; Sharma, Ruchi; George, Aman; Singh, Manoj K; Manik, Radhey S; Palta, Prabhat; Singla, Suresh K; Chauhan, Manmohan S

    2012-12-01

    In this study, we describe the production of buffalo parthenogenetic blastocysts and subsequent isolation of parthenogenetic embryonic stem cell (PGESC)-like cells. PGESC colonies exhibited dome-shaped morphology and were clearly distinguishable from the feeder layer cells. Different stages of development of parthenogenetic embryos and derived embryonic stem cell (ESC)-like cells expressed key ESC-specific markers, including OCT-4, NANOG, SOX-2, FOXD3, REX-1, STAT-3, TELOMERASE, NUCLEOSTEMIN, and cMYC. Immunofluorescence-based studies revealed that the PGESCs were positive for surface-based pluripotent markers, viz., SSEA-3, SSEA-4, TRA 1-80, TRA 1-60, CD-9, and CD-90 and exhibited high alkaline phosphatase (ALP) activity. PGEC cell-like cells formed embryoid body (EB)-like structures in hanging drop cultures and when cultured for extended period of time spontaneously differentiated into derivatives of three embryonic germ layers as confirmed by RT-PCR for ectodermal (CYTOKERATIN8, NF-68), mesodermal (MSX1, BMP-4, ASA), and endodermal markers (AFP, HNF-4, GATA-4). Differentiation of PGESCs toward the neuronal lineage was successfully directed by supplementation of serum-containing media with retinoic acid. Our results indicate that the isolated ESC-like cells from parthenogenetic blastocyst hold properties of ESCs and express markers of pluripotency. The pluripotency markers were also expressed by early cleavage-stage of buffalo embryos.

  15. Inhibition of SRY-calmodulin complex formation induces ectopic expression of ovarian cell markers in developing XY gonads.

    PubMed

    Sim, Helena; Argentaro, Anthony; Czech, Daniel P; Bagheri-Fam, Stefan; Sinclair, Andrew H; Koopman, Peter; Boizet-Bonhoure, Brigitte; Poulat, Francis; Harley, Vincent R

    2011-07-01

    The transcription factor sex-determining region of the Y chromosome (SRY) plays a key role in human sex determination, because mutations in SRY cause disorders of sex development in XY individuals. During gonadal development, Sry in pre-Sertoli cells activates Sox9 gene transcription, committing the fate of the bipotential gonad to become a testis rather than an ovary. The high-mobility group domain of human SRY contains two independent nuclear localization signals, one bound by calmodulin (CaM) and the other by importin-β. Although XY females carry SRY mutations in these nuclear localization signals that affect SRY nuclear import in transfected cells, it is not known whether these transport mechanisms are essential for gonadal development and sex determination. Here, we show that mouse Sry protein binds CaM and that a CaM antagonist reduces CaM binding, nuclear accumulation, and transcriptional activity of Sry in transfected cells. CaM antagonist treatment of cultured, sexually indifferent XY mouse fetal gonads led to reduced expression of the Sry target gene Sox9, defects in testicular cord formation, and ectopic expression of the ovarian markers Rspondin1 and forkhead box L2. These results indicate the importance of CaM for SRY nuclear import, transcriptional activity, testis differentiation, and sex determination.

  16. Expression of VEGF-A, HIF-1 A, CD34 and Ki67 in clear cell renal cell carcinomas and their relationship with conventional prognostic markers.

    PubMed

    Bürgesser, María Virginia; Riva, Verónica; Ojeda, Silvia María; Muñoz Morales, Duberney; Calafat, Patricia; Diller, Ana

    2014-01-01

    Clear cell renal carcinoma is the most frequent type of renal carcinoma. Recently, attention has been focused in the expression of angiogenic factors by these tumors, which would justify in part their capacity to grow, invade and disseminate, stating a worse evolution of those patients with an unfavorable angiogenic profile. 83 samples of nephrectomy with a diagnosis of clear cell renal cell carcinoma were studied. Clinical and pathological data were collected. Tumors were studied to assess immunohistochemical expression of the following markers: VEGF-A, HIF-1α, CD34 and Ki67. Results indicated a direct linear relationship between expressions of these four markers. Besides, the expression of HIF-1α was directly related to Furhman grade, invasion of the renal vein and tumor stage. Likewise, tumor proliferation index, assessed with Ki67, was directly related to the presence of necrosis, capsular invasion and advanced tumor stage. Regarding the expression of CD34, vascular density was inversely related to tumor necrosis and overall survival. These findings are controversial compared with the available literature. Then, a research scenery would be open, where the importance of generating prospective and more standardized studies are highlighted to determine the role of these angiogenic factors in tumor evolution and prognostic evaluation of these tumors.

  17. High level of perforin expression in T cells: An early prognostic marker of the severity of herpesvirus reactivation after allogeneic stem cell transplantation in adults.

    PubMed

    Pietersma, F L; van Dorp, S; Jacobi, R; Ran, L; Nanlohy, N M; Schuurman, R; Minnema, M C; Meijer, E; van Baarle, D

    2010-03-01

    Epstein-Barr virus (EBV) and cytomegalovirus reactivations are frequent complications of hematopoeitic allogeneic stem cell transplantation (SCT) because of a lack of T cell control after immunosuppression. Early diagnosis of reactivation and subsequent preemptive therapy relies on frequent viral load measurement. Additional virus-specific T cell reconstitution data could improve the predictive value of viral load detection for viral complications after transplantation. Here, we studied perforin expression in CD8(+) T cells as a measure of cytotoxic T cell capacity in relation to the occurrence of viral reactivation. In a prospective study, we monitored 40 patients during the first 3 months after transplantation and measured viral loads in combination with intracellular perforin expression in CD8(+) T cells. Median perforin expression in CD8(+) T cells throughout follow-up was higher in patients with viral reactivations than in patients without viral reactivations (4.9% vs 2.3%; P = .001). The median percentage of perforin-expressing CD8(+) T cells in patients with high viral reactivations exceeding 1000 copies/mL (10.7%) was statistically significantly higher than that in patients with minor reactivations of 50-1000 copies (4.0%), that in patients with detectable EBV loads that did not exceed the detection limit of 50 copies/mL (2.9%), and that in patients without reactivations (0.8%). Patients with high viral reactivations reached a high percentage of perforin-expressing CD8(+) T cells (>10.2%) more often and faster than did patients with low viral loads (1000 copies/mL) or without viral reactivations. High perforin expression preceded high viral loads. Perforin-expressing CD8(+) T cells may be useful as an easy-to-measure prognostic marker for identifying patients at risk for severe viral reactivation very soon after SCT.

  18. The fibroblast surface markers FAP, anti-fibroblast, and FSP are expressed by cells of epithelial origin and may be altered during epithelial-to-mesenchymal transition.

    PubMed

    Kahounová, Zuzana; Kurfürstová, Daniela; Bouchal, Jan; Kharaishvili, Gvantsa; Navrátil, Jiří; Remšík, Ján; Šimečková, Šárka; Študent, Vladimír; Kozubík, Alois; Souček, Karel

    2017-04-06

    The identification of fibroblasts and cancer-associated fibroblasts from human cancer tissue using surface markers is difficult, especially because the markers used currently are usually not expressed solely by fibroblasts, and the identification of fibroblast-specific surface molecules is still under investigation. It was aimed to compare three commercially available antibodies in the detection of different surface epitopes of fibroblasts (anti-fibroblast, fibroblast activation protein α, and fibroblast surface protein). The specificity of their expression, employing fibroblast cell lines and tumor-derived fibroblasts from breast and prostate tissues was investigated. Both the established fibroblast cell line HFF-1 and ex vivo primary fibroblasts isolated from breast and prostate cancer tissues expressed the tested surface markers to different degrees. Surprisingly, those markers were expressed also by permanent cell lines of epithelial origin, both benign and cancer-derived (breast-cell lines MCF 10A, HMLE and prostate-cell lines BPH-1, DU 145, and PC-3). The expression of fibroblast activation protein α increased on the surface of previously described models of epithelial cells undergoing epithelial-to-mesenchymal transition in response to treatment with TGF-β1. To prove the co-expression of the fibroblast markers on cells of epithelial origin, we used freshly dissociated human prostate and breast cancer tissues. The results confirmed the co-expression of anti-fibroblast and fibroblast surface protein on CD31/CD45-negative/EpCAM-positive epithelial cells. In summary, our data support the findings that the tested fibroblast markers are not fibroblast specific and may be expressed also by cells of epithelial origin (e.g., cells undergoing EMT). Therefore, the expression of these markers should be interpreted with caution, and the combination of several epitopes for both positive (anti-fibroblast or fibroblast activation protein α) and negative (Ep

  19. Tumor endothelial marker 8 expression levels in dendritic cell-based cancer vaccines are related to clinical outcome.

    PubMed

    Venanzi, Franco Maria; Petrini, Massimiliano; Fiammenghi, Laura; Bolli, Elisabetta; Granato, Anna Maria; Ridolfi, Laura; Gabrielli, Federica; Barucca, Alessandra; Concetti, Antonio; Ridolfi, Ruggero; Riccobon, Angela

    2010-01-01

    Previous studies have shown that tumor endothelial markers (TEMs 1-9) are up modulated in immunosuppressive, pro-angiogenic dendritic cells (DCs) found in tumor microenvironments. We recently reported that monocyte-derived DCs used for vaccination trials may accumulate high levels of TEM8 gene transcripts. Here, we investigate whether TEM8 expression in DC preparations represents a specific tumor-associated change of potential clinical relevance. TEM8 expression at the mRNA and protein level was evaluated by quantitative real-time RT-PCR and cytofluorimetric analysis in human clinical grade DCs utilized for the therapeutic vaccination of 17 advanced cancer patients (13 melanoma and 4 renal cell carcinoma). The analyses revealed that DCs from patients markedly differ in their ability to up-modulate TEM8. Indeed, mDCs from eight non-progressing patients [median overall survival (OS) = 32 months, all positive to the delayed-type hypersensitivity test (DTH)], had similar TEM8 mRNA expression levels [mDCs vs. immature iDCs; mean fold increase (mfi) = 1.97] to those found in healthy donors (mfi = 2.7). Conversely, mDCs from nine progressing patients (OS < 5 months, all but one with negative DTH) showed an increase in TEM8 mRNA levels (mfi = 12.88, p = 0.0018). The present observations suggest that TEM8 expression levels in DC-based therapeutic vaccines would allow the selection of a subgroup of patients who are most likely to benefit from therapeutic vaccination.

  20. The Transcription Factor MIST1 Is a Novel Human Gastric Chief Cell Marker Whose Expression Is Lost in Metaplasia, Dysplasia, and Carcinoma

    PubMed Central

    Lennerz, Jochen K. M.; Kim, Seok-Hyung; Oates, Edward L.; Huh, Won Jae; Doherty, Jason M.; Tian, Xiaolin; Bredemeyer, Andrew J.; Goldenring, James R.; Lauwers, Gregory Y.; Shin, Young-Kee; Mills, Jason C.

    2010-01-01

    The lack of reliable molecular markers for normal differentiated epithelial cells limits understanding of human gastric carcinogenesis. Recognized precursor lesions for gastric adenocarcinoma are intestinal metaplasia and spasmolytic polypeptide expressing metaplasia (SPEM), defined here by ectopic CDX2 and TFF2 expression, respectively. In mice, expression of the bHLH transcription factor MIST1, normally restricted to mature chief cells, is down-regulated as chief cells undergo experimentally induced metaplasia. Here, we show MIST1 expression is also a specific marker of human chief cells. SPEM, with and without MIST1, is present in human lesions and, akin to murine data, likely represents transitional (TFF2+/MIST1+ = “hybrid”-SPEM) and established (TFF2+/MIST1− = SPEM) stages. Co-visualization of MIST1 and CDX2 shows similar progressive loss of MIST1 with a transitional, CDX2+/MIST1− hybrid-intestinal metaplasia stage. Interinstitutional analysis and comparison of findings in tissue microarrays, resection specimens, and biopsies (n > 400 samples), comprising the entire spectrum of recognized stages of gastric carcinogenesis, confirm MIST1 expression is restricted to the chief cell compartment in normal oxyntic mucosa, rare in established metaplastic lesions, and lost in intraepithelial neoplasia/dysplasia and carcinoma of various types with the exception of rare chief cell carcinoma (∼1%). Our findings implicate MIST1 as a reliable marker of mature, healthy chief cells, and we provide the first evidence that metaplasia in humans arises at least in part from the chief cell lineage. PMID:20709804

  1. Expression of SHP2 and related markers in non-small cell lung cancer: a tissue microarray study of 80 cases.

    PubMed

    Tang, Chunlan; Luo, Dan; Yang, Heping; Wang, Qingliang; Zhang, Rong; Liu, Guoxiang; Zhou, Xiangdong

    2013-10-01

    The purpose of this study was to assess the relationships between the expression of SHP2 and VEGF, VEGFR-1, VEGFR-2, MMP-2, MMP-9, TIMP-1, TIMP-2, and microvessel density (MVD), as well as the clinicopathologic parameters of these markers in non-small cell lung cancer (NSCLC). Using a tissue microarray, the expression of these 8 markers in 80 NSCLC cases was detected by immunohistochemistry. The expression of the markers was higher in cancer tissues when compared with the surrounding tissues. The MVD was lower in the CD34-positive cancer tissues than in the surrounding tissues. Significantly higher positive rates of expression for SHP2, MMP-9, and MMP-2 were observed in patients with lymph node metastases. The later the clinical stage was, the higher the expression of MMP-9 and MMP-2. The expression of VEGF in patients with lung squamous cell carcinomas was significantly higher than in patients with lung adenocarcinomas. The positive expression of SHP2 correlated significantly with that of VEGFR-2 and the MVD and a survival disadvantage was noted in the patients with SHP2-positive tumors. Therefore, our data suggest that the expression of SHP2 in NSCLC has high specificity and sensitivity and is closely related to lymph node metastasis and the expression of VEGFR-2 and the MVD in patients with NSCLC. SHP2 expression may promote the invasion and metastasis of NSCLC through angiogenesis and the lymphatic system.

  2. Major Neutrophilia Observed in Acute Phase of Human Leptospirosis Is Not Associated with Increased Expression of Granulocyte Cell Activation Markers

    PubMed Central

    Raffray, Loic; Giry, Claude; Vandroux, David; Kuli, Barbara; Randrianjohany, Andry; Pequin, Anne-Marie; Renou, Frédéric; Jaffar-Bandjee, Marie-Christine; Gasque, Philippe

    2016-01-01

    It has long been known that pathogenic Leptospira can mobilize the immune system but the specific contribution of neutrophils to control the infectious challenge remains to be clarified. We herein analyzed the phenotype of circulating neutrophils of patients with leptospirosis and healthy controls for the expression of toll-like receptor (TLR) type 2 (TLR2, to sense the leptospiral LPS) and several activation markers: interleukin 8 chemokine receptor CD182 (CXCR2), CD11b of the integrin/opsonin complement receptor type 3 (CR3) and CD15 (ligand of the selectin). The plasmatic level of the main CD182 ligand, interleukin 8 (CXCL8), was measured by ELISA. Hospitalized leptospirosis cases showed marked neutrophilia, particularly in the most severe cases. Interestingly, TLR2 was significantly increased in leptospirosis but identical levels of CD182 and CD11b were detected when compared to controls. CD15 was significantly decreased on neutrophils in leptospirosis but returned to normal within 1 month. Basal levels of IL-8 were measured in control subjects and were not increased in leptospirosis cases at the initial stage of the disease. In conclusion, we observed that neutrophils failed to regulate the expression of several of the receptors involved in cell activation and recruitment. This study further emphasizes the paradigm that neutrophils may be impaired in their overall capacity to thwart bacterial infection in leptospirosis patients. PMID:27802348

  3. Major Neutrophilia Observed in Acute Phase of Human Leptospirosis Is Not Associated with Increased Expression of Granulocyte Cell Activation Markers.

    PubMed

    Raffray, Loic; Giry, Claude; Vandroux, David; Kuli, Barbara; Randrianjohany, Andry; Pequin, Anne-Marie; Renou, Frédéric; Jaffar-Bandjee, Marie-Christine; Gasque, Philippe

    2016-01-01

    It has long been known that pathogenic Leptospira can mobilize the immune system but the specific contribution of neutrophils to control the infectious challenge remains to be clarified. We herein analyzed the phenotype of circulating neutrophils of patients with leptospirosis and healthy controls for the expression of toll-like receptor (TLR) type 2 (TLR2, to sense the leptospiral LPS) and several activation markers: interleukin 8 chemokine receptor CD182 (CXCR2), CD11b of the integrin/opsonin complement receptor type 3 (CR3) and CD15 (ligand of the selectin). The plasmatic level of the main CD182 ligand, interleukin 8 (CXCL8), was measured by ELISA. Hospitalized leptospirosis cases showed marked neutrophilia, particularly in the most severe cases. Interestingly, TLR2 was significantly increased in leptospirosis but identical levels of CD182 and CD11b were detected when compared to controls. CD15 was significantly decreased on neutrophils in leptospirosis but returned to normal within 1 month. Basal levels of IL-8 were measured in control subjects and were not increased in leptospirosis cases at the initial stage of the disease. In conclusion, we observed that neutrophils failed to regulate the expression of several of the receptors involved in cell activation and recruitment. This study further emphasizes the paradigm that neutrophils may be impaired in their overall capacity to thwart bacterial infection in leptospirosis patients.

  4. Expression of Intermediate Filaments and Germ Cell Markers in the Developing Bovine Ovary: An Immunohistochemical and Laser-Assisted Microdissection Study.

    PubMed

    Kenngott, Rebecca Anna-Maria; Sauer, Ulrich; Vermehren, Margarete; Sinowatz, Fred

    2014-01-01

    In the present investigation, bovine ovary prenatal development was studied using immunohistochemistry and laser-assisted microdissection (LAM). A major aim of this study was to evaluate the protein expression pattern of intermediate filaments (IF) and distinguish S100 protein (S100 alpha and S100 beta protein) isoforms during prenatal follicle differentiation, subsequently correlating them with germ cell marker expression. A development-specific expression pattern of different keratins as well as vimentin was detected in the prenatal bovine ovary; K18-specific expression was found during all developmental stages (i.e. in surface epithelium, germ cell cord somatic cells, and follicle cells), and keratins 5, 7, 8, 14, and 19 and vimentin had a stage-specific expression pattern in the different cell populations of the prenatal ovaries. Additionally, our results represent new data on the expression pattern of germ cell markers during bovine ovary prenatal development. S100 alpha and beta protein was localized to oocyte cytoplasm of different follicle stages, and S100 alpha staining could be observed in granulosa cells. Furthermore, through isolation of characteristic ovary cell populations using LAM, specific confirmation of some genes of interest (KRT8, KRT18, S100 alpha, S100 beta, and OCT4, DDX4) could be obtained by RT-PCR in single cell groups of the developing bovine ovary. © 2015 S. Karger AG, Basel.

  5. Expression of novel, putative stem cell markers in prepubertal and lactating mammary glands of bovine

    USDA-ARS?s Scientific Manuscript database

    Mammary stem cells (MaSC) are essential for growth and maintenance of the mammary epithelium. Two main phases of mammary growth include ductal elongation prior to puberty and lobulo-alveolar growth and development during pregnancy. Some studies have utilized morphological characteristics and retenti...

  6. Tumor endothelial marker 5 expression in endothelial cells during capillary morphogenesis is induced by the small GTPase Rac and mediates contact inhibition of cell proliferation

    SciTech Connect

    Vallon, Mario; Rohde, Franziska; Janssen, Klaus-Peter; Essler, Markus

    2010-02-01

    Tumor endothelial marker (TEM) 5 is an adhesion G-protein-coupled receptor upregulated in endothelial cells during tumor and physiologic angiogenesis. So far, the mechanisms leading to upregulation of TEM5 and its function during angiogenesis have not been identified. Here, we report that TEM5 expression in endothelial cells is induced during capillary-like network formation on Matrigel, during capillary morphogenesis in a three-dimensional collagen I matrix, and upon confluence on a two-dimensional matrix. TEM5 expression was not induced by a variety of soluble angiogenic factors, including VEGF and bFGF, in subconfluent endothelial cells. TEM5 upregulation was blocked by toxin B from Clostridium difficile, an inhibitor of the small GTPases Rho, Rac, and Cdc42. The Rho inhibitor C3 transferase from Clostridium botulinum did not affect TEM5 expression, whereas the Rac inhibitor NSC23766 suppressed TEM5 upregulation. An excess of the soluble TEM5 extracellular domain or an inhibitory monoclonal TEM5 antibody blocked contact inhibition of endothelial cell proliferation resulting in multilayered islands within the endothelial monolayer and increased vessel density during capillary formation. Based on our results we conclude that TEM5 expression during capillary morphogenesis is induced by the small GTPase Rac and mediates contact inhibition of proliferation in endothelial cells.

  7. Cell adhesion markers are expressed by a stable human endothelial cell line transformed by the SV40 large T antigen under vimentin promoter control.

    PubMed

    Vicart, P; Testut, P; Schwartz, B; Llorens-Cortes, C; Perdomo, J J; Paulin, D

    1993-10-01

    Markers of endothelium have been studied in a new endothelial cell line derived from human umbilical cord vein cells by microinjection of a recombinant gene that includes a deletion mutant of the human vimentin gene regulatory region controlling the large T and small t antigen coding region of the SV40 virus. In culture, this immortalized venous endothelial cell line (IVEC) demonstrated morphological characteristics of endothelium; uptake of acetylated low density lipoprotein and presence of the Factor VIII-related antigen. Treatment of IVEC cells with Interleukin-1 beta (IL-1 beta) at 10 U.ml-1 activates the expression of cell adhesion molecules such as endothelial leucocyte adhesion molecule (ELAM-1), intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1), as observed in primary culture. Prostacyclin secretion was induced in the IVEC cells by 100 nM PMA treatment and thrombin at 0.5 U/ml. Angiotensin converting enzyme (ACE) activity detected in IVEC cells was present but lower than ACE activity in primary endothelial cells and was completely blocked by enalaprilat (1 microM), a specific ACE inhibitor. The presence of ACE mRNA was also demonstrated in IVEC cells by RT-PCR amplification. Our data demonstrate that endothelial cells immortalized by use of this recombinant gene retain the morphological organization and numerous differentiated properties of endothelium.

  8. A Multiplex High-Throughput Gene Expression Assay to Simultaneously Detect Disease and Functional Markers in Induced Pluripotent Stem Cell-Derived Retinal Pigment Epithelium

    PubMed Central

    Ferrer, Marc; Corneo, Barbara; Davis, Janine; Wan, Qin; Miyagishima, Kiyoharu Joshua; King, Rebecca; Maminishkis, Arvydas; Marugan, Juan; Sharma, Ruchi; Shure, Michael; Temple, Sally; Miller, Sheldon

    2014-01-01

    There is continuing interest in the development of lineage-specific cells from induced pluripotent stem (iPS) cells for use in cell therapies and drug discovery. Although in most cases differentiated cells show features of the desired lineage, they retain fetal gene expression and do not fully mature into “adult-like” cells. Such cells may not serve as an effective therapy because, once implanted, immature cells pose the risk of uncontrolled growth. Therefore, there is a need to optimize lineage-specific stem cell differentiation protocols to produce cells that no longer express fetal genes and have attained “adult-like” phenotypes. Toward that goal, it is critical to develop assays that simultaneously measure cell function and disease markers in high-throughput format. Here, we use a multiplex high-throughput gene expression assay that simultaneously detects endogenous expression of multiple developmental, functional, and disease markers in iPS cell-derived retinal pigment epithelium (RPE). We optimized protocols to differentiate iPS cell-derived RPE that was then grown in 96- and 384-well plates. As a proof of principle, we demonstrate differential expression of eight genes in iPS cells, iPS cell-derived RPE at two different differentiation stages, and primary human RPE using this multiplex assay. The data obtained from the multiplex gene expression assay are significantly correlated with standard quantitative reverse transcription-polymerase chain reaction-based measurements, confirming the ability of this high-throughput assay to measure relevant gene expression changes. This assay provides the basis to screen for compounds that improve RPE function and maturation and target disease pathways, thus providing the basis for effective treatments of several retinal degenerative diseases. PMID:24873859

  9. Smooth Muscle-Like Cells Generated from Human Mesenchymal Stromal Cells Display Marker Gene Expression and Electrophysiological Competence Comparable to Bladder Smooth Muscle Cells

    PubMed Central

    Brun, Juliane; Lutz, Katrin A.; Neumayer, Katharina M. H.; Klein, Gerd; Seeger, Tanja; Uynuk-Ool, Tatiana; Wörgötter, Katharina; Schmid, Sandra; Kraushaar, Udo; Guenther, Elke; Rolauffs, Bernd; Aicher, Wilhelm K.; Hart, Melanie L.

    2015-01-01

    The use of mesenchymal stromal cells (MSCs) differentiated toward a smooth muscle cell (SMC) phenotype may provide an alternative for investigators interested in regenerating urinary tract organs such as the bladder where autologous smooth muscle cells cannot be used or are unavailable. In this study we measured the effects of good manufacturing practice (GMP)-compliant expansion followed by myogenic differentiation of human MSCs on the expression of a range of contractile (from early to late) myogenic markers in relation to the electrophysiological parameters to assess the functional role of the differentiated MSCs and found that differentiation of MSCs associated with electrophysiological competence comparable to bladder SMCs. Within 1–2 weeks of myogenic differentiation, differentiating MSCs significantly expressed alpha smooth muscle actin (αSMA; ACTA2), transgelin (TAGLN), calponin (CNN1), and smooth muscle myosin heavy chain (SM-MHC; MYH11) according to qRT-PCR and/or immunofluorescence and Western blot. Voltage-gated Na+ current levels also increased within the same time period following myogenic differentiation. In contrast to undifferentiated MSCs, differentiated MSCs and bladder SMCs exhibited elevated cytosolic Ca2+ transients in response to K+-induced depolarization and contracted in response to K+ indicating functional maturation of differentiated MSCs. Depolarization was suppressed by Cd2+, an inhibitor of voltage-gated Ca2+-channels. The expression of Na+-channels was pharmacologically identified as the Nav1.4 subtype, while the K+ and Ca2+ ion channels were identified by gene expression of KCNMA1, CACNA1C and CACNA1H which encode for the large conductance Ca2+-activated K+ channel BKCa channels, Cav1.2 L-type Ca2+ channels and Cav3.2 T-type Ca2+ channels, respectively. This protocol may be used to differentiate adult MSCs into smooth muscle-like cells with an intermediate-to-late SMC contractile phenotype exhibiting voltage-gated ion channel

  10. A mature T lymphocyte marker closely linked to Igh-1 that is expressed on the precursor for the suppressor T cell regulating a primary response to SRBC.

    PubMed

    Owen, F L

    1980-03-01

    The product of a new genetic locus closely linked to Igh-1 in the mouse has previously been shown to be expressed on T cells by using an in vitro fluorescent antibody assay. In vivo studies reported here show the determinant coded for by that locus is expressed in unimmunized animals on the precursor for a suppressor T cell regulating a primary response to SRBC. Antisera raised in allotype congenic animals by immunization with suppressor T cells (Ts) for the ARS.CRI stimulate Lyt2+3+ suppressor cells for unrelated antigens. The suppressive effects of the serum can be absorbed with T cells but not with B cells. Suppression of an IgM PFC response in allotype congenic animals confirms close linkage of the marker to Igh-1 but not to H-2. This determinant is expressed on Ts cells for many antigens and may represent a heavy chain linked "constant" portion of the antigen-specific receptor.

  11. Neural Stem Cells Injected into the Sound-Damaged Cochlea Migrate Throughout the Cochlea and Express Markers of Hair Cells, Supporting Cells, and Spiral Ganglion Cells

    PubMed Central

    Corliss, Deborah A.; Gray, Brianna; Anderson, Julia K.; Bobbin, Richard P.; Snyder, Evan Y.; Cotanche, Douglas A.

    2007-01-01

    Most cases of hearing loss are caused by the death or dysfunction of one of the many cochlear cell types. We examined whether cells from a neural stem cell line could replace cochlear cell types lost after exposure to intense noise. For this purpose, we transplanted a clonal stem cell line into the scala tympani of sound damaged mice and guinea pigs. Utilizing morphological, protein expression and genetic criteria, stem cells were found with characteristics of both neural tissues (satellite, spiral ganglion and Schwann cells) and cells of the organ of Corti (hair cells, supporting cells). Additionally, noise-exposed, stem cell-injected animals exhibited a small but significant increase in the number of satellite cells and Type I spiral ganglion neurons compared to non-injected noise-exposed animals. These results indicate that cells of this neural stem cell line migrate from the scala tympani to Rosenthal's canal and the organ of Corti. Moreover, it suggests that cells of this neural stem cell line may derive some information needed from the microenvironment of the cochlea to differentiate into replacement cells in the cochlea. PMID:17659854

  12. Expression of Cell-Surface Marker ABCB5 Causes Characteristic Modifications of Glucose, Amino Acid and Phospholipid Metabolism in the G3361 Melanoma-Initiating Cell Line

    PubMed Central

    Lutz, Norbert W.; Banerjee, Pallavi; Wilson, Brian J.; Ma, Jie; Cozzone, Patrick J.; Frank, Markus H.

    2016-01-01

    We present a pilot study aimed at determining the effects of expression of ATP-binding cassette member B5 (ABCB5), a previously described marker for melanoma-initiating cells, on cellular metabolism. Metabolic profiles for two groups of human G3361 melanoma cells were compared, i.e. wildtype melanoma cells with intact ABCB5 expression (ABCB5-WT) and corresponding melanoma cell variants with inhibited ABCB5 expression, through shRNA-mediated gene knockdown (ABCB5-KD). A comprehensive metabolomic analysis was performed by using proton and phosphorus NMR spectroscopy of cell extracts to examine water-soluble metabolites and lipids. Parametric and non-parametric statistical analysis of absolute and relative metabolite levels yielded significant differences for compounds involved in glucose, amino acid and phospholipid (PL) metabolism. By contrast, energy metabolism was virtually unaffected by ABCB5 expression. The sum of water-soluble metabolites per total protein was 17% higher in ABCB5-WT vs. ABCB5-KD G3361 variants, but no difference was found for the sum of PLs. Enhanced abundance was particularly pronounced for lactate (+ 23%) and alanine (+ 26%), suggesting an increase in glycolysis and potentially glutaminolysis. Increases in PL degradation products, glycerophosphocholine and glycerophosphoethanolamine (+ 85 and 123%, respectively), and redistributions within the PL pool suggested enhanced membrane PL turnover as a consequence of ABCB5 expression. The possibility of glycolysis modulation by an ABCB5-dependent IL1β-mediated mechanism was supported by functional studies employing monoclonal antibody (mAb)-dependent ABCB5 protein inhibition in wildtype G3361 melanoma cells. Our metabolomic results suggest that the underlying biochemical pathways may offer targets for melanoma therapy, potentially in combination with other treatment forms. PMID:27560924

  13. Impaired Cytokine but Enhanced Cytotoxic Marker Expression in Mycobacterium tuberculosis-Induced CD8+ T Cells in Individuals With Type 2 Diabetes and Latent Mycobacterium tuberculosis Infection.

    PubMed

    Kumar, Nathella Pavan; Moideen, Kadar; George, Parakkal Jovvian; Dolla, Chandrakumar; Kumaran, Paul; Babu, Subash

    2016-03-01

    Type 2 diabetes mellitus (DM) is a risk factor for tuberculosis among individuals with latent Mycobacterium tuberculosis infection. To explore the influence of DM on CD8(+) T-cell responses during latent M. tuberculosis infection, we estimated the cytokine and cytotoxic marker expression pattern in individuals with latent M. tuberculosis infection with DM and those with latent M. tuberculosis infection without DM. Among individuals with latent M. tuberculosis infection, those with DM had diminished frequencies of CD8(+) T-helper type 1 (Th1), Th2, and Th17 cells following stimulation by M. tuberculosis antigen and enhanced frequencies of CD8(+) T cells expressing cytotoxic markers, compared with those without DM. Thus, our results suggest that coincident DM modulates CD8(+) T-cell function during latent M. tuberculosis infection.

  14. The TRA-1-60 and TRA-1-81 human pluripotent stem cell markers are expressed on podocalyxin in embryonal carcinoma.

    PubMed

    Schopperle, William M; DeWolf, William C

    2007-03-01

    We have previously identified the cell adhesion protein podocalyxin expressed in a human pluripotent stem cell, embryonal carcinoma (EC), which is a malignant germ cell. Podocalyxin is a heavily glycosylated membrane protein with amino acid sequence homology to the hematopoietic stem cell marker CD34. Since the initial discovery of podocalyxin in a cancerous stem cell, numerous new studies have identified podocalyxin in many different human cancers and in embryonic stem cells lines (ES) derived from human embryos. Embryonal carcinoma, as do all human pluripotent stem cells, expresses TRA-1-60 and TRA-1-81 antigens, and although their molecular identities are unknown, they are commonly used as markers of undifferentiated pluripotent human stem cells. We report here that purified podocalyxin from embryonal carcinoma has binding activity with the TRA-1-60 and TRA-1-81 antibodies. Embryonal carcinoma cells treated with retinoic acid undergo differentiation and lose the TRA-1-60/TRA-1-81 markers from their plasma membrane surface. We show that podocalyxin is modified in the retinoic acid-treated cells and has an apparent molecular mass of 170 kDa on protein blots as compared with the apparent 200-kDa molecular weight form of podocalyxin expressed in untreated cells. Furthermore, the modified form of podocalyxin no longer reacts with the TRA-1-60/TRA-1-81 antibodies. Thus, embryonal carcinoma expresses two distinct forms of podocalyxin, and the larger version is a molecular carrier of the human stem cell-defining antigens TRA-1-60 and TRA-1-81.

  15. Association between expression of cumulus expansion markers and real-time proliferation of porcine follicular granulosa cells in a primary cell culture model.

    PubMed

    Ciesiółka, S; Budna, J; Bryja, A; Kranc, W; Chachuła, A; Dyszkiewicz-Konwińska, M; Piotrowska, H; Bukowska, D; Antosik, P; Bruska, M; Brüssow, K P; Nowicki, M; Zabel, M; Kempisty, B

    2016-01-01

    Folliculogenesis is a compound process that involves both ovarian follicle growth and oocyte development, which is tightly attached to the follicular wall. During this process, cells that form the follicle structure undergo substantial morphological and molecular modifications that finally lead to differentiation and specialization of ovarian follicular cells. The differentiation of ovarian cells encompasses formation of follicle, which is composed of theca (TCs), mural granulosa (GCs), and cumulus cells (CCs). It was previously hypothesized that GCs and CCs represent undifferentiated and highly specialized follicular cells, respectively, which may have similar primordial cell origins. In this study, we investigated the expression pattern of cumulus expansion markers such as COX2, HAS2, PTX3, and TSG6 in porcine GCs during short-term, in vitro culture. We hypothesized that these genes may display an important function in GCs in relation to cellular real-time proliferation. The expression pattern of COX2, HAS2, PTX3, and TSG6 was evaluated after using RT-qPCR in relation to confocal microscopy observations of protein expression and distribution during real-time proliferation of porcine follicular GCs. The COX2 and HAS2 mRNAs were highly expressed after 120 h of in vitro culture (IVC), whereas PTX3 and TSG6 mRNAs were increased during the first 24-48 h of IVC (P less than 0.001, P less than 0.01). Conversely, all of the encoded proteins were highly expressed after 144-168 h of IVC as compared to other culture periods (P less than 0.001, P less than 0.01). When analyzing the realtime proliferation of GCs in vitro, we observed a logarithmic increase of cell proliferation between 0 h and 120 h of IVC. However, after 120-168 h of IVC, the cells reached the lag phase of proliferation. Since it is well accepted that porcine GCs undergo luteinization shortly after 24-48 h of IVC, the expression pattern of investigated genes indicated that Cox2 and Has2 are independent from

  16. Differential association of S100A9, an inflammatory marker, and p53, a cell cycle marker, expression with epicardial adipocyte size in patients with cardiovascular disease.

    PubMed

    Agra, Rosa María; Fernández-Trasancos, Ángel; Sierra, Juan; González-Juanatey, José Ramón; Eiras, Sonia

    2014-10-01

    S100A9 (calgranulin B) has inflammatory and oxidative stress properties and was found to be associated with atherosclerosis and obesity. One of the proteins that can regulate S100A9 transcription is p53, which is involved in cell cycle, apoptosis and adipogenesis. Thus, it triggers adipocyte enlargement and finally obesity. Because epicardial adipose tissue (EAT) volume and thickness is related to coronary artery disease (CAD), we studied the gene expression of this pathway in patients with cardiovascular disease and its association with obesity. Adipocytes and stromal cells from EAT and subcutaneous adipose tissue (SAT) from 48 patients who underwent coronary artery bypass graft and/or valve replacement were obtained after collagenase digestion and differential centrifugation. The expression levels of the involved genes on adipogenesis and cell cycle like fatty acid-binding protein (FABP) 4, retinol-binding protein (RBP)4, p53 and S100A9 were determined by real-time polymerase chain reaction (PCR). Adipocyte diameter was measured by optical microscopy. We found that epicardial adipocytes expressed significantly lower levels of adipogenic genes (FABP4 and RBP4) and cell cycle-related genes (S100A9 and p53) than subcutaneous adipocytes. However, in obese patients, upregulation of adipogenic and cell cycle-related genes in subcutaneous and epicardial adipocytes, respectively, was observed. The enlargement of adipocyte size was related to FABP4, S100A9 and p53 expression levels in stromal cells. But only the p53 association was maintained in epicardial stromal cells from obese patients (p=0.003). The expression of p53, but not S100A9, in epicardial stromal cells is related to adipocyte enlargement in obese patients with cardiovascular disease. These findings suggest new mechanisms for understanding the relationship between epicardial fat thickness, obesity and cardiovascular disease.

  17. TRPV1, ASICs and P2X2/3 expressed in bone cells simultaneously regulate bone metabolic markers in ovariectomized mice

    PubMed Central

    Kanaya, K.; Iba, K.; Dohke, T.; Okazaki, S.; Yamashita, T.

    2016-01-01

    Objectives: Nociceptors are expressed at peripheral terminals of neurons. Recent studies have shown that TRPV1, a nociceptor, is expressed in bone tissue and regulates bone metabolism. We have demonstrated that a TRPV1 antagonist improved pain-like behavior in ovariectomized (OVX) mice. The aim of this study was to determine whether nociceptors, including TRPV1, acid-sensing ion channel (ASIC) and P2X2/3 are expressed in bone cells, and to examine the effects of nociceptor antagonists on bone metabolism. Methods: The expression of nociceptors in femoral bone tissue and cultured bone marrow cells in OVX and sham-operated mice were examined. The effects of nociceptor antagonists on the up-regulated expression of bone metabolic markers, Runx2, Osterix, osteocalcin and RANKL, were also examined. Results: TRPV1, ASIC 2 and 3, and P2X2 and 3, were expressed in bone tissue and bone marrow cells, and the expression levels of ASIC1 and 2, and P2X2 were significantly increased in OVX mice in comparison with those in sham mice. Treatment with nociceptor antagonists significantly inhibited the expression of bone metabolic markers in OVX mice. Conclusion: An array of nociceptors, TRPV1, ASICs and P2X2/3, could simultaneously regulate not only increases in skeletal pain but also bone turnover in OVX mice. PMID:27282458

  18. Adenoviral Gene Therapy for Diabetic Keratopathy: Effects on Wound Healing and Stem Cell Marker Expression in Human Organ-cultured Corneas and Limbal Epithelial Cells.

    PubMed

    Kramerov, Andrei A; Saghizadeh, Mehrnoosh; Ljubimov, Alexander V

    2016-04-07

    The goal of this protocol is to describe molecular alterations in human diabetic corneas and demonstrate how they can be alleviated by adenoviral gene therapy in organ-cultured corneas. The diabetic corneal disease is a complication of diabetes with frequent abnormalities of corneal nerves and epithelial wound healing. We have also documented significantly altered expression of several putative epithelial stem cell markers in human diabetic corneas. To alleviate these changes, adenoviral gene therapy was successfully implemented using the upregulation of c-met proto-oncogene expression and/or the downregulation of proteinases matrix metalloproteinase-10 (MMP-10) and cathepsin F. This therapy accelerated wound healing in diabetic corneas even when only the limbal stem cell compartment was transduced. The best results were obtained with combined treatment. For possible patient transplantation of normalized stem cells, an example is also presented of the optimization of gene transduction in stem cell-enriched cultures using polycationic enhancers. This approach may be useful not only for the selected genes but also for the other mediators of corneal epithelial wound healing and stem cell function.

  19. Expression of Th1, Th2, lymphocyte trafficking and activation markers on CD4+ T-cells of Hymenoptera allergic subjects and after venom immunotherapy.

    PubMed

    Cabrera, Carmen M; Urra, José M; Alfaya, Teresa; Roca, Federico De La; Feo-Brito, Francisco

    2014-11-01

    Systemic reactions to Hymenoptera stings can be fatal and represent a reduction in the quality of life. The immune mechanisms involved in venom allergic subjects are barely known. Nevertheless, a shift towards a Th1-type response with an increase in IFNγ levels has been observed after venom immunotherapy (VIT). There is currently no information available about the expression of markers on CD4+ T-cells or their involvement in venom allergy, nor following VIT. For this, we have studied the expression of Th1 and Th2-cell markers, homing receptors and activation markers on CD4+ T-cells of subjects who presented systemic allergic reactions, mainly to Polistes dominulus, and after receiving a 4-month conventional VIT protocol. The markers studied were: CD26 (Th1), CD30 (Th2), CXCR4, CXCR3 (Th1), CCR4 (Th2), CD154 (CD40L), CD152 (CTLA-A), and ICOS. We also determined the IL-4 (Th2) and IFNγ (Th1) intracellular cytokine levels in T-cells and carried out a basophil activation test (BAT). Comparing venom allergic subjects with non-allergic healthy controls, we have found up-regulation of CD26, CXCR4, CXCR3, CD154 and ICOS. Conversely, a down-regulation of CD30, CD154 and CD152 occurred upon immune intervention, whereas the remaining markers were not affected. Equally, VIT has been shown to be effective, as evidenced by the decrease of basophil degranulation and increase of IFNγ levels in T-cells after the fourth month of treatment. These new findings highlight the possible application of these surface molecules as markers to distinguish between symptomatic and asymptomatic subjects sensitized to Hymenoptera venom, as well as revealing information about the immune changes associated with VIT.

  20. Stem cell marker TRA-1-60 is expressed in foetal and adult kidney and upregulated in tubulo-interstitial disease.

    PubMed

    Fesenko, Irina; Franklin, Danielle; Garnett, Paul; Bass, Paul; Campbell, Sara; Hardyman, Michelle; Wilson, David; Hanley, Neil; Collins, Jane

    2010-10-01

    The kidney has an intrinsic ability to repair itself when injured. Epithelial cells of distal tubules may participate in regeneration. Stem cell marker, TRA-1-60 is linked to pluripotency in human embryonic stem cells and is lost upon differentiation. TRA-1-60 expression was mapped and quantified in serial sections of human foetal, adult and diseased kidneys. In 8- to 10-week human foetal kidney, the epitope was abundantly expressed on ureteric bud and structures derived therefrom including collecting duct epithelium. In adult kidney inner medulla/papilla, comparisons with reactivity to epithelial membrane antigen, aquaporin-2 and Tamm-Horsfall protein, confirmed extensive expression of TRA-1-60 in cells lining collecting ducts and thin limb of the loop of Henle, which may be significant since the papillae were proposed to harbour slow cycling cells involved in kidney homeostasis and repair. In the outer medulla and cortex there was rare, sporadic expression in tubular cells of the collecting ducts and nephron, with positive cells confined to the thin limb and thick ascending limb and distal convoluted tubules. Remarkably, in cortex displaying tubulo-interstitial injury, there was a dramatic increase in number of TRA-1-60 expressing individual cells and in small groups of cells in distal tubules. Dual staining showed that TRA-1-60 positive cells co-expressed Pax-2 and Ki-67, markers of tubular regeneration. Given the localization in foetal kidney and the distribution patterns in adults, it is tempting to speculate that TRA-1-60 may identify a population of cells contributing to repair of distal tubules in adult kidney.

  1. Stem cell marker TRA-1-60 is expressed in foetal and adult kidney and upregulated in tubulo-interstitial disease

    PubMed Central

    2012-01-01

    The kidney has an intrinsic ability to repair itself when injured. Epithelial cells of distal tubules may participate in regeneration. Stem cell marker, TRA-1-60 is linked to pluripotency in human embryonic stem cells and is lost upon differentiation. TRA-1-60 expression was mapped and quantified in serial sections of human foetal, adult and diseased kidneys. In 8- to 10-week human foetal kidney, the epitope was abundantly expressed on ureteric bud and structures derived therefrom including collecting duct epithelium. In adult kidney inner medulla/papilla, comparisons with reactivity to epithelial membrane antigen, aquaporin-2 and Tamm–Horsfall protein, confirmed extensive expression of TRA-1-60 in cells lining collecting ducts and thin limb of the loop of Henle, which may be significant since the papillae were proposed to harbour slow cycling cells involved in kidney homeostasis and repair. In the outer medulla and cortex there was rare, sporadic expression in tubular cells of the collecting ducts and nephron, with positive cells confined to the thin limb and thick ascending limb and distal convoluted tubules. Remarkably, in cortex displaying tubulo-interstitial injury, there was a dramatic increase in number of TRA-1-60 expressing individual cells and in small groups of cells in distal tubules. Dual staining showed that TRA-1-60 positive cells co-expressed Pax-2 and Ki-67, markers of tubular regeneration. Given the localization in foetal kidney and the distribution patterns in adults, it is tempting to speculate that TRA-1-60 may identify a population of cells contributing to repair of distal tubules in adult kidney. PMID:20853169

  2. Severe polyposis in Apc1322T mice is associated with submaximal Wnt signalling and increased expression of the stem cell marker Lgr5

    PubMed Central

    Segditsas, Stefania; Deheragoda, Maesha; Pollard, Patrick; Jeffery, Rosemary; Nye, Emma; Lockstone, Helen; Davis, Hayley; Clark, Susan; Stamp, Gordon; Poulsom, Richard; Wright, Nicholas; Tomlinson, Ian

    2010-01-01

    Background and aims Adenomatous polyposis coli (APC) is a tumour suppressor gene mutated in the germline of patients with familial adenomatous polyposis (FAP) and somatically in most colorectal cancers. APC mutations impair β-catenin degradation, resulting in increased Wnt signalling. The most frequent APC mutation is a codon 1309 truncation that is associated with severe FAP. A previous study compared two mouse models of intestinal tumorigenesis, ApcR850X (Min) and Apc1322T (1322T), the latter a model of human codon 1309 changes. 1322T mice had more severe polyposis but, surprisingly, these tumours had lower levels of nuclear β-catenin than Min tumours. The consequences of these different β-catenin levels were investigated. Methods Enterocytes were isolated from 1322T and Min tumours by microdissection and gene expression profiling was performed. Differentially expressed Wnt targets and other stem cell markers were validated using quantitative PCR, in situ hybridisation and immunohistochemistry. Results As expected, lower nuclear β-catenin levels in 1322T lesions were associated with generally lower levels of Wnt target expression. However, expression of the Wnt target and stem cell marker Lgr5 was significantly higher in 1322T tumours than in Min tumours. Other stem cell markers (Musashi1, Bmi1 and the Wnt target Cd44) were also at higher levels in 1322T tumours. In addition, expression of the Bmp antagonist Gremlin1 was higher in 1322T tumours, together with lower Bmp2 and Bmp4 expression. Conclusions The severe phenotype caused by truncation of Apc at codon 1322 is associated with an increased number of stem cells. Thus, a submaximal level of Wnt signalling favours the stem cell phenotype and this may promote tumorigenesis. A level of Wnt signalling exists that is too high for optimal tumour growth. PMID:20926645

  3. Expression of cancer stem markers could be influenced by silencing of p16 gene in HeLa cervical carcinoma cells.

    PubMed

    Wu, H; Zhang, J; Shi, H

    2016-01-01

    Effect of the tumor suppression gene p16 on the biological characteristics of HeLa cervical carcinoma cells was explored. The expression of p16 protein was increased in HeLa tumor sphere cells, and no significant difference in tumor spheres from the first to the fourth passages. Compared with those of parental HeLa cells, the proportion of CD44+/CD24- and ABCG2+ cells increased significantly in tumor spheres. However after the cells were silenced by the p16-sh289 vector, expression of P16 protein and the cell number of CD44+/CD24- and ABCG2+ decreased. Moreover, HeLa cells with p16 gene silencing showed decreased abilities of sphere formation and matrigel invasion. More HeLa cells with p16 gene silence were needed for tumor formation in nude mice. Tumor size and weight in mouse model established with p16 gene silenced HeLa cells were less than those with HeLa parental cell model. The present results indicate that silencing of the p16 gene inhibits expression of cancer stem cell markers and tumorigenic ability of HeLa cells.

  4. Expression of Lymphatic Markers in the Adult Rat Spinal Cord.

    PubMed

    Kaser-Eichberger, Alexandra; Schroedl, Falk; Bieler, Lara; Trost, Andrea; Bogner, Barbara; Runge, Christian; Tempfer, Herbert; Zaunmair, Pia; Kreutzer, Christina; Traweger, Andreas; Reitsamer, Herbert A; Couillard-Despres, Sebastien

    2016-01-01

    Under physiological conditions, lymphatic vessels are thought to be absent from the central nervous system (CNS), although they are widely distributed within the rest of the body. Recent work in the eye, i.e., another organ regarded as alymphatic, revealed numerous cells expressing lymphatic markers. As the latter can be involved in the response to pathological conditions, we addressed the presence of cells expressing lymphatic markers within the spinal cord by immunohistochemistry. Spinal cord of young adult Fisher rats was scrutinized for the co-expression of the lymphatic markers PROX1 and LYVE-1 with the cell type markers Iba1, CD68, PGP9.5, OLIG2. Rat skin served as positive control for the lymphatic markers. PROX1-immunoreactivity was detected in many nuclei throughout the spinal cord white and gray matter. These nuclei showed no association with LYVE-1. Expression of LYVE-1 could only be detected in cells at the spinal cord surface and in cells closely associated with blood vessels. These cells were found to co-express Iba1, a macrophage and microglia marker. Further, double labeling experiments using CD68, another marker found in microglia and macrophages, also displayed co-localization in the Iba1+ cells located at the spinal cord surface and those apposed to blood vessels. On the other hand, PROX1-expressing cells found in the parenchyma were lacking Iba1 or PGP9.5, but a significant fraction of those cells showed co-expression of the oligodendrocyte lineage marker OLIG2. Intriguingly, following spinal cord injury, LYVE-1-expressing cells assembled and reorganized into putative pre-vessel structures. As expected, the rat skin used as positive controls revealed classical lymphatic vessels, displaying PROX1+ nuclei surrounded by LYVE-1-immunoreactivity. Classical lymphatics were not detected in adult rat spinal cord. Nevertheless, numerous cells expressing either LYVE-1 or PROX1 were identified. Based on their localization and overlapping expression with

  5. Expression of Lymphatic Markers in the Adult Rat Spinal Cord

    PubMed Central

    Kaser-Eichberger, Alexandra; Schroedl, Falk; Bieler, Lara; Trost, Andrea; Bogner, Barbara; Runge, Christian; Tempfer, Herbert; Zaunmair, Pia; Kreutzer, Christina; Traweger, Andreas; Reitsamer, Herbert A.; Couillard-Despres, Sebastien

    2016-01-01

    Under physiological conditions, lymphatic vessels are thought to be absent from the central nervous system (CNS), although they are widely distributed within the rest of the body. Recent work in the eye, i.e., another organ regarded as alymphatic, revealed numerous cells expressing lymphatic markers. As the latter can be involved in the response to pathological conditions, we addressed the presence of cells expressing lymphatic markers within the spinal cord by immunohistochemistry. Spinal cord of young adult Fisher rats was scrutinized for the co-expression of the lymphatic markers PROX1 and LYVE-1 with the cell type markers Iba1, CD68, PGP9.5, OLIG2. Rat skin served as positive control for the lymphatic markers. PROX1-immunoreactivity was detected in many nuclei throughout the spinal cord white and gray matter. These nuclei showed no association with LYVE-1. Expression of LYVE-1 could only be detected in cells at the spinal cord surface and in cells closely associated with blood vessels. These cells were found to co-express Iba1, a macrophage and microglia marker. Further, double labeling experiments using CD68, another marker found in microglia and macrophages, also displayed co-localization in the Iba1+ cells located at the spinal cord surface and those apposed to blood vessels. On the other hand, PROX1-expressing cells found in the parenchyma were lacking Iba1 or PGP9.5, but a significant fraction of those cells showed co-expression of the oligodendrocyte lineage marker OLIG2. Intriguingly, following spinal cord injury, LYVE-1-expressing cells assembled and reorganized into putative pre-vessel structures. As expected, the rat skin used as positive controls revealed classical lymphatic vessels, displaying PROX1+ nuclei surrounded by LYVE-1-immunoreactivity. Classical lymphatics were not detected in adult rat spinal cord. Nevertheless, numerous cells expressing either LYVE-1 or PROX1 were identified. Based on their localization and overlapping expression with

  6. Gene Expression Profiling Supports the Neural Crest Origin of Adult Rodent Carotid Body Stem Cells and Identifies CD10 as a Marker for Mesectoderm-Committed Progenitors.

    PubMed

    Navarro-Guerrero, Elena; Platero-Luengo, Aida; Linares-Clemente, Pedro; Cases, Ildefonso; López-Barneo, José; Pardal, Ricardo

    2016-06-01

    Neural stem cells (NSCs) are promising tools for understanding nervous system plasticity and repair, but their use is hampered by the lack of markers suitable for their prospective isolation and characterization. The carotid body (CB) contains a population of peripheral NSCs, which support organ growth during acclimatization to hypoxia. We have set up CB neurosphere (NS) cultures enriched in differentiated neuronal (glomus) cells versus undifferentiated progenitors to investigate molecular hallmarks of cell classes within the CB stem cell (CBSC) niche. Microarray gene expression analysis in NS is compatible with CBSCs being neural crest derived-multipotent progenitor cells able to sustain CB growth upon exposure to hypoxia. Moreover, we have identified CD10 as a marker suitable for isolation of a population of CB mesectoderm-committed progenitor cells. CD10 + cells are resting in normoxia, and during hypoxia they are activated to proliferate and to eventually complete maturation into mesectodermal cells, thus participating in the angiogenesis necessary for CB growth. Our results shed light into the molecular and cellular mechanisms involved in CBSC fate choice, favoring a potential use of these cells for cell therapy. Stem Cells 2016;34:1637-1650. © 2016 AlphaMed Press.

  7. Global microRNA expression profiling uncovers molecular markers for classification and prognosis in aggressive B-cell lymphoma

    PubMed Central

    Shen, Yulei; Huang, Xin; Liu, Yanyan; Wake, Laura; Liu, Cuiling; Deffenbacher, Karen; Lachel, Cynthia M.; Wang, Chao; Rohr, Joseph; Guo, Shuangping; Smith, Lynette M.; Wright, George; Bhagavathi, Sharathkumar; Dybkaer, Karen; Fu, Kai; Greiner, Timothy C.; Vose, Julie M.; Jaffe, Elaine; Rimsza, Lisa; Rosenwald, Andreas; Ott, German; Delabie, Jan; Campo, Elias; Braziel, Rita M.; Cook, James R.; Tubbs, Raymond R.; Armitage, James O.; Weisenburger, Dennis D.; Staudt, Louis M.; Gascoyne, Randy D.; McKeithan, Timothy W.; Chan, Wing C.

    2015-01-01

    We studied the global microRNA (miRNA) expression in diffuse large B-cell lymphoma (DLBCL; n = 79), Burkitt lymphoma (BL; n = 36), primary mediastinal B-cell lymphoma (PMBL; n = 12), B-cell lines (n = 11), and normal subsets of naïve B cells, centroblasts (CBs), and peripheral blood B cells along with their corresponding gene expression profiles (GEPs). The normal B-cell subsets have well-defined miRNA signatures. The CB miRNA signature was significantly associated with germinal center B-cell (GCB)–DLBCL compared with activated B-cell (ABC)–DLBCL (P = .002). We identified a 27-miRNA signature that included v-myc avian myelomatosis viral oncogene homolog (MYC) targets and enabled the differentiation of BL from DLBCL, a distinction comparable with the “gold standard” GEP-defined diagnosis. Distinct miRNA signatures were identified for DLBCL subgroups, including GCB-DLBCL, activated B-cell (ABC)-DLBCL, and PMBL. Interestingly, most of the unclassifiable-DLBCL by GEP showed a strong similarity to the ABC-DLBCL by miRNA expression profiling. Consistent results for BL and DLBCL subgroup classification were observed in formalin-fixed, paraffin-embedded tissue, making such tests practical for clinical use. We also identified predictive miRNA biomarker signatures in DLBCL, including high expression of miR-155, which is significantly associated with rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) treatment failure. This finding was further supported by the observation that high expression of miR-155 sensitizes cells to v-akt murine thymoma viral oncogene homolog-1 inhibitors in vitro, suggesting a novel treatment option for resistant DLBCL. PMID:25498913

  8. Differential Expression of Conserved Germ Line Markers and Delayed Segregation of Male and Female Primordial Germ Cells in a Hermaphrodite, the Leech Helobdella

    PubMed Central

    Cho, Sung-Jin; Vallès, Yvonne; Weisblat, David A.

    2014-01-01

    In sexually reproducing animals, primordial germ cells (PGCs) are often set aside early in embryogenesis, a strategy that minimizes the risk of genomic damage associated with replication and mitosis during the cell cycle. Here, we have used germ line markers (piwi, vasa, and nanos) and microinjected cell lineage tracers to show that PGC specification in the leech genus Helobdella follows a different scenario: in this hermaphrodite, the male and female PGCs segregate from somatic lineages only after more than 20 rounds of zygotic mitosis; the male and female PGCs share the same (mesodermal) cell lineage for 19 rounds of zygotic mitosis. Moreover, while all three markers are expressed in both male and female reproductive tissues of the adult, they are expressed differentially between the male and female PGCs of the developing embryo: piwi and vasa are expressed preferentially in female PGCs at a time when nanos is expressed preferentially in male PGCs. A priori, the delayed segregation of male and female PGCs from somatic tissues and from one another increases the probability of mutations affecting both male and female PGCs of a given individual. We speculate that this suite of features, combined with a capacity for self-fertilization, may contribute to the dramatically rearranged genome of Helobdella robusta relative to other animals. PMID:24217283

  9. Evaluation of the expression of stem cell markers in human breast cancer reveals a correlation with clinical progression and metastatic disease in ductal carcinoma.

    PubMed

    Martin, Tracey Amanda; Jiang, Wen Guo

    2014-01-01

    The tumor stem cell theory could explain how patients with metastatic disease show clinical relapse several months after starting treatment due to the survival of a small group of cells with unique characteristics. We examined the distribution and expression of a panel of stem cell markers in human breast cancer primary tumors. Human breast tissues were processed for immunohistochemistry, and RNA was extracted for analysis by quantitative-PCR. Immunohistochemical assay revealed that CD44 was strongly expressed in background endothelia and epithelia. CD133 expression was lost in tumor-associated endothelial cells. Conversely, CD49b was strongly stained in the tumors, associated vessels and ducts but was weakly stained in the background epithelia. q-PCR analysis revealed that CD44 and PSCA were reduced in patients with poor outcome (metastatic disease and death from breast cancer), with a marked reduction in ductal carcinoma, particularly with metastasis to bone although these did not reach significant difference. CD133 was significantly reduced in patients with metastatic disease and was also significantly reduced in patients with ductal carcinoma/bone metastasis. Conversely, CD49F was increased in patients with a poor outcome and those with ductal cancer and bone metastases. This is the first study to determine the distribution and expression pattern of these stem cell markers in human breast cancer. There was a significant association between loss of expression and metastatic disease in patients with breast cancer. Such differential expression may play a part in breast cancer disease progression, and suggests that the current stem cell theory may not hold true for all cancer types.

  10. Differential expression of nucleostemin, a stem cell marker, and its variants in different types of brain tumors.

    PubMed

    Malakootian, Mahshid; Mowla, Seyed Javad; Saberi, Hooshang; Asadi, Malek Hossein; Atlasi, Yaser; Shafaroudi, Afsaneh Malekzadeh

    2010-09-01

    Nucleostemin (NS) is implicated in the control of stem and cancer cell proliferation. In the present study, we have examined the expression of NS and its spliced variants in various brain tumors. Total RNA was extracted from 59 brain tumor samples, and the expression of different NS spliced variants was measured by semi-quantitative RT-PCR. The subcellular distribution of NS protein in brain tumors was further examined by immunohistochemistry. Furthermore, to decipher the potential involvement of NS in brain tumorogenesis, its expression was knocked-down by means of RNA interference (RNAi) in two malignant glioma (U-87MG and A172), one astrocytoma (1321N1) and one medulloblastoma (DAOY) cell lines. The alterations in cell-cycle progression of the treated cells were then analyzed by flow cytometry. Our data revealed that NS and its variants are widely expressed in different types of brain tumors. Among the NS spliced variants, variant "1" and variant "3" were detected in the majority of tumor samples, whereas variant "2" was only detectable in few samples. Moreover, the intensity of the expression was correlated with the grade of the tumors (P < 0.05). Accordingly, the expression was much higher in glial tumors compared to that of meningiomas. As expected, a nucleolar/nucleoplasmic localization of NS protein was observed in the examined tumor samples. RNAi results revealed a significant reduction of NS expression along with a moderate blockade of the cell cycle in G(2)/M and S phases of NS-siRNA treated cells. All in all, our data suggest a potential role for NS in tumorogenesis of brain cancers.

  11. Primitive stem cells derived from bone marrow express glial and neuronal markers and support revascularization in injured retina exposed to ischemic and mechanical damage.

    PubMed

    Goldenberg-Cohen, Nitza; Avraham-Lubin, Bat-Chen R; Sadikov, Tamilla; Goldstein, Ronald S; Askenasy, Nadir

    2012-06-10

    Ischemic or mechanical injury to the optic nerve is an irreversible cause of vision loss, associated with limited regeneration and poor response to neuroprotective agents. The aim of this study was to assess the capacity of adult bone marrow cells to participate in retinal regeneration following the induction of anterior ischemic optic neuropathy (AION) and optic nerve crush (ONC) in a rodent model. The small-sized subset of cells isolated by elutriation and lineage depletion (Fr25lin(-)) was found to be negative for the neuroglial markers nestin and glial fibrillary acidic protein (GFAP). Syngeneic donor cells, identified by genomic marker in sex-mismatched transplants and green fluorescent protein, incorporated into the injured retina (AION and ONC) at a frequency of 0.35%-0.45% after intravenous infusion and 1.8%-2% after intravitreous implantation. Perivascular cells with astrocytic morphology expressing GFAP and vimentin were of the predominant lineage that engrafted after AION injury; 10%-18% of the donor cells incorporated in the retinal ganglion cell layer and expressed NeuN, Thy-1, neurofilament, and beta-tubulin III. The Fr25lin(-) cells displayed an excellent capacity to migrate to sites of tissue disruption and developed coordinated site-specific morphological and phenotypic neural and glial markers. In addition to cellular reconstitution of the injured retinal layers, these cells contributed to endothelial revascularization and apparently supported remodeling by secretion of insulin-like growth factor-1. These results suggest that elutriated autologous adult bone marrow-derived stem cells may serve as an accessible source for cellular reconstitution of the retina following injury.

  12. Inhibition of extracellular matrix assembly induces the expression of osteogenic markers in skeletal muscle cells by a BMP-2 independent mechanism

    PubMed Central

    Osses, Nelson; Casar, Juan Carlos; Brandan, Enrique

    2009-01-01

    Background The conversion of one cell type into another has been suggested to be, at the molecular level, the consequence of change(s) in the expression level of key developmental genes. Myoblasts have the ability to differentiate either to skeletal muscle or osteogenic lineage depending of external stimuli. Extracellular matrix (ECM) has been shown to be essential for skeletal muscle differentiation, through its direct interaction with myoblasts' cell receptors. We attempt to address if ECM also plays a role in the osteogenic differentiation of skeletal muscle cells. Results Inhibition of proteoglycan sulfation by sodium chlorate in myoblast cultures strongly affects ECM synthesis and deposition and induces the expression of the osteogenic lineage markers alkaline phosphatase (ALP) and osteocalcin in mononuclear cells. Induction of ALP by sodium chlorate does not affect the expression of specific muscle determination transcription factors, such as MyoD and Myf-5, in the same cells. The osteogenic transcription factor Cbfa-1 expression is also unaffected. Induction of ALP is not inhibited by a soluble form of BMP receptor IA. This suggests that the deviation of the myogenic pathway of C2C12 myoblasts into the osteogenic lineage by inhibitors of proteoglycan sulfation is BMP-2 independent. The increase of osteogenic markers expression can be totally prevented by an exogenous ECM. Interestingly, a similar BMP-2-independent ALP activity induction can be observed in myoblasts cultured on an ECM previously synthesized by BMP-2 treated myoblasts. Under in vivo conditions of increased ECM turn-over and deposition, as in the mdx dystrophic muscle and during skeletal muscle regeneration, an induction and relocalization of ALP is observed in a subpopulation of skeletal muscle fibers, whereas in normal skeletal muscle, ALP expression is restricted to blood vessels and some endomysial mononuclear cells. Conclusion These results suggest that signals arising from the ECM induce

  13. Expression status of CD44 and CD133 as a prognostic marker in esophageal squamous cell carcinoma treated with neoadjuvant chemotherapy followed by radical esophagectomy.

    PubMed

    Okamoto, Koichi; Ninomiya, Itasu; Ohbatake, Yoshinao; Hirose, Atsushi; Tsukada, Tomoya; Nakanuma, Shinichi; Sakai, Seisho; Kinoshita, Jun; Makino, Isamu; Nakamura, Keishi; Hayashi, Hironori; Oyama, Katsunobu; Inokuchi, Masafumi; Nakagawara, Hisatoshi; Miyashita, Tomoharu; Hidehiro, Tajima; Takamura, Hiroyuki; Fushida, Sachio; Ohta, Tetsuo

    2016-12-01

    Cancer stem cells (CSCs) have self-renewal and pluripotency capabilities and contribute to cancer progression and chemoresistance. It has been proposed that the treatment resistance and heterogeneity of CSCs are deeply involved in the prognosis of patients with esophageal squamous cell carcinoma (ESCC). The objective of this study was to identify the influence of the expression status of the CSC markers CD44 and CD133 on chemotherapeutic efficacy and prognosis in ESCC patients who underwent radical esophagectomy after neoadjuvant chemotherapy (NAC). Endoscopically biopsied specimens taken before NAC and surgically resected specimens after NAC were immunohistochemically assessed for CD44 and CD133 expression for 47 ESCC patients who underwent NAC followed by radical esophagectomy. The correlation between CD44 and CD133 expression status and clinicopathological findings and the prognosis of ESCC patients after NAC followed by esophagectomy were analyzed. The percentages of CD44-positive cells and CD133-positive cells in specimens were increased after NAC. CD44 and CD133 expression status before NAC did not correlate with the degree of tumor progression and had no impact on the chemotherapeutic effect. However, strong expression of CD44 or CD133 and a high proportion of CD133-expressing cells before NAC were significantly associated with poorer esophageal cancer-specific survival. Patients with strong expression of CD44 or CD133 and those with a high ratio of CD133-positive tumor cells showed significantly poor prognosis regardless of the effect of chemotherapy. Multivariate analysis showed that simultaneous strong expression of CD44 and CD133 before NAC, a high rate of CD133-positive tumor cells before NAC, and primary tumor remission assessed by preoperative endoscopy were significant independent prognostic factors for ESCC. Our data indicate that CD44 and CD133 expression status prior to treatment dictates the malignant potential of ESCC and may be a novel

  14. Human dental pulp stem cells respond to cues from the rat retina and differentiate to express the retinal neuronal marker rhodopsin.

    PubMed

    Bray, A F; Cevallos, R R; Gazarian, K; Lamas, M

    2014-11-07

    Human adult dental pulp stem cells (DPSCs) are self-renewing stem cells that originate from the neural crest during development and remain within the dental pulp niche through adulthood. Due to their multi-lineage differentiation potential and their relative ease of access they represent an exciting alternative for autologous stem cell-based therapies in neurodegenerative diseases. In animal models, DPSCs transplanted into the brain differentiate into functional neurons or astrocytes in response to local environmental cues that appear to influence the fate of the surviving cells. Here we tested the hypothesis that DPSCs might be able to respond to factors present in the retina enabling the regenerative potential of these cells. We evaluated the response of DPSCs to conditioned media from organotypic explants from control and chemically damaged rat retinas. To evaluate cell differentiation, we analyzed the expression of glial fibrillary acidic protein (GFAP), early neuronal and retinal markers (polysialic acid-neural cell adhesion molecule (PSA-NCAM); Pax6; Ascl1; NeuroD1) and the late photoreceptor marker rhodopsin, by immunofluorescence and reverse transcription polymerase chain reaction (RT-PCR). Exposure of DPSC cultures to conditioned media from control retinas induced a 39% reduction on the number of DPSCs that expressed GFAP; the expression of Pax6, Ascl1, PSA-NCAM or NeuroD1 was undetectable or did not change significantly. Expression of rhodopsin was not detectable in control or after exposure of the cultures with retinal conditioned media. By contrast, 44% of DPSCs exposed to conditioned media from damaged retinas were immunopositive to this protein. This response could not be reproduced when conditioned media from Müller-enriched primary cultures was used. Finally, quantitative RT-PCR was performed to compare the relative expression of glial cell-derived neurotrophic factor (GDNF), nerve growth factor (NGF), ciliary neurotrophic factor (CNTF) and brain

  15. Inhibition of Six1 affects tumour invasion and the expression of cancer stem cell markers in pancreatic cancer.

    PubMed

    Lerbs, Tristan; Bisht, Savita; Schölch, Sebastian; Pecqueux, Mathieu; Kristiansen, Glen; Schneider, Martin; Hofmann, Bianca T; Welsch, Thilo; Reissfelder, Christoph; Rahbari, Nuh N; Fritzmann, Johannes; Brossart, Peter; Weitz, Jürgen; Feldmann, Georg; Kahlert, Christoph

    2017-04-07

    Epithelial-to-mesenchymal transition (EMT) and cancer stem cells (CSC) contribute to tumour progression and metastasis. Assessment of transcription factors involved in these two mechanisms can help to identify new targets for an oncological therapy. In this study, we focused on the evaluation of the transcription factor Six1 (Sine oculis 1). This protein is involved in embryologic development and its contribution to carcinogenesis has been described in several studies. Immunohistochemistry against Six1 was performed on a tissue microarray containing specimens of primary pancreatic ductal adenocarcinomas (PDAC) of 139 patients. Nuclear and cytoplasmic expression was evaluated and correlated to histopathological parameters. Expression of Six1 was inhibited transiently by siRNA in Panc1 and BxPc3 cells and stably by shRNA in Panc1 cells. Expression analysis of CDH1 and Vimentin mRNA was performed and cell motility was tested in a migration assay. Panc1 cells transfected with Six1 shRNA or scrambled shRNA were injected subcutaneously into nude mice. Tumour growth was observed for four weeks. Afterwards, tumours were stained against Six1, CD24 and CD44. Six1 was overexpressed in the cytoplasm and cellular nuclei in malignant tissues (p < 0.0001). No correlation to histopathological parameters could be detected. Six1 down-regulation decreased pancreatic cancer cell motility in vitro. CDH1 and vimentin expression was decreased after inhibition of the expression of Six1. Pancreatic tumours with impaired expression of Six1 showed significantly delayed growth and displayed loss of the CD24(+)/CD44(+) phenotype. We show that Six1 is overexpressed in human PDAC and that its inhibition results in a decreased tumour progression in vitro and in vivo. Therefore, targeting Six1 might be a novel therapeutic approach in patients with pancreatic cancer.

  16. The expression of pluripotency genes and neuronal markers after neurodifferentiation in fibroblasts co-cultured with human umbilical cord blood mononuclear cells.

    PubMed

    Marinowic, D R; Domingues, M F; Machado, D C; DaCosta, J C

    2015-01-01

    Human umbilical cord blood is an attractive source of stem cells; however, it has a heterogeneous cell population with few mesenchymal stem cells. Cell reprogramming induced by different methodologies can confer pluripotency to differentiated adult cells. The objective of this study was to evaluate the reprogramming of fibroblasts and their subsequent neural differentiation after co-culture with umbilical cord blood mononuclear cells. Cells were obtained from four human umbilical cords. The mononuclear cells were cultured for 7 d and subsequently co-cultured with mouse fibroblast NIH-3T3 cells for 6 d. The pluripotency of the cells was evaluated by RT-PCR using primers specific for pluripotency marker genes. The pluripotency was also confirmed by adipogenic and osteogenic differentiation. Neural differentiation of the reprogrammed cells was evaluated by immunofluorescence. All co-cultured cells showed adipogenic and osteogenic differentiation capacity. After co-cultivation, cells expressed the pluripotency gene KLF4. Statistically significant differences in cell area, diameter, optical density, and fractal dimension were observed by confocal microscopy in the neurally differentiated cells. Contact in the form of co-cultivation of fibroblasts with umbilical cord blood mononuclear fraction for 6 d promoted the reprogramming of these cells, allowing the later induction of neural differentiation.

  17. Brain Region–Specific Alterations in the Gene Expression of Cytokines, Immune Cell Markers and Cholinergic System Components during Peripheral Endotoxin–Induced Inflammation

    PubMed Central

    Silverman, Harold A; Dancho, Meghan; Regnier-Golanov, Angelique; Nasim, Mansoor; Ochani, Mahendar; Olofsson, Peder S; Ahmed, Mohamed; Miller, Edmund J; Chavan, Sangeeta S; Golanov, Eugene; Metz, Christine N; Tracey, Kevin J; Pavlov, Valentin A

    2014-01-01

    Inflammatory conditions characterized by excessive peripheral immune responses are associated with diverse alterations in brain function, and brain-derived neural pathways regulate peripheral inflammation. Important aspects of this bidirectional peripheral immune–brain communication, including the impact of peripheral inflammation on brain region–specific cytokine responses, and brain cholinergic signaling (which plays a role in controlling peripheral cytokine levels), remain unclear. To provide insight, we studied gene expression of cytokines, immune cell markers and brain cholinergic system components in the cortex, cerebellum, brainstem, hippocampus, hypothalamus, striatum and thalamus in mice after an intraperitoneal lipopolysaccharide injection. Endotoxemia was accompanied by elevated serum levels of interleukin (IL)-1β, IL-6 and other cytokines and brain region–specific increases in Il1b (the highest increase, relative to basal level, was in cortex; the lowest increase was in cerebellum) and Il6 (highest increase in cerebellum; lowest increase in striatum) mRNA expression. Gene expression of brain Gfap (astrocyte marker) was also differentially increased. However, Iba1 (microglia marker) mRNA expression was decreased in the cortex, hippocampus and other brain regions in parallel with morphological changes, indicating microglia activation. Brain choline acetyltransferase (Chat ) mRNA expression was decreased in the striatum, acetylcholinesterase (Ache) mRNA expression was decreased in the cortex and increased in the hippocampus, and M1 muscarinic acetylcholine receptor (Chrm1) mRNA expression was decreased in the cortex and the brainstem. These results reveal a previously unrecognized regional specificity in brain immunoregulatory and cholinergic system gene expression in the context of peripheral inflammation and are of interest for designing future antiinflammatory approaches. PMID:25299421

  18. A modular and optimized single marker system for generating Trypanosoma brucei cell lines expressing T7 RNA polymerase and the tetracycline repressor.

    PubMed

    Poon, S K; Peacock, L; Gibson, W; Gull, K; Kelly, S

    2012-02-01

    Here, we present a simple modular extendable vector system for introducing the T7 RNA polymerase and tetracycline repressor genes into Trypanosoma brucei. This novel system exploits developments in our understanding of gene expression and genome organization to produce a streamlined plasmid optimized for high levels of expression of the introduced transgenes. We demonstrate the utility of this novel system in bloodstream and procyclic forms of Trypanosoma brucei, including the genome strain TREU927/4. We validate these cell lines using a variety of inducible experiments that recapture previously published lethal and non-lethal phenotypes. We further demonstrate the utility of the single marker (SmOx) TREU927/4 cell line for in vivo experiments in the tsetse fly and provide a set of plasmids that enable both whole-fly and salivary gland-specific inducible expression of transgenes.

  19. Neuronal markers expression of NGF-primed bone marrow cells (BMCs) transplanted in the brain of 6-hydroxydopamine and ibotenic acid lesioned littermate mice.

    PubMed

    Triaca, Viviana; Aloe, Luigi

    In the present study, we aim to show that non-adherent bone marrow cells (BMCs) express TrkA, the nerve growth factor (NGF) receptor, and that addition of NGF promotes the survival and neuronal commitment of BMC transplanted into the experimentally injured brain of littermates mice. Immunohistochemical analysis revealed that transplanted BMCs express tyrosine hydroxylase (TH) in proximity of the damaged dopaminergic tissues and choline acetyltransferase (ChAT) in the lesioned cholinergic regions. These results suggest that NGF supports the survival and differentiation of uncommitted BMCs and concurs with other local environmental signals to promote the expression of neuronal markers in these cells. The possible functional significance of these observations will be discussed.

  20. Increased expression of senescence markers in cystic fibrosis airways

    PubMed Central

    Wong, Jessica K.; Degan, Simone; Kummarapurugu, Apparao B.; Zheng, Shuo; Haridass, Prashamsha; Voynow, Judith A.

    2013-01-01

    Cystic Fibrosis (CF) is a chronic lung disease characterized by chronic neutrophilic airway inflammation and increased levels of neutrophil elastase (NE) in the airways. We have previously reported that NE treatment triggers cell cycle arrest. Cell cycle arrest can lead to senescence, a complete loss of replicative capacity. Importantly, senescent cells can be proinflammatory and would perpetuate CF chronic inflammation. By immunohistochemistry, we evaluated whether airway sections from CF and control subjects expressed markers of senescence, including p16INK4a (p16), a cyclin-dependent kinase inhibitor, phospho-Histone H2A.X (γH2A.X), and phospho-checkpoint 2 kinase (phospho-Chk2), which are also DNA damage response markers. Compared with airway epithelium from control subjects, CF airway epithelium had increased levels of expression of all three senescence markers. We hypothesized that the high load of NE in the CF airway triggers epithelial senescence by upregulating expression of p16, which inhibits cyclin-dependent kinase 4 (CDK4). Normal human bronchial epithelial (NHBE) cells, cultured in air-liquid interface were treated with NE (0, 200, and 500 nM) to induce visible injury. Total cell lysates were collected and evaluated by Western analysis for p16 protein expression and CDK4 kinase activity. NE significantly increased p16 expression and decreased CDK4 kinase activity in NHBE cells. These results support the concept that NE triggers expression of senescence markers in CF airway epithelial cells. PMID:23316069

  1. Co-expression of putative stemness and epithelial-to-mesenchymal transition markers on single circulating tumour cells from patients with early and metastatic breast cancer.

    PubMed

    Papadaki, Maria A; Kallergi, Galatea; Zafeiriou, Zafeiris; Manouras, Lefteris; Theodoropoulos, Panayiotis A; Mavroudis, Dimitris; Georgoulias, Vassilis; Agelaki, Sofia

    2014-09-03

    The detection of circulating tumor cells (CTCs) in peripheral blood (PB) of patients with breast cancer predicts poor clinical outcome. Cancer cells with stemness and epithelial-to-mesenchymal transition (EMT) features display enhanced malignant and metastatic potential. A new methodology was developed in order to investigate the co-expression of a stemness and an EMT marker (ALDH1 and TWIST, respectively) on single CTCs of patients with early and metastatic breast cancer. Triple immunofluorescence using anti-pancytokeratin (A45-B/B3), anti-ALDH1 and anti-TWIST antibodies was performed in cytospins prepared from hepatocellular carcinoma HepG2 cells and SKBR-3, MCF-7 and MDA.MB.231 breast cancer cell lines. Evaluation of ALDH1 expression levels (high, low or absent) and TWIST subcellular localization (nuclear, cytoplasmic or absent) was performed using the ARIOL system. Cytospins prepared from peripheral blood of patients with early (n = 80) and metastatic (n = 50) breast cancer were analyzed for CTC detection (based on pan-cytokeratin expression and cytomorphological criteria) and characterized according to ALDH1 and TWIST. CTCs were detected in 13 (16%) and 25 (50%) patients with early and metastatic disease, respectively. High ALDH1 expression (ALDH1high) and nuclear TWIST localization (TWISTnuc) on CTCs was confirmed in more patients with metastatic than early breast cancer (80% vs. 30.8%, respectively; p = 0.009). In early disease, ALDH1low/neg CTCs (p = 0.006) and TWISTcyt/neg CTCs (p = 0.040) were mainly observed. Regarding co-expression of these markers, ALDH1high/TWISTnuc CTCs were more frequently evident in the metastatic setting (76% vs. 15.4% of patients, p = 0.001; 61.5% vs. 12.9% of total CTCs), whereas in early disease ALDH1low/neg/TWISTcyt/neg CTCs were mainly detected (61.5% vs. 20% of patients, p = 0.078; 41.9% vs. 7.7% of total CTCs). A new assay is provided for the evaluation of ALDH1 and TWIST co-expression at the

  2. CD26 expression in mature B-cell neoplasia: its possible role as a new prognostic marker in B-CLL.

    PubMed

    Cro, Lilla; Morabito, Fortunato; Zucal, Nadia; Fabris, Sonia; Lionetti, Marta; Cutrona, Giovanna; Rossi, Francesca; Gentile, Massimo; Ferrario, Andrea; Ferrarini, Manlio; Molica, Stefano; Neri, Antonino; Baldini, Luca

    2009-09-01

    CD26 (dipeptidyl peptidase IV, DPP IV) is widely expressed by T and natural killer (NK) cells, epithelial and endothelial cells of different tissues, and it is strongly upregulated in activated B-cells; moreover it plays a regulatory role in the neoplastic transformation and progression of various types of tumours. CD26 expression was evaluated by means of flow cytometry in various peripheral B-cell lymphoid tumours: 12 follicular and 12 mantle cell lymphomas, 20 multiple myelomas (MMs), 12 hairy cell leukaemias (HCLs), 112 chronic lymphocytic leukaemias (CLLs), 20 CD5(negative) B-cell chronic lymphoproliferative diseases (CD5(neg) B-CLPDs) and 12 diffuse large cell lymphomas (DLCLs). CD26 expression was absent or barely detectable in follicular and mantle cell lymphomas, high in MMs and HCLs, and variable in CLLs, in CD5(neg) B-CLPDs and in DLCLs. CD26 significantly correlated with CD49d and CD38 expressions (p < 0.0001) in B-CLLs, and there was a significant correlation between CD26 and ZAP-70 expressions or IgVH mutational status (p < 0.0001). After a median follow-up of 36 months, 65 B-CLL patients were treated; taking 10% as the best CD26 cut-off value, Kaplan-Meier curves revealed a significantly shorter time to treatment in the CD26-positive cases (p < 0.0001). Overall, our data indicate that CD26 expression may identify subsets of B-CLL patients with an unfavourable clinical outcome in terms of therapeutic need, thus suggesting its potential role as a marker (together with CD38 and CD49d) in a future routine cytofluorimetric panel to be validated for the prognostic stratification of B-CLLs.

  3. The pan-B cell marker CD22 is expressed on gastrointestinal eosinophils and negatively regulates tissue eosinophilia¶

    PubMed Central

    Wen, Ting; Mingler, Melissa K.; Blanchard, Carine; Wahl, Benjamin; Pabst, Oliver; Rothenberg, Marc E.

    2011-01-01

    CD22 is currently recognized as a B cell-specific Siglec and has been exploited therapeutically with humanized anti-CD22 monoclonal antibody having been used against B cell leukemia. Herein, tissue-specific eosinophil mRNA microarray analysis identified that CD22 transcript levels of murine gastrointestinal (GI) eosinophils are 10-fold higher than those of lung eosinophils. In order to confirm the mRNA data at the protein level, we developed a FACS-based protocol designed to phenotype live GI eosinophils isolated from the murine lamina propria. Indeed, we found that jejunum eosinophils expressed remarkably high levels of surface CD22, similar to levels found in B cells across multiple mouse strains. In contrast, CD22 was undetectable on eosinophils from the colon, blood, thymus, spleen, uterus, peritoneal cavity and allergen-challenged lung. Eosinophils isolated from newborn mice did not express CD22 but subsequently upregulated CD22 expression to adult levels within the first 10 days after birth. The GI lamina propria from CD22 gene-targeted mice harbored more eosinophils than wild-type control mice, while the GI eosinophil turnover rate was unaltered in the absence of CD22. Our findings identify a novel expression pattern and tissue eosinophilia-regulating function for the “B cell-specific” inhibitory molecule CD22 on GI eosinophils. PMID:22190185

  4. Up-regulation of BMP2/4 signaling increases both osteoblast-specific marker expression and bone marrow adipogenesis in Gja1Jrt/+ stromal cell cultures.

    PubMed

    Zappitelli, Tanya; Chen, Frieda; Aubin, Jane E

    2015-03-01

    Gja1(Jrt)/+ mice carry a mutation in one allele of the gap junction protein α1 gene (Gja1), resulting in a G60S connexin 43 (Cx43) mutant protein that is dominant negative for Cx43 protein production of <50% of wild-type (WT) levels and significantly reduced gap junction formation and function in osteoblasts and other Cx43-expressing cells. Previously we reported that Gja1(Jrt)/+ mice exhibited early-onset osteopenia caused by activation of osteoclasts secondary to activation of osteoblast lineage cells, which expressed increased RANKL and produced an abnormal resorption-stimulating bone matrix high in BSP content. Gja1(Jrt)/+ mice also displayed early and progressive bone marrow atrophy, with a significant increase in bone marrow adiposity versus WT littermates but no increase in adipose tissues elsewhere in the body. BMP2/4 production and signaling were increased in Gja1(Jrt)/+ trabecular bone and osteogenic stromal cell cultures, which contributed to the up-regulated expression of osteoblast-specific markers (e.g., Bsp and Ocn) in Gja1(Jrt)/+ osteoblasts and increased Pparg2 expression in bone marrow-derived adipoprogenitors in vitro. The elevated levels of BMP2/4 signaling in G60S Cx43-containing cells resulted at least in part from elevated levels of cAMP. We conclude that up-regulation of BMP2/4 signaling in trabecular bone and/or stromal cells increases osteoblast-specific marker expression in hyperactive Gja1(Jrt)/+ osteoblasts and may also increase bone marrow adipogenesis by up-regulation of Pparg2 in the Cx43-deficient Gja1(Jrt)/+ mouse model.

  5. Cell-surface marker analysis of rat thymic dendritic cells.

    PubMed Central

    Bañuls, M P; Alvarez, A; Ferrero, I; Zapata, A; Ardavin, C

    1993-01-01

    Rat thymic dendritic cells have been isolated by collagenase digestion, separation of the low-density cell fraction by centrifugation on metrizamide, and differential adherence. The resulting dendritic cell preparation had a purity of > 90%, and has been analysed by flow cytometry (FCM) using a large panel of monoclonal antibodies (mAb). Dendritic cells expressed major histocompatibility (MHC) class I and class II molecules, the leucocyte common antigen CD45, the rat leucocyte antigen OX44, the rat macrophage marker ED1, and the adhesion molecules Mac-1, LFA-1 and ICAM-1. They were negative for the T- and B-cell-specific forms of CD45, CD45R and B220, and the B-cell marker OX12. Concerning T-cell marker expression, they were negative for T-cell receptor (TcR) and OX40, but they expressed CD2, CD4 and CD8, and interestingly, 50% of DC were CD5+, 50% expressed the alpha-chain of interleukin-2 receptor (IL-2R), and 80% were positive for the T-cell activation antigen recognized by the mAb OX48. Moreover, 60% of DC expressed high levels of Thy-1, whereas 40% displayed intermediate levels of this T-cell marker. PMID:8102122

  6. Gene expression profiling of single circulating tumor cells in ovarian cancer - Establishment of a multi-marker gene panel.

    PubMed

    Blassl, Christina; Kuhlmann, Jan Dominik; Webers, Alessandra; Wimberger, Pauline; Fehm, Tanja; Neubauer, Hans

    2016-08-01

    The presence of circulating tumor cells (CTCs) in the blood of ovarian cancer patients was shown to correlate with decreased overall survival, whereby CTCs with epithelial-mesenchymal-transition (EMT) or stem-like traits are supposed to be involved in metastatic progression and recurrence. Thus, investigating the transcriptional profiles of CTCs might help to identify therapy resistant tumor cells and to overcome treatment failure. For this purpose, we established a multi-marker panel for the molecular characterization of single CTCs, detecting epithelial (EpCAM, Muc-1, CK5/7), EMT (N-cadherin, Vimentin, Snai1/2, CD117, CD146, CD49f) and stem cell (CD44, ALDH1A1, Nanog, SOX2, Notch1/4, Oct4, Lin28) associated transcripts. First primer specificity and PCR-performance of the multiplex-RT-PCRs were successfully validated on genomic DNA and cDNA isolated from OvCar3 cells. The assay sensitivity of the epithelial panel was evaluated by adding defined numbers of tumor cells into the blood of healthy donors and performing a subsequent immunomagnetic tumor cell enrichment (AdnaTest OvarianCancerSelect), resulting in a 100% concordance for the epithelial markers EpCAM and Muc-1 to the AdnaTest OvarianCancerDetect. Additionally, by processing blood from ovarian cancer patients, high assay sensitivity could be verified. In blood of healthy donors no signals for epithelial markers were detected, for EMT and stem cell markers, however, signals were obtained mainly originating from leukocytes which calls for single cell analysis. To that aim by using the ovarian cancer cell line OvCar3, we successfully established a workflow enabling the characterization of single CTCs. It consists of a density gradient-dependent enrichment for nucleated cells, a depletion of CD45-positive cells of hematopoietic origin followed by immunofluorescent labeling of CTCs by EpCAM and Muc-1. Single CTCs are then isolated by micromanipulation and processed for panel gene expression profiling. Finally

  7. The number of cells expressing the myelin-supporting oligodendrocyte marker PLP-exon 3b remains unchanged in Wallerian degeneration.

    PubMed

    Li, G; Blakemore, W F

    2004-08-01

    Following spinal cord trauma there is controversy as to whether myelin-supporting oligodendrocytes at a distance from areas of spinal cord damage undergo apoptosis. To examine the response of oligodendrocytes to axon degeneration, we counted the number of oligodendrocytes and oligodendrocyte precursors in the dorsal funiculi during the course of Wallerian degeneration. Axons were disrupted at T13 and the number of labelled cells in the dorsal funiculi counted at T12, 4 days and 2, 4, and 8 weeks after injury using riboprobes to exon-3b of the PLP gene whose expression is considered to restricted to myelin-supporting oligodendrocytes, PDGFRalpha which is regarded as a marker of oligodendrocyte precursors, and MOG a marker previously used to identify myelin-supporting oligodendrocytes. We found that the number of PLP-exon-3b labelled cells remained constant during the course of Wallerian degeneration while the number of cells labelled with the riboprobes to PDGFRalpha and MOG increased. Significantly the number of MOG-positive cells was increased at times when the number of PDGFRalpha labelled cells was highest. The number of PDGFRalpha labelled cells decreased with time while the number PLP-exon-3b labelled cells remained constant. It is therefore possible that the apoptotic oligodendrocytes identified in previous studies could represent degenerating oligodendrocyte precursors or their progeny rather than degenerating myelin-supporting oligodendrocytes.

  8. Expression of dendritic cell markers CD11c/BDCA-1 and CD123/BDCA-2 in coronary artery disease upon activation in whole blood.

    PubMed

    Van Brussel, Ilse; Van Vré, Emily A; De Meyer, Guido R Y; Vrints, Christiaan J; Bosmans, Johan M; Bult, Hidde

    2010-10-31

    Previous in vivo studies on dendritic cell (DC) enumeration in coronary artery disease (CAD) were not always consistent. Therefore, we investigated by flow cytometry whether this was due to CAD-related differences in expression of subset markers for myeloid (m)DCs (blood DC antigen (BDCA)-1, CD11c) and plasmacytoid (p)DCs (BDCA-2, CD123), before and after in vitro stimulation with Toll-like receptor ligands. Our data showed that circulating DCs decline in CAD, irrespective of the DC subset marker that was used for enumeration. Upon in vitro activation, BDCA-2 was downregulated, whereas CD11c and CD123 were upregulated. This implies that the expression ratios CD11c/BDCA-1 and CD123/BDCA-2 can assess DC activation. Comparing these ratios between controls and CAD patients showed no differences in blood DC activation in both groups. This study suggests that when different DC numbers are found between two study populations, the DC activation status from both groups always needs to be verified, since a decrease in BDCA-2(+) pDCs or an increase in CD11c(+) mDCs or CD123(+) pDCs can be due to the altered expression of these markers during activation. Given that CD11c, BDCA-1, CD123 and BDCA-2 are more abundantly expressed on blood DCs than typical activation markers like CD83, CD86 or CCR-7, the use of the ratios is an easy and reliable way to determine DC activation in whole blood assays. Copyright © 2010 Elsevier B.V. All rights reserved.

  9. KAI-1 and p53 expression in oral squamous cell carcinomas: Markers of significance in future diagnostics and possibly therapeutics

    PubMed Central

    Patil, Namrata N; Wadhwan, Vijay; Chaudhary, Minal; Nayyar, Abhishek Singh

    2016-01-01

    Context: KAI-1/CD82 is a tumor suppressor gene with decreased gene expression being associated with increased invasive ability of oral squamous cell carcinomas (OSCCs). p53 protein functions in the G1-S phase of the cell cycle to allow repair of damaged DNA. In the present study, p53 and KAI-1 expression was investigated using monoclonal antibodies in OSCC. Aims: The aim of this study was to detect KAI-1 and p53 expression in OSCCs and to assess the relation between both in OSCCs. Materials and Methods: The present study included histopathologically diagnosed thirty cases of well- and moderately differentiated OSCCs to study the expression of KAI-1 and p53 antibodies. Statistical Analysis: The results obtained were tabulated and statistically analyzed using descriptive statistical analysis; one-way ANOVA; least square difference method and independent t-test. Results: OSCCs exhibited 41.62% positivity for KAI-1 while p53 positive cells were recorded to an extent of 60.82%. A significant positive correlation was observed between KAI-1 and p53 expression in OSCCs. Conclusions: Although a significant amount of work is still required to uncover the mechanisms of action and regulation of KAI-1 and p53 expression, control of the complex metastatic processes would be of interest in controlling the tumor biology in OSCCs as well as other types of malignancies to enhance prognosis in the affected patients and to help protect against future metastasis in the going to be treated and treated patients. PMID:27721601

  10. Liver-enriched transcription factors are critical for the expression of hepatocyte marker genes in mES-derived hepatocyte-lineage cells.

    PubMed

    Kheolamai, Pakpoom; Dickson, Alan J

    2009-04-23

    Induction of stem cell differentiation toward functional hepatocytes is hampered by lack of knowledge of the hepatocyte differentiation processes. The overall objective of this project is to characterize key stages in the hepatocyte differentiation process. We established a mouse embryonic stem (mES) cell culture system which exhibited changes in gene expression profiles similar to those observed in the development of endodermal and hepatocyte-lineage cells previously described in the normal mouse embryo. Transgenic mES cells were established that permitted isolation of enriched hepatocyte-lineage populations. This approach has isolated mES-derived hepatocyte-lineage cells that express several markers of mature hepatocytes including albumin, glucose-6-phosphatase, tyrosine aminotransferase, cytochrome P450-3a, phosphoenolpyruvate carboxykinase and tryptophan 2,3-dioxygenase. In addition, our results show that the up-regulation of the expression levels of hepatocyte nuclear factor-3alpha, -4alpha, -6, and CCAAT-enhancer binding protein-beta might be critical for passage into late-stage differentiation towards functional hepatocytes. These data present important steps for definition of regulatory phenomena that direct specific cell fate determination. The mES cell culture system generated in this study provides a model for studying transition between stages of the hepatocyte development and has significant potential value for studying the molecular basis of hepatocyte differentiation in vitro.

  11. 195 EXPRESSION OF MESENCHYMAL STROMAL CELL (MSC) MARKERS IN THE EQUINE ENDOMETRIUM AND IN VITRO INFLUENCE OF STEROID HORMONES ON ENDOMETRIAL-DERIVED MSC.

    PubMed

    Rink, E; Kuhl, J; Aurich, C; French, H; Nino-Fong, R; Watson, E; Donadeu, F X

    2016-01-01

    Mesenchymal stromal cell (MSC) are multipotent precursor cells that have been isolated from many tissues, including endometrium in some species. These cells are necessary for tissue homeostasis, which in the cycling equine endometrium is regulated in part by changes in concentration of steroid hormones. The expression of oestrogen and progesterone receptors during the oestrous cycle has been studied before, but MSC gene expression is not reported as well as the effects of steroid hormones on in vitro proliferation of endometrial MSC. This study was designed to investigate the influence of steroid hormones on endometrial MSC proliferation in vitro and to examine mRNA expression of MSC markers (CD29, CD44, CD73, CD90, and CD105) in the healthy equine endometrium during the oestrous cycle. Equine endometrial tissue was collected postmortem (n=6) and digested using a dissociation medium and mucin-1-bound magnetic beads were utilised to remove epithelial cells from the resulting single-cell solution. The cells were expanded in culture and, at passage 4, incubated with 3 different concentrations of oestradiol and progesterone for 5 days. For the proliferation analysis the Alamar Blue® assay was used according to manufacturer instructions. Endometrial biopsies, for quantitative RT-PCR analysis, were taken from healthy mares (n=5) on Day 5 and 13 post-ovulation, during oestrus (1 follicle >3.5cm, pronounced uterine oedema), and seasonal anestrous (seasonal anovulation). The ΔCt values were used for statistical analysis using SPSS Statistics 22 (IBM Corp., Armonk, NY). Data for quantitative PCR are presented as gene expression relative to the mean of 18S and GAPDH. No significant differences in proliferation could be detected in the various groups incubated with steroid hormones compared with the controls supplemented with charcoal-stripped fetal bovine serum. Detectable levels of mRNA for all 5 MSC markers analysed were present throughout the oestrous cycle. While the

  12. A Study on the Mechanism of Low-Expressed Cancer Stem Cell Marker Lgr5 in Inhibition of the Proliferation and Invasion of Colorectal Carcinoma.

    PubMed

    Jia, Houjun; Xiang, Lin; Wang, Ziwei; Zhou, Qipeng

    2015-11-01

    The present study intends to explore the influence of Lgr5 as a marker of tumor stem cells after siRNA interference on the proliferation and invasion of colorectal carcinoma (CRC) and its mechanism. The tissue samples were taken for biopsy from 32 cases of patients and 32 cases of normal subjects by colonoscopy. Real-time quantitative PCR was used to detect the differential expression of Lgr5. After siRNA interference of Lgr5 in CRC cell line CT-26 cells, RT-PCR method was used to detect the mRNA expression level of Lgr5 after interference of CT-26 cells. CCK8 method was used to observe the influence of Lgr5 interference on the proliferation, colony formation, and invasion of CT-26 cells. RT-PCR and Western blot were used to detect the E-cadherin mRNA and protein levels in CT-26 cells. Lgr5 expression level in CRC tissue was significantly higher than that in the corresponding para-carcinoma tissue and the control group, and the differences were statistically significant (P < 0.05). Lgr5 mRNA expression level in tissue with lymph node metastasis was significantly higher than that in the tissue without lymph node metastasis, and the difference was statistically significant (P < 0.05). Compared with the control group, CT-26 cell proliferation, colony formation, and migration capability after Lgr5 siRNA transfection were all significantly reduced, and the differences were statistically significant (P < 0.05). CT-26 cells after Lgr5 interference were found with significantly reduced E-cadherin mRNA and protein levels. Lgr5 facilitates the cell proliferation, colony formation, and migration of colorectal carcinoma, which may be closely related to the expression level of E-cadherin.

  13. Expression of apoptosis markers on peripheral blood lymphocytes from patients with cutaneous T-cell lymphoma during extracorporeal photochemotherapy.

    PubMed

    Osella-Abate, S; Zaccagna, A; Savoia, P; Quaglino, P; Salomone, B; Bernengo, M G

    2001-01-01

    The mechanisms of extracorporeal photochemotherapy (ExP) therapeutic activity in cutaneous T-cell lymphomas (CTCLs) are not yet well understood, even though it has been suggested that a major mechanism may be induction of apoptosis. In vitro studies demonstrate that UVA-induced apoptosis is mediated by CD95-Fas expression and inhibited by Bcl-2 up-regulation and that UVA irradiation is able to down-regulate Bcl-2 expression. High-resolution multiparameter flow-cytometric analyses were used to evaluate Bcl-2/CD95-Fas expression on phenotypically identifiable circulating clonal T cells from 7 patients with CTCL (4 with Sézary syndrome and 3 with mycosis fungoides with peripheral involvement) before and during ExP, in an attempt to ascertain whether Bcl-2/CD95-Fas status can be related to the hematologic response. A Bcl-2 normal phenotype before ExP or a normalization in Bcl-2 expression during ExP were related to a better clinical response, whereas a persistent Bcl-2 high expression was a negative prognostic factor. On the other hand, no response was found in patients with a CD95-Fas-negative phenotype, whereas the expression of CD95-Fas was associated with hematologic remission. Although further studies are needed to confirm these preliminary results, this study suggests that Bcl-2 and CD95-Fas expression could be evaluated, together with the other known clinical and immunologic factors, as additional parameters related to clinical response in patients with CTCL undergoing ExP.

  14. MTAP is an independent prognosis marker and the concordant loss of MTAP and p16 expression predicts short survival in non-small cell lung cancer patients.

    PubMed

    Su, C-Y; Chang, Y-C; Chan, Y-C; Lin, T-C; Huang, M-S; Yang, C-J; Hsiao, M

    2014-09-01

    Methylthioadenosine phosphorylase (MTAP), a ubiquitously expressed protein, plays important roles in purine biosynthesis. Locating near to each other on chromosome 9p21-22, codeletion of the MTAP and p16(Ink4A) genes have been reported in non-small cell lung cancer (NSCLC). The aim of this study is to determine the respective prognostic value of MTAP and p16 by considering their correlation in NSCLC patients. We analyzed MTAP and p16 protein expression by immunohistochemical staining on 99 NSCLC tissue microarray samples. The association between MTAP and p16 expression levels and prognosis were analyzed using the Kaplan-Meier method and Cox proportional hazards model for prognosis. Patients with a low MTAP expression level had poor overall survival (P = 0.010) and disease-free survival (P = 0.002). Low p16 expression indicated a trend toward poor overall survival (P = 0.138) and disease-free survival (P = 0.199). There was a significant positive correlation between MTAP and p16 expression levels (Spearman's ρ = 0.402, P < 0.001). By multivariate analyses, the MTAP expression level retained its independent prognostic power and p16 expression loss of the correlation with prognosis. Concordant loss of MTAP and p16 expression was observed in 24 out of 99 patients (24.2%). Patients with concordant loss of MTAP and p16 expression had the worst prognosis compared to patients with high expression of both markers. MTAP expression is an independent prognostic factor and has greater prognostic significance than p16 expression in NSCLC. Concordant loss of MTAP and p16 expression indicates poor outcomes in lung cancer patients. Copyright © 2014 Elsevier Ltd. All rights reserved.

  15. Frequency and pattern of expression of the stem cell marker CD133 have strong prognostic effect on the surgical outcome of colorectal cancer patients.

    PubMed

    Takahashi, Shusaku; Kamiyama, Toshiya; Tomaru, Utano; Ishizu, Akihiro; Shida, Toshiyuki; Osaka, Mineji; Sato, Yutaka; Saji, Yutaka; Ozaki, Michitaka; Todo, Satoru

    2010-11-01

    CD133 has been reported to be a cancer stem cell marker in colorectal cancer (CRC). The aim of this study was to examine the frequency and pattern of CD133 expression by immunohistochemical methods and evaluate their correlation with clinicopathological features, including patient survival (PS) and recurrence. Tissue specimens of 151 CRC patients who underwent surgical treatment for well-differentiated/moderately differentiated adenocarcinoma and stage I-IV tumors (TNM classification) were immunostained for analyzing CD133 expression. The frequency of CD133 expression was 91.4% (138/151), and the pattern of expression was divided into membranous and cytoplasmic expression. Of the 151 patients, 136 (90.1%) showed membranous expression, whereas 44 (29.1%) showed cytoplasmic expression. Both expression patterns were seen in 42 (27.8%) patients. The frequency of CD133 overexpression (>50% of stained cells) was 27.2% (41/151); univariate analysis showed CD133 overexpression to be significantly associated with PS, but not recurrence, and multivariate analysis indicated it to be an independent prognostic factor. Multivariate analysis showed membranous overexpression (>50% of stained tumor cells on the membrane), which significantly correlated with histology and chemoresistance of recurrent and stage IV tumors, to be an independent prognostic factor for PS and recurrence. However, multivariate analysis did not indicate cytoplasmic expression, which significantly correlated with histology, lymph node metastasis, TNM stage and lymphatic invasion, as an independent prognostic factor for PS and recurrence. Our results demonstrated that evaluation of the frequency and pattern of CD133 expression is useful for predicting prognosis, recurrence, and chemosensitivity in CRC patients.

  16. The Fc Receptor Polymorphisms and Expression of Neutrophil Activation Markers in Patients with Sickle Cell Disease from Western India

    PubMed Central

    Kangne, Harshada K.; Jijina, Farah F.; Italia, Yazdi M.; Jain, Dipti L.; Nadkarni, Anita H.; Gupta, Maya; Pradhan, Vandana; Mukesh, Rati D.; Ghosh, Kanjaksha K.; Colah, Roshan B.

    2013-01-01

    Objective. Sickle cell disease has variable clinical manifestations. Activation of neutrophils plays an important role in the initiation and propagation of vaso occlusive crises which can be analysed by determining the expression of neutrophil antigens such as CD16, CD32, and CD62L. The common FcγR polymorphisms (FcγRIIA and FcγRIIIB) are considered to influence clinical presentation. This study focuses on distribution of FcγR polymorphisms and their association with neutrophil activity among the patients from western India. Methods. In this paper 127 sickle cell anemia patients and 58 patients with sickle-β-thalassemia (median age 12 ± 8.58 years) with variable clinical phenotypes along with 175 normals were investigated. FcγRs polymorphisms were analysed by RFLP and AS-PCR. Activation of neutrophils was measured by flow cytometry. Results. The genotypic frequency of the H/R genotype of FcγRIIA and the NA1/NA1 genotype of FcγRIIIB was significantly decreased in patients compared to normals (P-0.0074, P-0.0471, resp.). We found a significant difference in the expression of CD32 and CD62L among the patients as against normals. A significantly higher expression of CD32 was seen in the milder patients with the H/H genotype (P-0.0231), whereas the expression of CD16 was higher in severe patients with the NA2/NA2 genotype (P-0.0312). Conclusion. The two FcγR polymorphisms had significant association with variable phenotypes of sickle cell disease. The expression of CD62L decreased in our patients indicating activation of neutrophils. PMID:24191245

  17. Somatostatin receptor expression in small cell lung cancer as a prognostic marker and a target for peptide receptor radionuclide therapy

    PubMed Central

    Lapa, Constantin; Hänscheid, Heribert; Wild, Vanessa; Pelzer, Theo; Schirbel, Andreas; Werner, Rudolf A.; Droll, Sabine; Herrmann, Ken; Buck, Andreas K.; Lückerath, Katharina

    2016-01-01

    Despite initial responsiveness to both chemotherapy and radiotherapy, small cell lung cancer (SCLC) commonly relapses within months. Although neuroendocrine characteristics may be difficult to demonstrate in individual cases, a relevant expression of somatostatin receptors (SSTR) on the cell surface has been described. We aimed to evaluate the prognostic value of SSTR-expression in advanced SCLC. We further examined pre-requisites for successful peptide receptor radionuclide therapy (PRRT). 21 patients with extensive stage SCLC were enrolled. All patients underwent positron emission tomography/computed tomography (PET/CT) with 68Ga-DOTATATE to select patients for SSTR-directed therapy. PET scans were visually and semi-quantitatively assessed and compared to SSTR2a and SSTR5 expression in biopsy samples. Peak standardized uptake values (SUVpeak) of tumors as well as tumor-to-liver ratios were correlated to progression-free (PFS) and overall survival (OS). In 4/21 patients all SCLC lesions were PET-positive. 6/21 subjects were rated “intermediate” with the majority of lesions positive, the remaining 11/21 patients were PET-negative. PET-positivity correlated well with histologic SSTR2a, but not with SSTR5 expression. Neither PET-positivity nor SUVpeak were predictors of PFS or OS. In 4 patients with intensive SSTR2a-receptor expression, PRRT was performed with one partial response and one stable disease, respectively. SSTR-expression as detected by 68Ga-DOTATATE-PET and/or histology is not predictive of PFS or OS in patients with advanced SCLC. However, in patients exhibiting sufficient tracer uptake, PRRT might be a treatment option given its low toxicity and the absence of effective alternatives. PMID:26936994

  18. The expressions of stem cell markers: Oct4, Nanog, Sox2, nucleostemin, Bmi, Zfx, Tcl1, Tbx3, Dppa4, and Esrrb in bladder, colon, and prostate cancer, and certain cancer cell lines.

    PubMed

    Amini, Sabrieh; Fathi, Fardin; Mobalegi, Jafar; Sofimajidpour, Heshmatollah; Ghadimi, Tayyeb

    2014-03-01

    Uncontrolled self-renewal plays a direct function in the progression of different types of carcinomas. The same molecular pathway that manages self-renewal in normal stem cells also seems to manage cancer stem cells. Here, we examine the expressions of self-renewal regulatory factors Oct4, Nanog, Sox2, nucleostemin, Zfx, Esrrb, Tcl1, Tbx3, and Dppa4 in tissue samples of colon, prostate, and bladder carcinomas as well as cancer cell lines HT-29, Caco-2, HT-1376, LNCaP, and HepG2. We used reverse transcriptase polymerase chain reaction to examine expressions of the above mentioned regulatory factors in cancer cell lines HT-29, Caco-2, HT-1376, LNCaP, and HepG2 and in 20 tumor tissue samples. Total RNA was isolated by the ISOGEN method. RNA integrity was checked by agarose gel electrophoresis and spectrophotometry. Expressions of Oct4 and nucleostemin at the protein level were determined by immunocytochemistry. A significant relationship was found between tumor grade and self-renewal gene expression. Expressions of stem cell specific marker genes were detected in all examined cancer cell lines, in 40% to 100% of bladder cancer samples, and in 60% to 100% of colon and prostate cancer samples. Oct4 expressed in 100% of tumor tissue samples. Our data show that stem cell markers Oct4, Nanog, Sox2, nucleostemin, Bmi, Zfx, Esrrb, Tcl1, Tbx3, and Dppa4 significantly express in cancer cell lines and cancer tissues. Hence, these markers might be useful as potential tumor markers in the diagnosis and/or prognosis of tumors.

  19. Association between Duffy antigen receptor for chemokines expression and levels of inflammation markers in sickle cell anemia patients.

    PubMed

    Nebor, Danitza; Durpes, Marie Claude; Mougenel, Danielle; Mukisi-Mukaza, Martin; Elion, Jacques; Hardy-Dessources, Marie-Dominique; Romana, Marc

    2010-07-01

    Since inflammation plays a prominent role in the pathogenesis of sickle cell anemia (SCA) and Duffy antigen receptor for chemokines (DARC) modulates the function of inflammatory processes, we analyzed the relationship between the erythrocyte DARC phenotype and clinical expression of SCA. DARC locus was genotyped in 212 SS adult patients followed by the sickle cell center of Guadeloupe (French West Indies). After patients' stratification according to RBC DARC expression, the prevalence of renal disease, leg ulcers, priapism and osteonecrosis was compared between patient groups as well as hematological variables and plasma levels of chemokines. Duffy-positive patients exhibited higher counts of white blood cells (9.95+/-2.36 vs 8.88+/-2.32 10(9)/L, p=0.0066), polynuclear neutrophils (5.1+/-1.73 vs 4.51+/-1.71 10(9)/L, p=0.0227), higher plasma levels of IL-8 (4.46+/-1.22 vs 1.47+/-0.5 pg/mL, p=0.0202) and RANTES (27.8+/-4.3 vs 18.1+/-2.3 ng/mL, p=0.04) than Duffy-negative patients. No association was detected between RBC expression of DARC and the studied complications.

  20. A Novel Marker for Purkinje Cells, Ribosomal Protein MPS1/S27: Expression of MPS1 in Human Cerebellum.

    PubMed

    Fernandez-Pol, J Alberto

    2016-01-01

    The ribosomal protein metallopanstimulin-1 (MPS1/S27) serves critical survival purposes in cell division, in normal and cancerous cells; for this reason, selective pressures of evolution have conserved the DNA sequences encoding MPS1/S27 in Archaea and Eukariotic cells. The expression of MPS1/S27 protein in human adult cerebellum has not been established. The presence of MPS1/S27, was screened in paraffin-embedded human adult brain specimens processed for tissue inmunohistochemistry. Affinity-purified specific antibodies were directed against the N-terminus of MPS1. The antibodies to MPS1 detected Purkinje cells (PC) and their dendrites. In PC, MPS1 antigen-positive staining was found in: the nucleolus, which was strongly stained; ribosomes attached to the external nuclear membrane; cytoplasm of PC, with strong staining in a punctuate fashion; the soma-attached large dendrite trunks of PC, which were MPS1 antigen-positive; and the granular cell layer, where cellular staining in a few cells that appeared to resemble smaller PC was observed. Since MPS1 is involved in cell division, DNA repair, and ribosomal biogenesis, it may be a useful antigen for studying processes such as protein synthesis, oncogenesis, regeneration, aging, and perhaps diseases of the human cerebellum. Copyright© 2016, International Institute of Anticancer Research (Dr. John G. Delinasios), All rights reserved.

  1. Aberrant expression of the dendritic cell marker TNFAIP2 by the malignant cells of Hodgkin lymphoma and primary mediastinal large B-cell lymphoma distinguishes these tumor types from morphologically and phenotypically similar lymphomas.

    PubMed

    Kondratiev, Svetlana; Duraisamy, Sekhar; Unitt, Christine L; Green, Michael R; Pinkus, Geraldine S; Shipp, Margaret A; Kutok, Jeffery L; Drapkin, Ronny I; Rodig, Scott J

    2011-10-01

    Tumor necrosis factor-α-inducible protein-2 (TNFAIP2) is a protein upregulated in cultured cells treated with tumor necrosis factor α (TNF), but its expression in normal and neoplastic tissues remains largely unknown. Here, we use standard immunohistochemical techniques to demonstrate that TNFAIP2 is normally expressed by follicular dendritic cells, interdigitating dendritic cells, and macrophages but not by lymphoid cells in secondary lymphoid tissues. Consistent with this expression pattern, we found strong TNFAIP2 staining of tumor cells in 4 of 4 cases (100%) of follicular dendritic cell sarcoma and in 3 of 3 cases (100%) of histiocytic sarcoma. Although TNFAIP2 is not expressed by the small and intermediate-sized neoplastic B cells comprising follicular lymphoma, small lymphocytic lymphoma, mantle cell lymphoma, or marginal zone lymphoma, we observed strong TNFAIP2 staining of the large, neoplastic cells in 31 of 31 cases (100%) of classical Hodgkin lymphoma, in 12 of 12 cases (100%) of nodular lymphocyte-predominant Hodgkin lymphoma, and in 27 of 31 cases (87%) of primary mediastinal (thymic) large B-cell lymphoma. In contrast, TNFAIP2 was expressed by malignant cells in only 2 of 45 cases (4%) of diffuse large B-cell lymphoma, not otherwise specified, in 2 of 18 cases (11%) of Burkitt lymphoma, and in 1 of 19 cases (5%) of anaplastic large cell lymphoma. Further analysis indicates that TNFAIP2, as a single diagnostic marker, is more sensitive (sensitivity=87%) and specific (specificity=96%) than TRAF1, nuclear cRel, or CD23 for distinguishing the malignant B cells of primary mediastinal (thymic) large B-cell lymphoma from those of its morphologic and immunophenotypic mimic, diffuse large B-cell lymphoma, not otherwise specified. Thus, TNFAIP2 may serve as a useful new marker of dendritic and histiocytic sarcomas, the aberrant expression of which in the malignant cells of classical Hodgkin lymphoma and primary mediastinal (thymic) large B-cell lymphoma

  2. Induction of dental epithelial cell differentiation marker gene expression in non-odontogenic human keratinocytes by transfection with thymosin beta 4.

    PubMed

    Kiyoshima, Tamotsu; Fujiwara, Hiroaki; Nagata, Kengo; Wada, Hiroko; Ookuma, Yukiko F; Shiotsuka, Maho; Kihara, Makiko; Hasegawa, Kana; Someya, Hirotaka; Sakai, Hidetaka

    2014-01-01

    Previous studies have shown that the recombination of cells liberated from developing tooth germs develop into teeth. However, it is difficult to use human developing tooth germ as a source of cells because of ethical issues. Previous studies have reported that thymosin beta 4 (Tmsb4x) is closely related to the initiation and development of the tooth germ. We herein attempted to establish odontogenic epithelial cells from non-odontogenic HaCaT cells by transfection with TMSB4X. TMSB4X-transfected cells formed nodules that were positive for Alizarin-red S (ALZ) and von Kossa staining (calcium phosphate deposits) when cultured in calcification-inducing medium. Three selected clones showing larger amounts of calcium deposits than the other clones, expressed PITX2, Cytokeratin 14, and Sonic Hedgehog. The upregulation of odontogenesis-related genes, such as runt-related transcription factor 2 (RUNX2), Amelogenin (AMELX), Ameloblastin (AMBN) and Enamelin (ENAM) was also detected. These proteins were immunohistochemically observed in nodules positive for the ALZ and von Kossa staining. RUNX2-positive selected TMSB4X-transfected cells implanted into the dorsal subcutaneous tissue of nude mice formed matrix deposits. Immunohistochemically, AMELX, AMBN and ENAM were observed in the matrix deposits. This study demonstrated the possibility of induction of dental epithelial cell differentiation marker gene expression in non-odontogenic HaCaT cells by TMSB4X. Copyright © 2013. Published by Elsevier B.V.

  3. Stem Cell Derived Retinal Pigment Epithelium: The Role of Pigmentation as Maturation Marker and Gene Expression Profile Comparison with Human Endogenous Retinal Pigment Epithelium.

    PubMed

    Bennis, A; Jacobs, J G; Catsburg, L A E; Ten Brink, J B; Koster, C; Schlingemann, R O; van Meurs, J; Gorgels, T G M F; Moerland, P D; Heine, V M; Bergen, A A

    2017-07-21

    In age-related macular degeneration (AMD) the retinal pigment epithelium (RPE) deteriorates, leading to photoreceptor decay and severe vision loss. New therapeutic strategies aim at RPE replacement by transplantation of pluripotent stem cell (PSC)-derived RPE. Several protocols to generate RPE have been developed where appearance of pigmentation is commonly used as indicator of RPE differentiation and maturation. It is, however, unclear how different pigmentation stages reflect developmental stages and functionality of PSC-derived RPE cells. We generated human embryonic stem cell-derived RPE (hESC-RPE) cells and investigated their gene expression profiles at early pigmentation (EP) and late pigmentation (LP) stages. In addition, we compared the hESC-RPE samples with human endogenous RPE. We used a common reference design microarray (44 K). Our analysis showed that maturing hESC-RPE, upon acquiring pigmentation, expresses markers specific for human RPE. Interestingly, our analysis revealed that EP and LP hESC-RPE do not differ much in gene expression. Our data further showed that pigmented hESC-RPE has a significant lower expression than human endogenous RPE in the visual cycle and oxidative stress pathways. In contrast, we observed a significantly higher expression of pathways related to the process adhesion-to-polarity model that is typical of developing epithelial cells. We conclude that, in vitro, the first appearance of pigmentation hallmarks differentiated RPE. However, further increase in pigmentation does not result in much significant gene expression changes and does not add important RPE functionalities. Consequently, our results suggest that the time span for obtaining differentiated hESC-RPE cells, that are suitable for transplantation, may be greatly reduced.

  4. Upregulation of osteoblastic differentiation marker mRNA expression in osteoblast-like UMR106 cells by puerarin and phytoestrogens from Pueraria mirifica.

    PubMed

    Tiyasatkulkovit, Wacharaporn; Charoenphandhu, Narattaphol; Wongdee, Kannikar; Thongbunchoo, Jirawan; Krishnamra, Nateetip; Malaivijitnond, Suchinda

    2012-10-15

    Phytoestrogens have attracted attention for their potential in the prevention of postmenopausal osteoporosis. Recently, phytoestrogen-rich herb Pueraria mirifica has been demonstrated to possess an osteogenic effect on bone in ovariectomized rats, but its underlying cellular mechanism was not known. Here, we investigated the effects of P. mirifica extract and its major isoflavone compound, puerarin, on cell viability, cell proliferation and the expression of differentiation markers in rat osteoblast-like UMR106 cells. After exposure to 17β-estradiol (E2), genistein, P. mirifica extract and puerarin, proliferation but not viability of UMR106 cells was markedly decreased. Quantitative real-time PCR revealed that P. mirifica extract and puerarin significantly increased the mRNA expression of alkaline phosphatase (ALP) and osteoprotegerin, but not Runx2, osterix or osteocalcin. Puerarin also decreased the mRNA expression of receptor activator of nuclear factor-κB ligand, an osteoclastogenic factor, suggesting that it could induce bone gain by enhancing osteoblast differentiation and suppressing osteoclast function. Furthermore, after an exposure to high affinity estrogen receptor (ER) antagonist (ICI182780), the E2-, genistein-, P. mirifica extract- and puerarin-induced upregulation of ALP expressions were completely abolished. It could be concluded that P. mirifica extract and puerarin induced osteoblast differentiation rather than osteoblast proliferation in an ER-dependent manner. The present findings, therefore, corroborated the potential benefit of P. mirifica extract and puerarin in the prevention and treatment of postmenopausal osteoporosis. Copyright © 2012 Elsevier GmbH. All rights reserved.

  5. The CD markers of camel (Camelus dromedarius) milk cells during mastitis: the LPAM-1 expression is an indication of possible mucosal nature of the cellular trafficking.

    PubMed

    Al-Ashqar, Roqaya A; Al-Mohammad Salem, Khadim M; Al Herz, Abdul Kareem M; Al-Haroon, Amal I; Alluwaimi, Ahmed M

    2015-04-01

    Studying the cellular populations of the camel mammary glands through the expression pattern of the CD markers and adhesion molecules is a mean to define whether the cellular trafficking pathway is peripheral or mucosal nature. Camel milk cells from 8 Gram-positive and 5 Gram-negative infected camels were examined with flow cytometry using cross-reacting antibodies like, anti-CD4(+), CD8(+), WC+1(+)γδ, CD62L, CD11a(+)/CD18, LPAM-1, CXCR2. The overall results indicated high flow cytometry output of most of the CD makers. The statistical analysis of the mean percentage of the expressed CD markers has shown that CD62L, CXCR-2, LPAM-1, CD11a/CD18, CD8(+), IL-6R and CD20(+) were expressed in significant differences in either type of the infection. The LPAM-1 expression has provided further support to the notion that the lymphocyte trafficking is of the mucosal nature. The mucosal origin of cellular trafficking has important implications on the vaccine design and therapeutical approaches to mastitis. Copyright © 2015 Elsevier Ltd. All rights reserved.

  6. Gene amplification and immunohistochemical expression of ERBB2 and EGFR in cervical carcinogenesis. Correlation with cell-cycle markers and HPV presence.

    PubMed

    Conesa-Zamora, Pablo; Torres-Moreno, Daniel; Isaac, María A; Pérez-Guillermo, Miguel

    2013-10-01

    Although the members of the epidermal growth factor receptor family ERBB2 and EGFR are important therapeutic targets in the treatment of malignant neoplasias, little is known about their role in cervical carcinogenesis. Our objective was to evaluate the dysfunction of ERBB2 and EGFR at the gene copy number and protein expression level in neoplastic lesions of the uterine cervix with the aim of obtaining information about its role in cervical carcinogenesis and their possible use as therapeutic targets in these diseases. We studied gene amplification and protein expression of ERBB2 and EGFR and their relationship with Ki67, p16 and p53 and HPV presence in 22 normal/benign (N/B) cervices, 20 low-grade squamous intraepithelial lesions (LSILs), 70 high-grade SILs (HSILs) and 32 invasive squamous cervical carcinomas (ISCCs). No cases showed selective amplification of ERBB2 or EGFR but corresponding chromosome-specific probes displayed chromosome 17 and 7 polyploidy associated with the grade of the lesion (p<0.0001 and p=0.004, respectively) and with the positive expression of Ki67 and p16 (p<0.01). Concurrent polyploidy for both chromosomes was statistically related (p<0.0001). ERBB2 immunohistochemical expression was not observed in any of the study cases except for one ISCC but EGFR was associated with higher-grade lesions (N/B plus LSIL 21.4% vs. HSIL plus ISCC 45.5%; p=0.007). No association was observed between EGFR expression and that of cell-cycle markers or HPV presence. Increased copy number of EGFR and ERBB2 is due to polyploidy of 7 and 17 chromosomes, this being a phenomenon associated with lesion severity and with an increase in the expression of cell-cycle markers. EGFR, but not ERBB2, is expressed in precursor lesions of squamous cervical neoplasia and is related to the neoplastic progression but not to proliferation marker expression and therefore ERBB2 and this calls into question the usefulness of ERBB2 as a therapeutic target.

  7. Peptide Agonists of Vasopressin V2 Receptor Reduce Expression of Neuroendocrine Markers and Tumor Growth in Human Lung and Prostate Tumor Cells

    PubMed Central

    Pifano, Marina; Garona, Juan; Capobianco, Carla S.; Gonzalez, Nazareno; Alonso, Daniel F.; Ripoll, Giselle V.

    2017-01-01

    Neuroendocrine tumors (NETs) comprise a heterogeneous group of malignancies that express neuropeptides as synaptophysin, chromogranin A (CgA), and specific neuronal enolase (NSE), among others. Vasopressin (AVP) is a neuropeptide with an endocrine, paracrine, and autocrine effect in normal and pathological tissues. AVP receptors are present in human lung, breast, pancreatic, colorectal, and gastrointestinal tumors. While AVP V1 receptors are associated with stimulation of cellular proliferation, AVP V2 receptor (V2r) is related to antiproliferative effects. Desmopressin (dDAVP) is a synthetic analog of AVP that acts as a selective agonist for the V2r, which shows antitumor properties in breast and colorectal cancer models. Recently, we developed a derivative of dDAVP named [V4Q5]dDAVP, which presents higher antitumor effects in a breast cancer model compared to the parental compound. The goal of present work was to explore the antitumor properties of the V2r agonist dDAVP and its novel analog [V4Q5]dDAVP on aggressive human lung (NCI-H82) and prostate cancer (PC-3) cell lines with neuroendocrine (NE) characteristics. We study the presence of specific NE markers (CgA and NSE) and V2r expression in NCI-H82 and PC-3. Both cell lines express high levels of NE markers NSE and CgA but then incubation with dDAVP diminished expression levels of both markers. DDAVP and [V4Q5]dDAVP significantly reduced proliferation, doubling time, and migration in both tumor cell cultures. [V4Q5]dDAVP analog showed a higher cytostatic effect than dDAVP, on cellular proliferation in the NCI-H82 cell line. Silencing of V2r using small interfering RNA significantly attenuated the inhibitory effects of [V4Q5]dDAVP on NCI-H82 cell proliferation. We, preliminarily, explored the in vivo effect of dDAVP and [V4Q5]dDAVP on NCI-H82 small cell lung cancer xenografts. Treated tumors (0.3 μg kg−1, thrice a week) grew slower in comparison to vehicle-treated animals. In this work, we demonstrated

  8. The heterogeneity of spermatogonia is revealed by their topology and expression of marker proteins including the germ cell-specific proteins Nanos2 and Nanos3.

    PubMed

    Suzuki, Hitomi; Sada, Aiko; Yoshida, Shosei; Saga, Yumiko

    2009-12-15

    Spermatogonial stem cells (SSCs) reside in undifferentiated type-A spermatogonia and contribute to continuous spermatogenesis by maintaining the balance between self-renewal and differentiation, thereby meeting the biological demand in the testis. Spermatogonia have to date been characterized principally through their morphology, but we herein report the detailed characterization of undifferentiated spermatogonia in mouse testes based on their gene expression profiles in combination with topological features. The detection of the germ cell-specific proteins Nanos2 and Nanos3 as markers of spermatogonia has enabled the clear dissection of complex populations of these cells as Nanos2 was recently shown to be involved in the maintenance of stem cells. Nanos2 is found to be almost exclusively expressed in A(s) to A(pr) cells, whereas Nanos3 is detectable in most undifferentiated spermatogonia (A(s) to A(al)) and differentiating A(1) spermatogonia. In our present study, we find that A(s) and A(pr) can be basically classified into three categories: (1) GFRalpha1(+)Nanos2(+)Nanos3(-)Ngn3(-), (2) GFRalpha1(+)Nanos2(+)Nanos3(+)Ngn3(-), and (3) GFRalpha1(-)Nanos2(+/-)Nanos3(+)Ngn3(+). We propose that the first of these groups is most likely to include the stem cell population and that Nanos3 may function in transit amplifying cells.

  9. Influence of suramin on the expression of Fc receptors and other markers on human monocytes and U937 cells, and on their phagocytic properties.

    PubMed Central

    Schiller, C; Spittler, A; Willheim, M; Szépfalusi, Z; Agis, H; Köller, M; Peterlik, M; Boltz-Nitulescu, G

    1994-01-01

    Suramin, a polyanionic and polycyclic compound, was initially used for the treatment of trypanosomiasis and onchocerciasis. In the last decade, it has been used in therapy of cancer and acquired immune deficiency syndrome (AIDS). The influence of suramin on the expression of various markers by human mononuclear phagocytes is not known and was, therefore, presently investigated. Suramin inhibited the proliferation of U937 cells and mitogen-induced T-cell proliferation in a dose-dependent manner. The constitutive and cytokine-driven expression of Fc receptors for IgG (Fc gamma RI and Fc gamma RII), IgE (Fc epsilon RII) and IgA (Fc alpha R) on blood monocytes and U937 cells was suppressed by suramin. The basal level, as well as cytokine-induced major histocompatibility complex (MHC) class II antigens, was markedly diminished on suramin-treated monocytes. Furthermore, suramin dramatically reduced expression of CD14 and partially reduced complement receptor type 3 (CR3) and CR4 expression on monocytes. In contrast, suramin slightly induced MHC class I antigens on monocytes and CD71 on U937 cells. The capacity of monocytes to phagocytose IgG-sensitized ox erythrocytes, opsonized Escherichia coli, or fluorescein isothiocyanate (FITC)-conjugated latex beads was significantly inhibited. Northern blot analysis showed that the amount of Fc epsilon RII-specific mRNA was only partially reduced, suggesting that other mechanisms may be involved in the regulation of Fc epsilon RII expression. Our data demonstrate that suramin suppresses the expression of various cell-surface structures on human mononuclear phagocytes and impairs their phagocytic capacity. Images Figure 2 PMID:8039810

  10. Expression of Leucine-rich Repeat-containing G-protein Coupled Receptor 5 and CD44: Potential Implications for Gastric Cancer Stem Cell Marker

    PubMed Central

    Choi, Yoon Jin; Kim, Nayoung; Lee, Hye Seung; Park, Seon Mee; Park, Ji Hyun; Yoon, Hyuk; Shin, Cheol Min; Park, Young Soo; Kim, Jin-Wook; Lee, Dong Ho

    2016-01-01

    Background The human leucine-rich repeat-containing G-protein coupled receptor (LGR) 5 and CD44 are one of the candidates for the marker of gastric cancer stem cells. We compared the expressions of two genes among control, dysplasia and cancer groups. Methods We compared the mRNA expression of LGR5, CD44 and CD44v8–10 and immunohistochemistry (IHC) of LGR5 and CD44 in gastric antral mucosa of 45 controls, 36 patients with gastric dysplasia, and 39 patients with early gastric cancer. Additionally, IHC of LGR5 in gastric body mucosa was analyzed. Normal mucosa adjacent to dysplastic or cancer lesions was used for the quantitative real-time–PCR and IHC. Results Immunoreactivity of LGR5 in base of antral mucosa was higher in non-cancerous tissues of cancer than those of control (P = 0.006), whereas the expression of LGR5 mRNA was not different among the three groups. Immunostaining of LGR5 was much stronger in the antrum than in the body of stomach (P < 0.001). Although there was no difference in antral immunointensity of LGR5 according to the severity of intestinal metaplasia, stronger immunostaining was found in the body with an aggravation of intestinal metaplasia (P trend < 0.001). The expression of CD44v8–10 mRNA was higher in cancer patients than control subjects and patients with dysplasia (P = 0.018 and 0.009) while the expression of CD44 mRNA was higher in the control groups than the others. Conclusions IHC of LGR5 in crypt base and CD44 may be used for gastric CSC markers. LGR5 expression may be associated with the developing of corporal intestinal metaplasia. The expression of CD44v8–10 mRNA would be more suitable for gastric cancer stem cell marker than CD44 or LGR5 mRNA. PMID:28053963

  11. 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.

  12. Gene expression analysis of Arc mRNA as a neuronal cell activity marker in the hippocampus and amygdala in two-way active avoidance test in rats.

    PubMed

    Yasuno, Kyohei; Takahashi, Erika; Igarashi, Isao; Iguchi, Takuma; Tsuchiya, Yoshimi; Kai, Kiyonori; Mori, Kazuhiko

    2017-09-27

    Immediate early genes are widely used as neuronal cell activity markers in neuroscience. The present study investigated the relationship between their expression and abnormality in context fear conditioning. The learning test (two-way active avoidance test) was conducted in male rats administered with nonselective muscarinic antagonist scopolamine or selective dopamine D1-like receptor antagonist SCH 23390 at a dose level of 2.0 or 0.1mg/kg, respectively, for 4days. Expression levels of Arc and Fos mRNA in the hippocampus and amygdala were also evaluated on the second day of dosing by fluorescent in situ hybridization (FISH) and reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR). Scopolamine had no effect on avoidance rate, but decreased freezing in the two-way active avoidance test. SCH 23390 decreased avoidance rate and increased freezing. In FISH and RT-qPCR assays, scopolamine decreased Arc mRNA in the hippocampus and amygdala, whereas SCH 23390 increased Arc mRNA in the hippocampus. By contrast, scopolamine and SCH 23390 did not change Fos mRNA expression compared to Arc mRNA expression. The results of the learning test indicated that scopolamine or SCH 23390 respectively inhibited fear or context conditioning in rats. Furthermore, alteration of the expression of Arc mRNA but not of Fos mRNA in the hippocampus and amygdala of the brain was suggested to be a sensitive neuronal cell activity marker to detect behavioral abnormality in the two-way active avoidance test. Copyright © 2017. Published by Elsevier Inc.

  13. Transcriptional expression levels of cell stress marker genes in the Pacific oyster Crassostrea gigas exposed to acute thermal stress

    PubMed Central

    Farcy, Émilie; Voiseux, Claire; Lebel, Jean-Marc

    2008-01-01

    During the annual cycle, oysters are exposed to seasonal slow changes in temperature, but during emersion at low tide on sunny summer days, their internal temperature may rise rapidly, resulting in acute heat stress. We experimentally exposed oysters to a 1-h acute thermal stress and investigated the transcriptional expression level of some genes involved in cell stress defence mechanisms, including chaperone proteins (heat shock proteins Hsp70, Hsp72 and Hsp90 (HSP)), regulation of oxidative stress (Cu-Zn superoxide dismutase, metallothionein (MT)), cell detoxification (glutathione S-transferase sigma, cytochrome P450 and multidrug resistance (MDR1)) and regulation of the cell cycle (p53). Gene mRNA levels were quantified by reverse transcription-quantitative polymerase chain reaction and expressed as their ratio to actin mRNA, used as a reference. Of the nine genes studied, HSP, MT and MDR1 mRNA levels increased in response to thermal stress. We compared the responses of oysters exposed to acute heat shock in summer and winter and observed differences in terms of magnitude and kinetics. A larger increase was observed in September, with recovery within 48 h, whereas in March, the increase was smaller and lasted more than 2 days. The results were also compared with data obtained from the natural environment. Though the functional molecule is the protein and information at the mRNA level only has limitations, the potential use of mRNAs coding for cell stress defence proteins as early sensitive biomarkers is discussed. PMID:19002605

  14. [Expression Level of Membrane-associated Proteins Numb in Epithelial Ovarian Carcinoma and Its Relationship with Ovarian Cancer Stem Cell Markers CD117, CD133, ALDH1.

    PubMed

    Jing, Hong; Liu, Xiao-Yu; Chen, Ya-Li; Bai, Li-Ping; Zheng, Ai

    2016-11-01

    To explore the expression level of membrane-associated protein Numb in epithelial ovarian carcinoma and its relationship with ovarian cancer stem cell markers CD117,CD133,acetaldehyde dehydrogenase 1(ALDH1). A total of 136 patients who had ovarian tumors and 22 patients who had not ovarian tumors in Department of Gynaecology and Obstetrics,West China Second University Hospital,Sichuan University were chosen as the study subjects.According to the histopathologic examination results,they were divided into epithelial ovarian carcinoma group (n=92),ovarian borderline tumor group (n=23),ovarian benign tumor group (n=21) and normal ovary group (n=22).Expression levels of Numb protein,CD117,CD133 and ALDH1 in ovarian tissue were detected by immunohistochemical SP method and these several kinds of protein expression differences and correlation statistical analysis were performend. 1 The positive expression rate of Numb protein in epithelial ovarian carcinoma group was higher than that in benign tumor or normal ovary group,also Numb protein positive expression rate in ovarian borderline tumor group was higher than that in normal ovary group,and the differences were statistically significant (P<0.05).2 Numb protein positive expression rate in ovarian tissue in patients with epithelial ovarian carcinoma FIGO stage1-2 was lower than that in stage 3-4,also the same in no lymph nodes metastasis compared with lymph nodes invasion,and the differences of positive expression rate were statistically significant (P<0.05).While there were no significant differences among different age,histopathological types,pathological grades and residual tumor size (P>0.05).3 There was no correlation between Numb protein and CD117 and CD133 positive expression rate in epithelial ovarian carcinoma tissue [correlation coefficient (r)=0.116,P=0.261; r=0.083,P=0.425].However,the positive expression rate of Numb protein and ALDH1 was positively correlated (r=0.296,P=0.261). The expression of Numb protein

  15. Expression of pluripotency markers in the bovine uterus with adenomyosis.

    PubMed

    Łupicka, Martyna; Socha, Barbara; Szczepańska, Agata; Korzekwa, Anna

    2015-09-29

    Adenomyosis is a proliferative uterine dysfunction with unknown aetiology. One possible mechanism of its development involves disturbances in stem cell differentiation in uterine tissue. Previously, we identified pluripotent/multipotent cells in the bovine uterus, therefore our present study focused on determining expression of pluripotency markers, NANOG, OCT4 and SOX2, in bovine adenomyotic tissues and cells. Immunolocalisation revealed protein expression of NANOG, OCT4 and SOX2 in both normal and adenomyotic uteri. mRNA expression for NANOG and OCT4 was increased in tissues obtained from uteri with adenomyosis compared to controls, but at the protein level there were no significant differences. mRNA expression for all three pluripotency markers was higher in myometrial cells isolated from uteri with adenomyotic lesions than in those isolated from normal uteri. The protein level of NANOG and SOX2 was decreased in stromal cells from adenomyotic tissues, whereas the level of OCT4 and SOX2 was increased in myometrial cells obtained from dysfunctional uteri. The results indicate significant changes in expression of pluripotency markers in adenomyotic compared to normal uteri, which suggest the involvement of uterine stem cells in adenomyosis.

  16. Maternal undernutrition does not alter Sertoli cell numbers or the expression of key developmental markers in the mid-gestation ovine fetal testis

    PubMed Central

    2013-01-01

    Background The aim of this study was to determine the effects of maternal undernutrition on ovine fetal testis morphology and expression of relevant histological indicators. Maternal undernutrition, in sheep, has been reported, previously, to alter fetal ovary development, as indicated by delayed folliculogenesis and the altered expression of ovarian apoptosis-regulating gene products, at day 110 of gestation. It is not known whether or not maternal undernutrition alters the same gene products in the day 110 fetal testis. Design and methods Mature Scottish Blackface ewes were fed either 100% (Control; C) or 50% (low; L) of estimated metabolisable energy requirements of a pregnant ewe, from mating to day 110 of gestation. All pregnant ewes were euthanized at day 110 and a sub-set of male fetuses was randomly selected (6 C and 9 L) for histology studies designed to address the effect of nutritional state on several indices of testis development. Sertoli cell numbers were measured using a stereological method and Ki67 (cell proliferation index), Bax (pro-apoptosis), Mcl-1 (anti-apoptosis), SCF and c-kit ligand (development and apoptosis) gene expression was measured in Bouins-fixed fetal testis using immunohistochemistry. Results No significant differences were observed in numbers of Sertoli cells or testicular Ki67 positive cells. The latter were localised to the testicular cords and interstitium. Bax and Mcl-1 were localised specifically to the germ cells whereas c-kit was localised to both the cords and interstitium. SCF staining was very sparse. No treatment effects were observed for any of the markers examined. Conclusions These data suggest that, unlike in the fetal ovary, maternal undernutrition for the first 110 days of gestation affects neither the morphology of the fetal testis nor the expression of gene products which regulate apoptosis. It is postulated that the effects of fetal undernutrition on testis function may be expressed through hypothalamic

  17. Moesin expression is a marker of basal breast carcinomas.

    PubMed

    Charafe-Jauffret, Emmanuelle; Monville, Florence; Bertucci, François; Esterni, Benjamin; Ginestier, Christophe; Finetti, Pascal; Cervera, Nathalie; Geneix, Jeannine; Hassanein, Mohamed; Rabayrol, Laetitia; Sobol, Hagay; Taranger-Charpin, Colette; Xerri, Luc; Viens, Patrice; Birnbaum, Daniel; Jacquemier, Jocelyne

    2007-10-15

    Basal breast cancers (BBCs) have a high risk of metastasis, recurrence and death. Formal subtype definition relies on gene expression but can be approximated by protein expression. New markers are needed to help in the management of the basal subtype of breast cancer. In a previous transcriptional analysis of breast cell lines we found that Moesin expression was a potential basal marker. We show here that Moesin protein expression is a basal marker in breast tumors. In a tissue microarray (TMA) containing 547 sporadic breast cancers, of which 108 were profiled for gene expression, Moesin was expressed in 31% of all tumors and in 82% of the basal tumors. To confirm that Moesin expression remained associated with the basal phenotype in specific types of BBCs, we analyzed Moesin expression in 2 other TMAs containing 40 medullary breast cancers (MBCs) and 27 BRCA1-associated breast cancers (BRCA1-BCs), respectively. Moesin was strongly expressed in MBCs (87%; p = 2.4 x 10(-5)) and in BRCA1-BCs (58%; p = 1.3 x 10(-5)) as compared with non-MBCs and sporadic cases. Moesin-expressing tumors display features of BBCs, such as high proliferation rate, hormone receptors negativity, expression of putative basal/myoepithelial markers (CAV1, CD10, CK5/6, CK14, EGFR, P53, P-cadherin and SMA). Survival analysis showed a reduced specific survival and metastasis-free survival in Moesin-expressing tumors by log-rank test (p(SS) = 0.014 and p(MFS) = 0.014). In multivariate analysis, Moesin expression was nearly an independent prognostic marker of poor outcome as shown by Cox proportional hazard model in patients without lymph node metastasis (p = 0.052, HR = 2.38, CI 95[0.99-5.69]).

  18. Connective tissue cells expressing fibro/adipogenic progenitor markers increase under chronic damage: relevance in fibroblast-myofibroblast differentiation and skeletal muscle fibrosis.

    PubMed

    Contreras, Osvaldo; Rebolledo, Daniela L; Oyarzún, Juan Esteban; Olguín, Hugo C; Brandan, Enrique

    2016-06-01

    Fibrosis occurs in skeletal muscle under various pathophysiological conditions such as Duchenne muscular dystrophy (DMD), a devastating disease characterized by fiber degeneration that results in progressive loss of muscle mass, weakness and increased extracellular matrix (ECM) accumulation. Fibrosis is also observed after skeletal muscle denervation and repeated cycles of damage followed by regeneration. The ECM is synthesized largely by fibroblasts in the muscle connective tissue under normal conditions. Myofibroblasts, cells that express α-smooth muscle actin (α-SMA), play a role in many tissues affected by fibrosis. In skeletal muscle, fibro/adipogenic progenitors (FAPs) that express cell-surface platelet-derived growth factor receptor-α (PDGFR-α) and the transcription factor Tcf4 seem to be responsible for connective tissue synthesis and are good candidates for the origin of myofibroblasts. We show that cells positive for Tcf4 and PDGFR-α are expressed in skeletal muscle under normal conditions and are increased in various skeletal muscles of mdx mice, a murine model for DMD, wild type muscle after sciatic denervation and muscle subjected to chronic damage. These cells co-label with the myofibroblast marker α-SMA in dystrophic muscle but not in normal tissue. The Tcf4-positive cells lie near macrophages mainly concentrated in dystrophic necrotic-regenerating foci. The close proximity of Tcf4-positive cells to inflammatory cells and their previously described role in muscle regeneration might reflect an active interaction between these cell types and growth factors, possibly resulting in a muscular regenerative or fibrotic condition.

  19. Effects of Silica and Titanium Oxide Particles on a Human Neural Stem Cell Line: Morphology, Mitochondrial Activity, and Gene Expression of Differentiation Markers

    PubMed Central

    Fujioka, Kouki; Hanada, Sanshiro; Inoue, Yuriko; Sato, Keisuke; Hirakuri, Kenji; Shiraishi, Kouichi; Kanaya, Fumihide; Ikeda, Keiichi; Usui, Ritsuko; Yamamoto, Kenji; Kim, Seung U.; Manome, Yoshinobu

    2014-01-01

    Several in vivo studies suggest that nanoparticles (smaller than 100 nm) have the ability to reach the brain tissue. Moreover, some nanoparticles can penetrate into the brains of murine fetuses through the placenta by intravenous administration to pregnant mice. However, it is not clear whether the penetrated nanoparticles affect neurogenesis or brain function. To evaluate its effects on neural stem cells, we assayed a human neural stem cell (hNSCs) line exposed in vitro to three types of silica particles (30 nm, 70 nm, and <44 μm) and two types of titanium oxide particles (80 nm and < 44 μm). Our results show that hNSCs aggregated and exhibited abnormal morphology when exposed to the particles at concentrations ≥ 0.1 mg/mL for 7 days. Moreover, all the particles affected the gene expression of Nestin (stem cell marker) and neurofilament heavy polypeptide (NF-H, neuron marker) at 0.1 mg/mL. In contrast, only 30-nm silica particles at 1.0 mg/mL significantly reduced mitochondrial activity. Notably, 30-nm silica particles exhibited acute membrane permeability at concentrations ≥62.5 μg/mL in 24 h. Although these concentrations are higher than the expected concentrations of nanoparticles in the brain from in vivo experiments in a short period, these thresholds may indicate the potential toxicity of accumulated particles for long-term usage or continuous exposure. PMID:24992594

  20. Effects of silica and titanium oxide particles on a human neural stem cell line: morphology, mitochondrial activity, and gene expression of differentiation markers.

    PubMed

    Fujioka, Kouki; Hanada, Sanshiro; Inoue, Yuriko; Sato, Keisuke; Hirakuri, Kenji; Shiraishi, Kouichi; Kanaya, Fumihide; Ikeda, Keiichi; Usui, Ritsuko; Yamamoto, Kenji; Kim, Seung U; Manome, Yoshinobu

    2014-07-02

    Several in vivo studies suggest that nanoparticles (smaller than 100 nm) have the ability to reach the brain tissue. Moreover, some nanoparticles can penetrate into the brains of murine fetuses through the placenta by intravenous administration to pregnant mice. However, it is not clear whether the penetrated nanoparticles affect neurogenesis or brain function. To evaluate its effects on neural stem cells, we assayed a human neural stem cell (hNSCs) line exposed in vitro to three types of silica particles (30 nm, 70 nm, and <44 µm) and two types of titanium oxide particles (80 nm and < 44 µm). Our results show that hNSCs aggregated and exhibited abnormal morphology when exposed to the particles at concentrations = 0.1 mg/mL for 7 days. Moreover, all the particles affected the gene expression of Nestin (stem cell marker) and neurofilament heavy polypeptide (NF-H, neuron marker) at 0.1 mg/mL. In contrast, only 30-nm silica particles at 1.0 mg/mL significantly reduced mitochondrial activity. Notably, 30-nm silica particles exhibited acute membrane permeability at concentrations =62.5 µg/mL in 24 h. Although these concentrations are higher than the expected concentrations of nanoparticles in the brain from in vivo experiments in a short period, these thresholds may indicate the potential toxicity of accumulated particles for long-term usage or continuous exposure.

  1. Effects of space flight on surface marker expression

    NASA Astrophysics Data System (ADS)

    Sonnenfeld, G.

    1999-01-01

    Space flight has been shown to affect expression of several cell surface markers. These markers play important roles in regulation of immune responses, including CD4 and CD8. The studies have involved flight of experimental animals and humans followed by analysis of tissue samples (blood in humans, rats and monkeys, spleen, thymus, lymph nodes and bone marrow in rodents). The degree and direction of the changes induced by space flight have been determined by the conditions of the flight. Also, there may be compartmentalization of the response of surface markers to space flight, with differences in the response of cells isolated from blood and local immune tissue. The same type of compartmentalization was also observed with cell adhesion molecules (integrins). In this case, the expression of integrins from lymph node cells differed from that of splenocytes isolated from rats immediately after space flight. Cell culture studies have indicated that there may be an inhibition in conversion of a precursor cell line to cells exhibiting mature macrophage characteristics after space flight, however, these experiments were limited as a result of technical difficulties. In general, it is clear that space flight results in alterations of cell surface markers. The biological significance of these changes remains to be established.

  2. microRNA expression profile and identification of miR-29 as a prognostic marker and pathogenetic factor by targeting CDK6 in mantle cell lymphoma

    PubMed Central

    Zhao, Jian-Jun; Lin, Jianhong; Lwin, Tint; Yang, Hua; Guo, Jianping; Kong, William; Dessureault, Sophie; Moscinski, Lynn C.; Rezania, Dorna; Dalton, William S.; Sotomayor, Eduardo

    2010-01-01

    Mantle cell lymphoma (MCL) is one of the most aggressive B-cell lymphomas. Although several protein-coding genes are altered, expression signature and importance of microRNA (miRNA) have not been well documented in this malignancy. Here, we performed miRNA expression profile in 30 patients with MCL using a platform containing 515 human miRNAs. Eighteen miRNAs were down-regulated and 21 were up-regulated in MCL compared with normal B lymphocytes. The most frequently altered miRNAs are decrease of miR-29a/b/c, miR-142-3p/5p, and miR-150 and increase of miR-124a and miR-155. Notably, expression levels of miR-29 family are associated with prognosis. The patients with significant down-regulated miR-29 had short survival compared with those who express relatively high levels of miR-29. The prognostic value of miR-29 is comparable with the Mantle Cell Lymphoma International Prognostic Index. Furthermore, we demonstrate miR-29 inhibition of CDK6 protein and mRNA levels by direct binding to 3′-untranslated region. Inverse correlation between miR-29 and CDK6 was observed in MCL. Because cyclin D1 overexpression is a primary event and exerts its function through activation of CDK4/CDK6, our results in primary MCL cells indicate that down-regulation of miR-29 could cooperate with cyclin D1 in MCL pathogenesis. Thus, our findings provide not only miRNA expression signature but also a novel prognostic marker and pathogenetic factor for this malignancy. PMID:20086245

  3. [The specific features of diagnosis of mixed-phenotype acute leukemia: A combination of B-cell antigen expressions according to the results of flow cytometry and morphological markers of myeloid differentiation in blast cells: A clinical case].

    PubMed

    Gritsaev, S V; Kostroma, I I; Ryadnova, G M; Tiranova, S A; Chubukina, Zh V; Balashova, V A; Zenina, M N; Martynkevich, I S; Potikhonova, N A; Abdulkadyrov, K M

    2015-01-01

    This rare type of acute leukemia, blast cells of which express myeloid and/or lymphoid markers, is mainly diagnosed using flow cytometric findings. The paper describes a clinical case of mixed-phenotype acute leukemia, in which B-cell lymphoid antigen expressions were revealed by a flow cytometric technique, while bone marrow morphological specimens showed the signs of myeloid differentiation specific to blast cells. It is concluded that there is a need for a comprehensive examination of patients with new-onset acute leukemia and for an aggregate analysis of flow cytometric results with morphological and cytochemical findings.

  4. Wilms tumor gene 1 expression as a predictive marker for relapse and survival after hematopoietic stem cell transplantation for myelodysplastic syndromes.

    PubMed

    Yoon, Jae-Ho; Jeon, Young-Woo; Yahng, Seung-Ah; Shin, Seung-Hwan; Lee, Sung-Eun; Cho, Byung-Sik; Lee, Dong-Gun; Eom, Ki-Seong; Kim, Hee-Je; Lee, Seok; Min, Chang-Ki; Cho, Seok-Goo; Kim, Yonggoo; Kim, Dong-Wook; Lee, Jong-Wook; Han, Kyungja; Min, Woo-Sung; Park, Chong-Won; Kim, Myungshin; Kim, Yoo-Jin

    2015-03-01

    Relapse after allogeneic hematopoietic stem cell transplantation (HSCT) is a major concern in myelodysplastic syndromes (MDS), but the role of Wilms tumor gene 1 (WT1) as a predictive marker for post-HSCT relapse remains to be validated. We measured WT1 transcript levels by real-time quantitative PCR from marrow samples of 82 MDS patients who underwent transplantation between 2009 and 2013. Pre-HSCT WT1 expression weakly correlated with marrow blast counts or International Prognostic Scoring System scores and failed to predict post-transplantation relapse. Regarding post-HSCT WT1, transcript levels of relapsed patients were significantly higher in comparison to those in remission. Further analysis using receiver operating characteristics curves showed that higher (>154 copies/10(4)ABL) 1-month post-HSCT WT1 resulted in a higher 3-year relapse rate (47.2% versus 6.9%, P < .001) with poorer disease-free survival (DFS) and overall survival at 3 years (41.7% versus 79.0% and 54.3% versus 82.1%, P = .003 and P = .033, respectively). Multivariate analysis after adjusting for pre-HSCT karyotype and chronic graft-versus-host disease (GVHD) also revealed that higher 1-month post-HSCT WT1 was an independent predictive marker for subsequent relapse (P = .002) and poorer DFS (P = .010). In the higher 1-month post-HSCT WT1 subgroup, patients with chronic GVHD showed lower relapse rate and favorable survival outcome. One month post-HSCT WT1 expression was a useful marker for minimal residual disease and relapse prediction in association with chronic GVHD in the context of HSCT for MDS. Copyright © 2015 American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.

  5. Hepatic expression of the proliferative marker Ki-67 and p53 protein in HBV or HCV cirrhosis in relation to dysplastic liver cell changes and hepatocellular carcinoma.

    PubMed

    Koskinas, J; Petraki, K; Kavantzas, N; Rapti, I; Kountouras, D; Hadziyannis, S

    2005-11-01

    To evaluate hepatic expression of the nuclear proliferative marker Ki-67 and the p53 oncoprotein in hepatitis B virus (HBV)/HCV cirrhosis in relation to dysplastic liver cell changes and hepatocellular carcinoma (HCC). We studied needle liver biopsies from 107 patients with cirrhosis and no HCC (52 HBV, 55 HCV) who had been assessed for protocol studies, and 57 cirrhotic patients with HCC (40 HBV, 17 HCV). We evaluated small and large cell dysplastic changes along with the expression of Ki-67 and p53 by immunohistochemistry. The labelling index (LI) was defined as the proportion (%) of positive-stained nuclei of the 500 measured. Large and small cell dysplastic changes were observed in 12 and 9% of specimens respectively. Only small cell changes were associated with Ki-67 expression. Ki-67 LI was 5.50 +/- 5.7 in cirrhosis (13.90 +/- 3.84 in those with small cell dysplastic changes vs 4.64 +/- 4.98 in those without, P < 0.01), 10.2 +/- 5.95 in cirrhosis with HCC (P < 0.05) and 18.56 +/- 10 in HCC (P < 0.01). Neither the presence of small cell dysplastic changes nor the expression of Ki-67 was related to severity or aetiology of cirrhosis. Expression of p53 was observed in 30% of the non-tumorous and in 53% of the neoplastic tissue obtained from patients with HCC, with no differences between HCV and HBV. Ki-67 and p53 expression was associated with the tumour grade (P < 0.001). Our observations clearly demonstrate the association between the proliferation activity and the morphological changes in the cirrhotic liver from the non-dysplastic to dysplastic lesion to HCC. They also support the hypothesis that p53 alterations are a rather late event in carcinogenesis and related to HCC grade. And finally, they suggest that the final steps of hepatocarcinogenesis are common and independent of the aetiology of the chronic viral infection.

  6. Delayed Cutaneous Wound Healing and Aberrant Expression of Hair Follicle Stem Cell Markers in Mice Selectively Lacking Ctip2 in Epidermis

    PubMed Central

    Bajaj, Gaurav; Guha, Gunjan; Wang, Zhixing; Jang, Hyo-Sang; Leid, Mark; Indra, Arup Kumar; Ganguli-Indra, Gitali

    2012-01-01

    Background COUP-TF interacting protein 2 [(Ctip2), also known as Bcl11b] is an important regulator of skin homeostasis, and is overexpressed in head and neck cancer. Ctip2ep−/− mice, selectively ablated for Ctip2 in epidermal keratinocytes, exhibited impaired terminal differentiation and delayed epidermal permeability barrier (EPB) establishment during development, similar to what was observed in Ctip2 null (Ctip2−/−) mice. Considering that as an important role of Ctip2, and the fact that molecular networks which underlie cancer progression partially overlap with those responsible for tissue remodeling, we sought to determine the role of Ctip2 during cutaneous wound healing. Methodology/Principal Findings Full thickness excisional wound healing experiments were performed on Ctip2L2/L2 and Ctip2ep−/− animals per time point and used for harvesting samples for histology, immunohistochemistry (IHC) and immunoblotting. Results demonstrated inherent defects in proliferation and migration of Ctip2 lacking keratinocytes during re-epithelialization. Mutant mice exhibited reduced epidermal proliferation, delayed keratinocyte activation, altered cell-cell adhesion and impaired ECM development. Post wounding, Ctip2ep−/− mice wounds displayed lack of E-Cadherin suppression in the migratory tongue, insufficient expression of alpha smooth muscle actin (alpha SMA) in the dermis, and robust induction of K8. Importantly, dysregulated expression of several hair follicle (HF) stem cell markers such as K15, NFATc1, CD133, CD34 and Lrig1 was observed in mutant skin during wound repair. Conclusions/Significance Results confirm a cell autonomous role of keratinocytic Ctip2 to modulate cell migration, proliferation and/or differentiation, and to maintain HF stem cells during cutaneous wounding. Furthermore, Ctip2 in a non-cell autonomous manner regulated granulation tissue formation and tissue contraction during wound closure. PMID:22383956

  7. Expression of the chemokine MIG is a sensitive and predictive marker for antigen-specific, genetically restricted IFN-gamma production and IFN-gamma-secreting cells.

    PubMed

    Brice, G T; Graber, N L; Hoffman, S L; Doolan, D L

    2001-11-01

    The evaluation of antigen-specific immune responses is critical for understanding the mechanisms of immune protection and for establishing the efficacy of candidate vaccines. Here, we describe a novel assay for IFN-gamma activity which is based on the flow cytometric detection of the chemokine, monokine induced by gamma interferon (MIG) as a sensitive and predictive measure of IFN-gamma-mediated effector function, and a surrogate marker for IFN-gamma-producing cells. Upregulation of MIG expression was demonstrated following in vitro activation of peripheral blood mononuclear cells (PBMCs) with defined CD8+ T-cell epitopes derived from influenza virus, cytomegalovirus (CMV), or Epstein-Barr virus (EBV) and was antigen-specific, genetically restricted and dependent on both CD8+ T cells and IFN-gamma. Furthermore, antigen-specific MIG expression was also demonstrated with Plasmodium falciparum circumsporozoite protein (CSP) peptides, using PBMCs from volunteers immunized with irradiated P. falciparum sporozoites. In multiple parallel experiments, the MIG assay was compared to conventional IFN-gamma ELISPOT, IFN-gamma ELISA, MIG ELISA and intracellular cytokine staining assays. The level of MIG expression was shown to be directly associated with the number of IFN-gamma spot-forming cells (SFCs) detected by ELISPOT (r2=0.94). Moreover, in all instances where cultures were considered positive by ELISPOT, a higher stimulation index was noted with the MIG assay as compared with the ELISPOT assay (on average at least threefold higher) and, in some cases, responses as detected by the MIG assay were significant, but the corresponding response as measured by ELISPOT was not significant. Finally, the flow-based MIG assay offers a number of practical and technical advantages over the ELISPOT assay. Our data validate this novel method for the detection of low as well as high levels of antigen-specific and genetically restricted IFN-gamma activity.

  8. Bortezomib resistance can be reversed by induced expression of plasma cell maturation markers in a mouse in vitro model of multiple myeloma.

    PubMed

    Stessman, Holly A F; Mansoor, Aatif; Zhan, Fenghuang; Linden, Michael A; Van Ness, Brian; Baughn, Linda B

    2013-01-01

    Multiple myeloma (MM), the second most common hematopoietic malignancy, remains an incurable plasma cell (PC) neoplasm. While the proteasome inhibitor, bortezomib (Bz) has increased patient survival, resistance represents a major treatment obstacle as most patients ultimately relapse becoming refractory to additional Bz therapy. Current tests fail to detect emerging resistance; by the time patients acquire resistance, their prognosis is often poor. To establish immunophenotypic signatures that predict Bz sensitivity, we utilized Bz-sensitive and -resistant cell lines derived from tumors of the Bcl-X(L)/Myc mouse model of PC malignancy. We identified significantly reduced expression of two markers (CD93, CD69) in "acquired" (Bz-selected) resistant cells. Using this phenotypic signature, we isolated a subpopulation of cells from a drug-naïve, Bz-sensitive culture that displayed "innate" resistance to Bz. Although these genes were identified as biomarkers, they may indicate a mechanism for Bz-resistance through the loss of PC maturation which may be induced and/or selected by Bz. Significantly, induction of PC maturation in both "acquired" and "innate" resistant cells restored Bz sensitivity suggesting a novel therapeutic approach for reversing Bz resistance in refractory MM.

  9. Prognostic investigations of B7-H1 and B7-H4 expression levels as independent predictor markers of renal cell carcinoma.

    PubMed

    Safaei, Hamid Reza; Rostamzadeh, Ayoob; Rahmani, Omid; Mohammadi, Mohsen; Ghaderi, Omar; Yahaghi, Hamid; Ahmadi, Koroosh

    2016-06-01

    In order to evaluate the correlation of B7-H4 and B7-H1 with renal cell carcinoma (RCC), we analyzed B7-H1 and B7-H4 expressions and their clinical significance by immunohistochemical method. Our result indicated that B7-H4-positive staining was detected in 58.13 % of RCC tissues (25 tissues tumors), and there were 18 tissues of patients without detectable B7-H4. Furthermore, 21 cases (48.83 %) were B7-H1-positive. Positive tumor expressions of B7-H4 and B7-H1 were markedly related to advanced TNM stage (P = 0.001; P = 0.014), high grade (P = 0.001; P = 002), and larger tumor size (P = 0.002; P = 024) in RCC tissues than patients with B7-H4-negative and B7-H1-negative in RCC tissues. The patients with B7-H1 and B7-H4-positive expressions were found to be markedly correlated with the overall survival of the patients (P < 0.05) and tended to have an increased risk of death when compared with negative expression groups. Univariate analysis showed that B7-H4 and B7-H1 expressions, TNM stage, high grade, and tumor size were significantly related to the prognosis of RCC. Furthermore, multivariate analysis showed that B7-H4 and B7-H1 expressions decreased overall survival. The adjusted HR for B7-H1 was 2.83 (95 % CI 1.210-2.971; P = 0.031) and also was 2.918 (95 % CI 1.243-3.102; P = 0.006) for B7-H4 that showed these markers were independent prognostic factors in RCC patients. The expressions of B7-H1 and B7-H4 in RCC patients indicate that these markers may be as a predictor of tumor development and death risk. Further investigations can be helpful to confirm B7-H1 and B7-H4 roles as an independent predictor of clinical RCC outcome.

  10. Dietary tocopherols inhibit cell proliferation, regulate expression of ERα, PPARγ and Nrf2, and decrease serum inflammatory markers during the development of mammary hyperplasia

    PubMed Central

    Smolarek, Amanda K.; So, Jae Young; Thomas, Paul E.; Lee, Hong Jin; Paul, Shiby; Dombrowski, Anne; Wang, Chung-Xiou; Saw, Constance Lay-Lay; Khor, Tin Oo; Kong, Ah-Ng Tony; Reuhl, Kenneth; Lee, Mao-Jung; Yang, Chung S.; Suh, Nanjoo

    2012-01-01

    Previous clinical and epidemiological studies of vitamin E have used primarily α-tocopherol for the prevention of cancer. However, γ-tocopherol has demonstrated greater anti-inflammatory and anti-tumor activity than α-tocopherol in several animal models of cancer. This study assessed the potential chemopreventive activities of a tocopherol mixture containing 58%γ-tocopherol (γ-TmT) in an established rodent model of mammary carcinogenesis. Female ACI rats were utilized due to their sensitivity to 17β-estradiol (E2) to induce mammary hyperplasia and neoplasia. The rats were implanted subcutaneously with sustained release E2 pellets and given dietary 0.3% or 0.5% γ-TmT for 2 or 10 weeks. Serum E2 levels were significantly reduced by the treatment with 0.5% γ-TmT. Serum levels of inflammatory markers, prostaglandin E2 and 8-isoprostane, were suppressed by γ-TmT treatment. Histology of mammary glands showed evidence of epithelial hyperplasia in E2 treated rats. Immunohistochemical analysis of the mammary glands revealed a decrease in proliferating cell nuclear antigen (PCNA), cyclooxygenase-2 (COX-2) and estrogen receptor α (ERα), while there was an increase in cleaved-caspase 3, peroxisome proliferator activated receptor γ (PPARγ), and nuclear factor (erythroid-derived 2)-like 2 (Nrf2) in γ-TmT treated rats. In addition, treatment with γ-TmT resulted in a decrease in the expression of ERα mRNA, whereas mRNA levels of ERβ and PPARγ were increased. In conclusion, γ-TmT was shown to suppress inflammatory markers, inhibit E2-induced cell proliferation, and upregulate PPARγ and Nrf2 expression in mammary hyperplasia, suggesting that γ-TmT may be a promising agent for human breast cancer prevention. PMID:22389237

  11. Co-expression of the Follicle Stimulating Hormone Receptor and Stem Cell Markers: A Novel Approach to Target Ovarian Cancer Stem Cells

    DTIC Science & Technology

    2013-09-01

    cautiously as a functional end point characteristic of ovarian cancer stem cells (Appendix I). 7 Year 2 1. Our veterinary staff has since...WTA system ® to amplify the RNA into a double stranded cDNA product. Real- time PCR using intron-spanning primers was carried out using a BioRad...mince, and digest in enzyme-free cell dissociation buffer (Gibco). This material has worked well for us with cultured cell expressing the FSHR and

  12. Expression of lymphatic markers during avian and mouse cardiogenesis

    PubMed Central

    Karunamuni, Ganga; Yang, Ke; Doughman, Yong Qiu; Wikenheiser, Jamie; Bader, David; Barnett, Joey; Austin, Anita; Parsons-Wingerter, Patricia; Watanabe, Michiko

    2013-01-01

    The adult heart has been reported to have an extensive lymphatic system, yet the development of this important system during cardiogenesis is still largely unexplored. The nuclear-localized transcription factor Prox-1 identified a sheet of Prox-1-positive cells on the developing aorta and pulmonary trunk in avian and murine embryos just prior to septation of the four heart chambers. The cells coalesced into a branching lymphatic network that spread within the epicardium to cover the heart. These vessels eventually expressed the lymphatic markers LYVE-1, VEGFR-3, and podoplanin. Before the Prox-1-positive cells were detected in the mouse epicardium, LYVE-1, a homologue of the CD44 glycoprotein, was primarily expressed in individual epicardial cells. Similar staining patterns were observed for CD44 in avian embryos. The proximity of these LYVE-1/CD44-positive mesenchymal cells to Prox-1-positive vessels suggests that they may become incorporated into the lymphatics. Unexpectedly, we detected LYVE-1/PECAM/VEGFR-3-positive vessels within the embryonic and adult myocardium which remained Prox-1/podoplanin-negative. Lymphatic markers were surprisingly found in adult rat and embryonic mouse epicardial cell lines, with Prox-1 also exhibiting nuclear-localized expression in primary cultures of embryonic avian epicardial cells. Our data identified three types of cells in the embryonic heart expressing lymphatic markers: (1) Prox-1-positive cells from an extracardiac source that migrate within the serosa of the outflow tract into the epicardium of the developing heart, (2) individual LYVE-1-positive cells in the epicardium that may be incorporated into the Prox-1-positive lymphatic vasculature, and (3) LYVE-1-positive cells/vessels in the myocardium that do not become Prox-1-positive even in the adult heart. PMID:19938109

  13. Ras protein expression as a marker for breast cancer

    PubMed Central

    CALAF, GLORIA M.; ABARCA-QUINONES, JORGE

    2016-01-01

    Breast cancer, the most common neoplasm in women of all ages, is the leading cause of cancer-related mortality in women worldwide. Markers to help to predict the risk of progression and ultimately provide non-surgical treatment options would be of great benefit. At present, there are no available molecular markers to predict the risk of carcinoma in situ progression to invasive cancer; therefore, all women diagnosed with this type of malignancy must undergo surgery. Breast cancer is a heterogeneous complex disease, and different patients respond differently to different treatments. In breast cancer, analysis using immunohistochemical markers remains an essential component of routine pathological examinations, and plays an import role in the management of the disease by providing diagnostic and prognostic strategies. The aim of the present study was to identify a marker that can be used as a prognostic tool for breast cancer. For this purpose, we firstly used an established breast cancer model. MCF-10F, a spontaneously immortalized breast epithelial cell line was transformed by exposure to estrogen and radiation. MCF-10F cells were exposed to low doses of high linear energy transfer (LET) α particles (150 keV/μm) of radiation, and subsequently cultured in the presence of 17β-estradiol. Three cell lines were used: i) MCF-10F cells as a control; ii) Alpha5 cells, a malignant and tumorigenic cell line; and iii) Tumor2 cells derived from Alpha5 cells injected into nude mice. Secondly, we also used normal, benign and malignant breast specimens obtained from biopsies. The results revealed that the MCF-10F cells were negative for c-Ha-Ras protein expression; however, the Alpha5 and Tumor2 cell lines were positive for c-Ha-Ras protein expression. The malignant breast samples were also strongly positive for c-Ha-Ras expression. The findings of our study indicate that c-Ha-Ras protein expression may be used as a marker to predict the progression of breast cancer; this

  14. Regulator of G-Protein Signaling-5 Is a Marker of Hepatic Stellate Cells and Expression Mediates Response to Liver Injury

    PubMed Central

    Bahrami, Arya J.; Gunaje, Jagadambika J.; Hayes, Brian J.; Riehle, Kimberly J.; Kenerson, Heidi L.; Yeung, Raymond S.; Stempien-Otero, April S.; Campbell, Jean S.; Mahoney, William M.

    2014-01-01

    Liver fibrosis is mediated by hepatic stellate cells (HSCs), which respond to a variety of cytokine and growth factors to moderate the response to injury and create extracellular matrix at the site of injury. G-protein coupled receptor (GPCR)-mediated signaling, via endothelin-1 (ET-1) and angiotensin II (AngII), increases HSC contraction, migration and fibrogenesis. Regulator of G-protein signaling-5 (RGS5), an inhibitor of vasoactive GPCR agonists, functions to control GPCR-mediated contraction and hypertrophy in pericytes and smooth muscle cells (SMCs). Therefore we hypothesized that RGS5 controls GPCR signaling in activated HSCs in the context of liver injury. In this study, we localize RGS5 to the HSCs and demonstrate that Rgs5 expression is regulated during carbon tetrachloride (CCl4)-induced acute and chronic liver injury in Rgs5LacZ/LacZ reporter mice. Furthermore, CCl4 treated RGS5-null mice develop increased hepatocyte damage and fibrosis in response to CCl4 and have increased expression of markers of HSC activation. Knockdown of Rgs5 enhances ET-1-mediated signaling in HSCs in vitro. Taken together, we demonstrate that RGS5 is a critical regulator of GPCR signaling in HSCs and regulates HSC activation and fibrogenesis in liver injury. PMID:25290689

  15. Inverse Relationship between Tumor Proliferation Markers and Connexin Expression in a Malignant Cardiac Tumor Originating from Mesenchymal Stem Cell Engineered Tissue in a Rat in vivo Model

    PubMed Central

    Spath, Cathleen; Schlegel, Franziska; Leontyev, Sergey; Mohr, Friedrich-Wilhelm; Dhein, Stefan

    2013-01-01

    Background: Recently, we demonstrated the beneficial effects of engineered heart tissues for the treatment of dilated cardiomyopathy in rats. For further development of this technique we started to produce engineered tissue (ET) from mesenchymal stem cells. Interestingly, we observed a malignant tumor invading the heart with an inverse relationship between proliferation markers and connexin expression. Methods: Commercial CD54+/CD90+/CD34−/CD45− bone marrow derived mesenchymal rat stem cells (cBM-MSC), characterized were used for production of mesenchymal stem-cell-ET (MSC-ET) by suspending them in a collagen I, matrigel-mixture and cultivating for 14 days with electrical stimulation. Three MSC-ET were implanted around the beating heart of adult rats for days. Another three MSC-ET were produced from freshly isolated rat bone marrow derived stem cells (sBM-MSC). Results: Three weeks after implantation of the MSC-ETs the hearts were surgically excised. While in 5/6 cases the ET was clearly distinguishable and was found as a ring containing mostly connective tissue around the heart, in 1/6 the heart was completely surrounded by a huge, undifferentiated, pleomorphic tumor originating from the cMSC-ET (cBM-MSC), classified as a high grade malignant sarcoma. Quantitatively we found a clear inverse relationship between cardiac connexin expression (Cx43, Cx40, or Cx45) and increased Ki-67 expression (Cx43: p < 0.0001, Cx45: p < 0.03, Cx40: p < 0.014). At the tumor-heart border there were significantly more Ki-67 positive cells (p = 0.001), and only 2% Cx45 and Ki-67-expressing cells, while the other connexins were nearly completely absent (p < 0.0001). Conclusion and Hypothesis: These observations strongly suggest the hypothesis, that invasive tumor growth is accompanied by reduction in connexins. This implicates that gap junction communication between tumor and normal tissue is reduced or absent, which could mean that growth and differentiation

  16. A new kymogram-based method reveals unexpected effects of marker protein expression and spatial anisotropy of cytoskeletal dynamics in plant cell cortex.

    PubMed

    Cvrčková, Fatima; Oulehlová, Denisa

    2017-01-01

    Cytoskeleton can be observed in live plant cells in situ with high spatial and temporal resolution using a combination of specific fluorescent protein tag expression and advanced microscopy methods such as spinning disc confocal microscopy (SDCM) or variable angle epifluorescence microscopy (VAEM). Existing methods for quantifying cytoskeletal dynamics are often either based on laborious manual structure tracking, or depend on costly commercial software. Current automated methods also do not readily allow separate measurements of structure lifetime, lateral mobility, and spatial anisotropy of these parameters. We developed a new freeware-based, operational system-independent semi-manual technique for analyzing VAEM or SDCM data, QuACK (Quantitative Analysis of Cytoskeletal Kymograms), and validated it on data from Arabidopsis thaliana fh1 formin mutants, previously shown by conventional methods to exhibit altered actin and microtubule dynamics compared to the wild type. Besides of confirming the published mutant phenotype, QuACK was used to characterize surprising differential effects of various fluorescent protein tags fused to the Lifeact actin probe on actin dynamics in A. thaliana cotyledon epidermis. In particular, Lifeact-YFP slowed down actin dynamics compared to Lifeact-GFP at marker expression levels causing no macroscopically noticeable phenotypic alterations, although the two fluorophores are nearly identical. We could also demonstrate the expected, but previously undocumented, anisotropy of cytoskeletal dynamics in elongated epidermal cells of A. thaliana petioles and hypocotyls. Our new method for evaluating plant cytoskeletal dynamics has several advantages over existing techniques. It is intuitive, rapid compared to fully manual approaches, based on the free ImageJ software (including macros we provide here for download), and allows measurement of multiple parameters. Our approach was already used to document unexpected differences in actin

  17. The effect of cancer procoagulant on expression of metastatic and angiogenic markers in breast cancer and embryonic stem cell lines.

    PubMed

    Kee, Nalise Low Ah; Naudé, Ryno J; Blatch, Gregory L; Frost, Carminita L

    2012-03-01

    Cancer procoagulant is present only in malignant tumours and the undifferentiated tissues of human placenta. Its possible role in angiogenesis and metastasis was investigated. Cancer procoagulant increased the steady-state mRNA level of L1 cell adhesion molecule (L1CAM) in MCF-7 breast cancer cells and E14 mouse embryonic stem cells (MESCs), while an increase in angiogenin mRNA was observed in MDA-MB-231 breast cancer cells. Furthermore, production of vascular endothelial growth factor (VEGF) protein in MCF-7 breast cancer cells and E14 MESCs, but decreased in MDA-MB-231 breast cancer cells. We conclude that cancer procoagulant could potentially play a part in angiogenesis in cancer and vascular development during embryonic development.

  18. Expression of Von Willebrand factor, an endothelial cell marker, is up-regulated by angiogenesis factors: a potential method for objective assessment of tumor angiogenesis.

    PubMed

    Zanetta, L; Marcus, S G; Vasile, J; Dobryansky, M; Cohen, H; Eng, K; Shamamian, P; Mignatti, P

    2000-01-15

    von Willebrand factor (vWF), a glycoprotein produced uniquely by endothelial cells and megakaryocytes, is routinely used to identify vessels in tissue sections. Vessel density in tumor specimens, as determined by immuno-histochemical staining for vWF or other endothelial cell markers, is a negative prognostic factor for many solid tumors. vWF is heterogeneously distributed throughout the vasculature, transcriptional control in response to the tissue microenvironment being responsible for local variations in endothelial cell levels of vWF. Here, we report that fibroblast growth factor-2 and vascular endothelial growth factor, potent angiogenesis inducers expressed in a variety of tumors, up-regulate expression of vWF mRNA and protein in cultured endothelial cells with a synergistic effect. Our data support the measurement of vWF mRNA in tumors to detect activated endothelium or angiogenesis. For this purpose, we developed a semi-quantitative RT-PCR for vWF mRNA. Preliminary results obtained with specimens from colon carcinoma and the corresponding normal colonic mucosa showed higher vWF mRNA levels in most tumors than in their normal counterparts. The differences in vWF mRNA levels were much larger than the differences in vessel counts between a tumor and the corresponding normal mucosa, indicating that high vWF mRNA levels in tumors may indeed be an early sign of activation of the endothelium. The rapidity, objectivity, sensitivity and specificity of this technique make it suitable for routine clinical application to identify aggressive, highly angiogenic tumors.

  19. Coordinated gene expression of neuroinflammatory and cell signaling markers in dorsolateral prefrontal cortex during human brain development and aging.

    PubMed

    Primiani, Christopher T; Ryan, Veronica H; Rao, Jagadeesh S; Cam, Margaret C; Ahn, Kwangmi; Modi, Hiren R; Rapoport, Stanley I

    2014-01-01

    Age changes in expression of inflammatory, synaptic, and neurotrophic genes are not well characterized during human brain development and senescence. Knowing these changes may elucidate structural, metabolic, and functional brain processes over the lifespan, as well vulnerability to neurodevelopmental or neurodegenerative diseases. Expression levels of inflammatory, synaptic, and neurotrophic genes in the human brain are coordinated over the lifespan and underlie changes in phenotypic networks or cascades. We used a large-scale microarray dataset from human prefrontal cortex, BrainCloud, to quantify age changes over the lifespan, divided into Development (0 to 21 years, 87 brains) and Aging (22 to 78 years, 144 brains) intervals, in transcription levels of 39 genes. Gene expression levels followed different trajectories over the lifespan. Many changes were intercorrelated within three similar groups or clusters of genes during both Development and Aging, despite different roles of the gene products in the two intervals. During Development, changes were related to reported neuronal loss, dendritic growth and pruning, and microglial events; TLR4, IL1R1, NFKB1, MOBP, PLA2G4A, and PTGS2 expression increased in the first years of life, while expression of synaptic genes GAP43 and DBN1 decreased, before reaching plateaus. During Aging, expression was upregulated for potentially pro-inflammatory genes such as NFKB1, TRAF6, TLR4, IL1R1, TSPO, and GFAP, but downregulated for neurotrophic and synaptic integrity genes such as BDNF, NGF, PDGFA, SYN, and DBN1. Coordinated changes in gene transcription cascades underlie changes in synaptic, neurotrophic, and inflammatory phenotypic networks during brain Development and Aging. Early postnatal expression changes relate to neuronal, glial, and myelin growth and synaptic pruning events, while late Aging is associated with pro-inflammatory and synaptic loss changes. Thus, comparable transcriptional regulatory networks that operate

  20. Coordinated Gene Expression of Neuroinflammatory and Cell Signaling Markers in Dorsolateral Prefrontal Cortex during Human Brain Development and Aging

    PubMed Central

    Primiani, Christopher T.; Ryan, Veronica H.; Rao, Jagadeesh S.; Cam, Margaret C.; Ahn, Kwangmi; Modi, Hiren R.; Rapoport, Stanley I.

    2014-01-01

    Background Age changes in expression of inflammatory, synaptic, and neurotrophic genes are not well characterized during human brain development and senescence. Knowing these changes may elucidate structural, metabolic, and functional brain processes over the lifespan, as well vulnerability to neurodevelopmental or neurodegenerative diseases. Hypothesis Expression levels of inflammatory, synaptic, and neurotrophic genes in the human brain are coordinated over the lifespan and underlie changes in phenotypic networks or cascades. Methods We used a large-scale microarray dataset from human prefrontal cortex, BrainCloud, to quantify age changes over the lifespan, divided into Development (0 to 21 years, 87 brains) and Aging (22 to 78 years, 144 brains) intervals, in transcription levels of 39 genes. Results Gene expression levels followed different trajectories over the lifespan. Many changes were intercorrelated within three similar groups or clusters of genes during both Development and Aging, despite different roles of the gene products in the two intervals. During Development, changes were related to reported neuronal loss, dendritic growth and pruning, and microglial events; TLR4, IL1R1, NFKB1, MOBP, PLA2G4A, and PTGS2 expression increased in the first years of life, while expression of synaptic genes GAP43 and DBN1 decreased, before reaching plateaus. During Aging, expression was upregulated for potentially pro-inflammatory genes such as NFKB1, TRAF6, TLR4, IL1R1, TSPO, and GFAP, but downregulated for neurotrophic and synaptic integrity genes such as BDNF, NGF, PDGFA, SYN, and DBN1. Conclusions Coordinated changes in gene transcription cascades underlie changes in synaptic, neurotrophic, and inflammatory phenotypic networks during brain Development and Aging. Early postnatal expression changes relate to neuronal, glial, and myelin growth and synaptic pruning events, while late Aging is associated with pro-inflammatory and synaptic loss changes. Thus, comparable

  1. Comparison of the gene expression profiles of human fetal cortical astrocytes with pluripotent stem cell derived neural stem cells identifies human astrocyte markers and signaling pathways and transcription factors active in human astrocytes.

    PubMed

    Malik, Nasir; Wang, Xiantao; Shah, Sonia; Efthymiou, Anastasia G; Yan, Bin; Heman-Ackah, Sabrina; Zhan, Ming; Rao, Mahendra

    2014-01-01

    Astrocytes are the most abundant cell type in the central nervous system (CNS) and have a multitude of functions that include maintenance of CNS homeostasis, trophic support of neurons, detoxification, and immune surveillance. It has only recently been appreciated that astrocyte dysfunction is a primary cause of many neurological disorders. Despite their importance in disease very little is known about global gene expression for human astrocytes. We have performed a microarray expression analysis of human fetal astrocytes to identify genes and signaling pathways that are important for astrocyte development and maintenance. Our analysis confirmed that the fetal astrocytes express high levels of the core astrocyte marker GFAP and the transcription factors from the NFI family which have been shown to play important roles in astrocyte development. A group of novel markers were identified that distinguish fetal astrocytes from pluripotent stem cell-derived neural stem cells (NSCs) and NSC-derived neurons. As in murine astrocytes, the Notch signaling pathway appears to be particularly important for cell fate decisions between the astrocyte and neuronal lineages in human astrocytes. These findings unveil the repertoire of genes expressed in human astrocytes and serve as a basis for further studies to better understand astrocyte biology, especially as it relates to disease.

  2. Effect of icariin on cell proliferation and the expression of bone resorption/formation-related markers in human periodontal ligament cells.

    PubMed

    Pei, Zhenhua; Zhang, Fengqiu; Niu, Zhongying; Shi, Shenggen

    2013-11-01

    Periodontitis is a common destructive inflammatory disease that leads to changes in the tooth-supporting tissues. Human periodontal ligament cells are essential in periodontal tissue regeneration. The traditional Chinese medicine icariin promoted bone formation, stimulated the osteogenic differentiation of preosteoblastic cells and inhibited osteoclast differentiation and bone resorption. Thus, in the present study, the effect of icariin on cell proliferation and the expression of osteoprotegerin (OPG), receptor activator of nuclear factor-κB ligand (RANKL), core binding factor α1 (Cbfa1) and osteocalcin (OC) was investigated in human periodontal ligament cells, by an MTT assay, qPCR and western blot analysis. The results demonstrated that icariin promoted cell proliferation in a dose- and time-dependent manner, upregulated OPG, Cbfa1 and OC expression, and downregulated RANKL production and the RANKL/OPG expression ratio. This suggested the potential value of icariin in treating alveolar bone resorption and promoting periodontal tissue regeneration, due to its ability to stimulate the proliferation and osteogenic differentiation of human periodontal ligament cells and inhibit osteoclast differentiation.

  3. Effects of the EVCAM chemical validation library on differentiation using marker gene expression in lmouse embryonic stem cells

    EPA Science Inventory

    The adherent cell differentiation and cytotoxicity (ACDC) assay was used to profile the effects of the ECVAM EST validation chemical library (19 compounds) on J1 mouse embryonic stem cells (mESC). PCR-based TaqMan Low Density Arrays (TLDA) provided a high-content assessment of al...

  4. Effects of the EVCAM chemical validation library on differentiation using marker gene expression in lmouse embryonic stem cells

    EPA Science Inventory

    The adherent cell differentiation and cytotoxicity (ACDC) assay was used to profile the effects of the ECVAM EST validation chemical library (19 compounds) on J1 mouse embryonic stem cells (mESC). PCR-based TaqMan Low Density Arrays (TLDA) provided a high-content assessment of al...

  5. Colon cancer: cancer stem cells markers, drug resistance and treatment.

    PubMed

    Kozovska, Zuzana; Gabrisova, Veronika; Kucerova, Lucia

    2014-10-01

    Malignant tumours consist of heterogeneous populations of tumour cells. Cancer stem cells (CSC) represent a population of cells within a tumour with highly tumorigenic and chemoresistant properties. These cells may be identified by the expression of CSC markers. There are several key stem cells markers specified for colon cancer: CD133, CD44, ALDH1, ALCAM. These days, a major obstacle to effective cancer management is development of a multidrug resistance (MDR). The principal mechanism responsible for development of MDR phenotype is the over-expression of ABC transporters. Tumours and relapsing tumours after therapy are drived by subpopulations of tumour cells with aggressive phenotype resistant to chemotherapeutics. These cells are called CSC or tumour-initiating cells (TIC). Here we outline recent information about MDR of colon cancer and CSC markers. We have focused on novel therapeutic strategies which have been developed to prevent or overcome MDR. One such strategy is a combination of chemotherapy and modulators of MDR pumps or chemotherapy and monoclonal antibodies against vascular endothelial growth factor VEGF. Colon cancer is characterized by the presence of colon CSC expressing specific stem cell markers. The divergent presence of these markers can help to adjust personalized therapy. The review provides a detailed overview of resistance of colon cancer cells and discusses how the presence of CSC markers can influence therapy and prognosis of patients.

  6. Comprehensive Screening of Cell Surface Markers Expressed by Adult-Derived Human Liver Stem/Progenitor Cells Harvested at Passage 5: Potential Implications for Engraftment

    PubMed Central

    Sokal, Etienne

    2016-01-01

    Mesenchymal stromal cells (MSCs) are known to have potential therapeutic benefits for a number of diseases. However, many studies report low engraftment levels, regardless of the target organ. One possible explanation could be that MSCs do not express the necessary receptors for engraftment. Indeed, MSCs appear to use a similar mechanism to leukocytes to engraft into injured organs, relying on various receptors for rolling, firm adhesion, and transmigration. In this study, we conducted an extensive surface molecule screening of adult-derived human liver stem/progenitor cells (ADHLSC) in an attempt to shed some light on this subject. We observed that ADHLSCs lack expression of most of the costimulatory molecules tested. Furthermore, study of the adhesion molecule profile of ADHLSCs revealed that they do not express selectin ligands or LFA-1 which are, respectively, involved in the rolling process and the firm adhesion. In addition, ADHLSCs slightly express VLA-4 and lose expression of CXCR4 altogether on their surface during culture expansion. However, ADHLSCs express all the integrin couples and matrix metalloproteinases needed to bind and integrate the extracellular matrix once the endothelial barrier is crossed. Collectively, these results suggest that binding to the endothelium may be the critical weak point in the engraftment process. PMID:27956903

  7. Prognostic Value of Cancer Stem Cell Marker ALDH1 Expression in Colorectal Cancer: A Systematic Review and Meta-Analysis

    PubMed Central

    Jiang, Bin; Chang, Weilong; Yuan, Wenzheng; Ma, Zhijun; Liu, Zhengyi; Shu, Xiaogang

    2015-01-01

    Objective Many studies have indicated the prognostic and clinicopathological value of aldehyde dehydrogenase 1 (ALDH1) in colorectal cancer (CRC) patients still remains controversial. Thus we performed this study to clarify the relationship between high ALDH1 expression in CRC and its impact on survival and clinicopathological features. Methods Publications for relevant studies in Pubmed, the Cochrane Library, Embase, and China National Knowledge Infrastructure (CNKI) through April 2015 were identified. Only articles describing ALDH1 antigen with immunohistochemistry in CRC were included. The software RevMan 5.1 was used to analyze the outcomes, including 5-year overall survival (OS), disease-free survival (DFS) and clinicopathological features. Results 9 studies with 1203 patients satisfying the criteria were included. The overall rate of high ALDH1 expression was 46.5% by immunohistochemical staining. High ALDH1 expression as an independent prognostic factor was significantly associated with the 5-year OS and DFS (OR = 0.42, 95%CI: 0.26–0.68, P = 0.0004; OR = 0.38, 95%CI: 0.24–0.59, P < 0.0001, respectively). High ALDH1 expression was highly correlated with the tumor (T) stage (T3 + T4 vs. T1 + T2; OR = 2.16, 95%CI: 1.09–4.28, P = 0.03), lymph node (N) stage (N1 + N2 vs. N0; OR = 1.8; 95%CI: 1.17–2.79, P = 0.008), and tumor differentiation (G3 vs. G1 + G2; OR = 1.88; 95%CI: 1.07–3.30, P = 0.03). However, high ALDH1 expression was not significantly correlated with the patient age (>60 years old vs. <60 years old; OR = 1.11, 95%CI: 0.63–1.94, P = 0.72). Conclusions High ALDH1 expression indicates a poor prognosis in CRC patients. Moreover, high ALDH1 expression correlates with the T stage, N stage, and tumor differentiation, but not with age. PMID:26682730

  8. Immunohistochemical investigation of cells expressing CD21, membrane IgM, CD32 and a follicular dendritic cell marker in the lymphoid tissues of neonatal calves.

    PubMed

    Chattha, Kuldeep S; Hodgins, Douglas C; DeLay, Josepha; Antoine, Nadine; Shewen, Patricia E

    2010-10-15

    Activation of B lymphocytes in the presence of passive maternal antibodies depends on expression of CD21, membrane IgM and CD32. On colligation with IgM, CD32 inhibits activation whereas CD21 enhances it. Recently, we assessed expression of CD21 and CD32 on IgM(+) cells from lymphoid tissues of newborn calves by flow cytometry, but this approach does not provide information about spatial distribution within lymphoid compartments. Therefore, histologic sections of lymphoid tissues from newborn and 7-month-old calves were examined using an immunoperoxidase technique. In all calves, CD21 and IgM stained cells were collocated in the cortex and paracortex of the retropharyngeal lymph node, in the marginal zone of the spleen and in lymphoid aggregates of palatine tonsils. Most CD32(+) cells were in the mantle zone of lymphoid follicles in 7-month-old calves, whereas only weak staining was observed in newborns. A few CD32(+) cells were also observed in the paracortex at both ages. Absence of CD32(+) cells in the center of follicles suggests that IgM(+)CD32(-) cells observed previously by flow cytometry were from germinal centers. Overall, there were few organized lymphoid aggregates within lymphoid tissues of newborn calves, and follicular dendritic cells were virtually undetectable. Their absence may be an important limitation for neonatal immunization.

  9. Runx2 expression: A mesenchymal stem marker for cancer

    PubMed Central

    Valenti, Maria Teresa; Serafini, Paola; Innamorati, Giulio; Gili, Anna; Cheri, Samuele; Bassi, Claudio; Dalle Carbonare, Luca

    2016-01-01

    The transcription factor runt-related transcription factor 2 (Runx2) is a master gene implicated in the osteogenic differentiation of mesenchymal stem cells, and thus serves a determinant function in bone remodelling and skeletal integrity. Various signalling pathways regulate Runx2 abundance, which requires a number of molecules to finely modulate its expression. Furthermore, this gene may be ectopically-expressed in cancer cells. Recent studies have reported the involvement of Runx2 in cell proliferation, epithelial-mesenchymal transition, apoptosis and metastatic processes, suggesting it may represent a useful therapeutic target in cancer treatment. However, studies evaluating this gene as a cancer marker are lacking. In the present study, Runx2 expression was analysed in 11 different cancer cell lines not derived from bone tumour. In addition, the presence of Runx2-related cell-free RNA was examined in the peripheral blood of 41 patients affected by different forms of tumours. The results demonstrated high expression levels of Runx2 in the cancer cell lines and identified the presence of Runx2-related cell-free RNA in the peripheral blood of patients with cancer. As compared with normal individuals, the expression level was increased by 14.2-fold in patients with bone metastases and by 4.01-fold in patients without metastases. The results of the present study therefore opens up the possibility to exploit Runx2 expression as a cancer biomarker allowing the use of minimally invasive approaches for diagnosis and follow-up. PMID:27895787

  10. Cancer stem cell marker ALDH1 expression is associated with lymph node metastasis and poor survival in esophageal squamous cell carcinoma: a study from high incidence area of northern China.

    PubMed

    Wang, Y; Zhe, H; Gao, P; Zhang, N; Li, G; Qin, J

    2012-08-01

    Tumor recurrence and metastasis is the leading cause of death in esophageal squamous cell carcinoma (ESCC). Cancer stem cell (CSC) may be responsible for tumor growth and maintenance of aggressive behavior. Aldehyde dehydrogenase 1 (ALDH1) has been proposed as one of the possible candidates for a CSC marker. The expression of ALDH1 may be correlated with the clinicopathologic factor and clinical outcome of patients with ESCC. The purpose of this study was to investigate the expression of ALDH1 protein in human ESCC tissues, and evaluated the clinical implication of ALDH1 expression for these patients. All 79 patients who underwent esophagectomy for ESCC between January 2005 and June 2006 were enrolled in this study. The expression of ALDH1 in ESCC and adjacent noncancerous tissues was analyzed by immunohistochemistry. ALDH1 was mainly expressed in ESCC cell nucleus. For the 79 ESCC patients, increased nuclear accumulation of ALDH1 was found in 12 (15.2%) specimens. ALDH1 expression was correlated with poor histological differentiation (P= 0.003), lymph node metastasis (P= 0.011), and late pathologic TNM classification (pTNM) staging (P= 0.003). Patients in ALDH1 positive group had a significantly poor 5-year overall survival than those in the negative group (8.3% vs. 52.2%, P= 0.025). We have demonstrated for the first time that the CSC marker, ALDH1, is expressed in human ESCC. The expression of ALDH1 protein in nucleus of the ESCC is significantly associated with lymph node metastasis and poor survival. Our results highly indicate the involvement of ALDH1 in the aggressive behavior of ESCC.

  11. Expression of LGR-5, MSI-1 and DCAMKL-1, putative stem cell markers, in the early phases of 1,2-dimethylhydrazine-induced rat colon carcinogenesis: correlation with nuclear β-catenin.

    PubMed

    Femia, Angelo Pietro; Dolara, Piero; Salvadori, Maddalena; Caderni, Giovanna

    2013-02-01

    Colon cancer stem cells may drive carcinogenesis and account for chemotherapeutic failure. Although many markers for these cells have been proposed, there is no complete agreement regarding them, nor has their presence in the early phases of carcinogenesis been characterized in depth. The expression of the putative markers LGR-5 (leucine-rich-repeat-containing G-protein-coupled receptor 5), MSI-1 (Musashi-1) and DCAMKL-1 (doublecortin and calcium/calmodulin-dependent protein kinase-like-1) was studied in normal colon mucosa (NM), in the precancerous lesions Mucin Depleted Foci (MDF) and in macroscopic tumours (adenomas) of 1,2-dimethylhydrazine-treated rats. Co-localization between these markers and nuclear β-catenin (NBC), an attributed feature of cancer stem cells, was also determined. Moreover, since PGE2 could increase NBC, we tested whether short-term treatment with celecoxib, a COX-2 inhibitor (2 weeks, 250 ppm in the diet) could reduce the expression of these markers. LGR-5 expression in NM was low (Labelling Index (LI): 0.22 ± 0.03 (means ± SE)) with positive cells located mainly at the base of the crypts. Compared to NM, LGR-5 was overexpressed in MDF and tumours (LI: 4.7 ± 2.0 and 2.9 ± 1.0 in MDF and tumours, respectively, P<0.01 compared to NM). DCAMKL-1 positive cells, distributed along the length of normal crypts, were reduced in MDF and tumours. Nuclear expression of MSI-1, located mainly at the base of normal crypts, was not observed in MDF or tumours. In both MDF and tumours, few cells co-expressed LGR-5 and NBC (LI: 1.0 ± 0.3 and 0.4 ± 0.2 in MDF and tumours, respectively). Notwithstanding the lower expression of DCAMKL-1 in tumours, the percentage of cells co-expressing DCAMKL-1 and NBC was higher than in NM (LI: 0.5 ± 0.1 and 0.04 ± 0.02 in tumours and NM, respectively). MSI-1 and NBC co-localization was not observed. Celecoxib did not reduce cells co-expressing LGR-5 and NBC. Based on its prevalent localization at the base of normal

  12. Expression of LGR-5, MSI-1 and DCAMKL-1, putative stem cell markers, in the early phases of 1,2-dimethylhydrazine-induced rat colon carcinogenesis: correlation with nuclear β-catenin

    PubMed Central

    2013-01-01

    Background Colon cancer stem cells may drive carcinogenesis and account for chemotherapeutic failure. Although many markers for these cells have been proposed, there is no complete agreement regarding them, nor has their presence in the early phases of carcinogenesis been characterized in depth. Methods The expression of the putative markers LGR-5 (leucine-rich-repeat-containing G-protein-coupled receptor 5), MSI-1 (Musashi-1) and DCAMKL-1 (doublecortin and calcium/calmodulin-dependent protein kinase-like-1) was studied in normal colon mucosa (NM), in the precancerous lesions Mucin Depleted Foci (MDF) and in macroscopic tumours (adenomas) of 1,2-dimethylhydrazine-treated rats. Co-localization between these markers and nuclear β-catenin (NBC), an attributed feature of cancer stem cells, was also determined. Moreover, since PGE2 could increase NBC, we tested whether short-term treatment with celecoxib, a COX-2 inhibitor (2 weeks, 250 ppm in the diet) could reduce the expression of these markers. Results LGR-5 expression in NM was low (Labelling Index (LI): 0.22±0.03 (means±SE)) with positive cells located mainly at the base of the crypts. Compared to NM, LGR-5 was overexpressed in MDF and tumours (LI: 4.7±2.0 and 2.9±1.0 in MDF and tumours, respectively, P<0.01 compared to NM). DCAMKL-1 positive cells, distributed along the length of normal crypts, were reduced in MDF and tumours. Nuclear expression of MSI-1, located mainly at the base of normal crypts, was not observed in MDF or tumours. In both MDF and tumours, few cells co-expressed LGR-5 and NBC (LI: 1.0±0.3 and 0.4±0.2 in MDF and tumours, respectively). Notwithstanding the lower expression of DCAMKL-1 in tumours, the percentage of cells co-expressing DCAMKL-1 and NBC was higher than in NM (LI: 0.5±0.1 and 0.04±0.02 in tumours and NM, respectively). MSI-1 and NBC co-localization was not observed. Celecoxib did not reduce cells co-expressing LGR-5 and NBC. Conclusions Based on its prevalent localization

  13. Cell-surface markers for colon adenoma and adenocarcinoma.

    PubMed

    Sewda, Kamini; Coppola, Domenico; Enkemann, Steven; Yue, Binglin; Kim, Jongphil; Lopez, Alexis S; Wojtkowiak, Jonathan W; Stark, Valerie E; Morse, Brian; Shibata, David; Vignesh, Shivakumar; Morse, David L

    2016-04-05

    Early detection of colorectal cancer (CRC) is crucial for effective treatment. Among CRC screening techniques, optical colonoscopy is widely considered the gold standard. However, it is a costly and invasive procedure with a low rate of compliance. Our long-term goal is to develop molecular imaging agents for the non-invasive detection of CRC by molecular imaging-based colonoscopy using CT, MRI or fluorescence. To achieve this, cell surface targets must be identified and validated. Here, we report the discovery of cell-surface markers that distinguish CRC from surrounding tissues that could be used as molecular imaging targets. Profiling of mRNA expression microarray data from patient tissues including adenoma, adenocarcinoma, and normal gastrointestinal tissues was used to identify potential CRC specific cell-surface markers. Of the identified markers, six were selected for further validation (CLDN1, GPR56, GRM8, LY6G6D/F, SLCO1B3 and TLR4). Protein expression was confirmed by immunohistochemistry of patient tissues. Except for SLCO1B3, diffuse and low expression was observed for each marker in normal colon tissues. The three markers with the greatest protein overexpression were CLDN1, LY6G6D/F and TLR4, where at least one of these markers was overexpressed in 97% of the CRC samples. GPR56, LY6G6D/F and SLCO1B3 protein expression was significantly correlated with the proximal tumor location and with expression of mismatch repair genes. Marker expression was further validated in CRC cell lines. Hence, three cell-surface markers were discovered that distinguish CRC from surrounding normal tissues. These markers can be used to develop imaging or therapeutic agents targeted to the luminal surface of CRC.

  14. Cell-surface markers for colon adenoma and adenocarcinoma

    PubMed Central

    Sewda, Kamini; Coppola, Domenico; Enkemann, Steven; Yue, Binglin; Kim, Jongphil; Lopez, Alexis S.; Wojtkowiak, Jonathan W.; Stark, Valerie E.; Morse, Brian; Shibata, David; Vignesh, Shivakumar; Morse, David L.

    2016-01-01

    Early detection of colorectal cancer (CRC) is crucial for effective treatment. Among CRC screening techniques, optical colonoscopy is widely considered the gold standard. However, it is a costly and invasive procedure with a low rate of compliance. Our long-term goal is to develop molecular imaging agents for the non-invasive detection of CRC by molecular imaging-based colonoscopy using CT, MRI or fluorescence. To achieve this, cell surface targets must be identified and validated. Here, we report the discovery of cell-surface markers that distinguish CRC from surrounding tissues that could be used as molecular imaging targets. Profiling of mRNA expression microarray data from patient tissues including adenoma, adenocarcinoma, and normal gastrointestinal tissues was used to identify potential CRC specific cell-surface markers. Of the identified markers, six were selected for further validation (CLDN1, GPR56, GRM8, LY6G6D/F, SLCO1B3 and TLR4). Protein expression was confirmed by immunohistochemistry of patient tissues. Except for SLCO1B3, diffuse and low expression was observed for each marker in normal colon tissues. The three markers with the greatest protein overexpression were CLDN1, LY6G6D/F and TLR4, where at least one of these markers was overexpressed in 97% of the CRC samples. GPR56, LY6G6D/F and SLCO1B3 protein expression was significantly correlated with the proximal tumor location and with expression of mismatch repair genes. Marker expression was further validated in CRC cell lines. Hence, three cell-surface markers were discovered that distinguish CRC from surrounding normal tissues. These markers can be used to develop imaging or therapeutic agents targeted to the luminal surface of CRC. PMID:26894861

  15. Spica prunellae and its marker compound rosmarinic acid induced the expression of efflux transporters through activation of Nrf2-mediated signaling pathway in HepG2 cells.

    PubMed

    Wu, Jinjun; Zhu, Yuanfeng; Li, Fangyuan; Zhang, Guiyu; Shi, Jian; Ou, Rilan; Tong, Yunli; Liu, Yuting; Liu, Liang; Lu, Linlin; Liu, Zhongqiu

    2016-12-04

    Spica prunellae (SP) is a well-known traditional Chinese medicinal herb with properties of antihypertensive, antihyperglycemic, antiviral, anti-inflammatory, and antitumor activities. This herb is also popularly consumed as a food additive in some drinks or other food forms for treating pyreticosis. Rosmarinic acid (RA) is the marker compound from SP, which possesses anti-oxidative and anti-inflammatory functions. This study aims to investigate the regulatory effect of the water extract of SP (WESP) and RA on efflux transports (ETs), including P-glycoprotein (p-gp), multidrug resistance-associated protein 2 (MRP2), and breast cancer resistance protein (BCRP) in HepG2 cell line. Results would provide beneficial information for the proper application of SP in clinics. HepG2 cells were treated with different doses of the tested drugs for 24 or 96h. MTT assay was used to examine cell viability. The protein and mRNA levels of the ETs were measured by using Western blot and real-time PCR, respectively. Reporter assay was used to study the antioxidant response element (ARE)-luciferin activity by using HepG2-C8 cells, which were generated by transfecting plasmid containing ARE-luciferin gene into HepG2 cells. The transport activities of ETs were tested by using substrate probes. WESP significantly (p<0.05) increased the expression of ETs in a dose-dependent manner. The increase caused by WESP was stronger than RA alone. Both WESP and RA promoted the translocation of nuclear factor E2-related factor-2 (Nrf2) from cytoplasm to the nucleus as well as significantly (p<0.05) enhanced the ARE-luciferin activity. WESP and RA also enhanced the efflux activity of P-gp and MRP2, accompanied by marked increase (p<0.05) in the intracellular ATP levels. WESP could significantly induce the expression of ETs through the activation of Nrf2-mediated signaling pathway in HepG2 cells. RA could be one of the active compounds responsible for the induction. WESP and RA also enhanced the efflux

  16. Identification and expression of troponin T, a new marker on the surface of cultured tumor endothelial cells by aptamer ligand

    PubMed Central

    Ara, Mst Naznin; Hyodo, Mamoru; Ohga, Noritaka; Akiyama, Kosuke; Hida, Kyoko; Hida, Yasuhiro; Shinohara, Nobuo; Harashima, Hideyoshi

    2014-01-01

    The identification of a specific biomarker involves the development of new clinical diagnostic tools, and an in-depth understanding of the disease at the molecular level. When new blood vessels form in tumor cells, endothelial cell production is induced, a process that plays a key role in disease progression and metastasis to distinct organs for solid tumor types. The present study reports on the identification of a new biomarker on primary cultured mouse tumor endothelial cells (mTECs) using our recently developed high-affinity DNA aptamer AraHH001 (Kd = 43 nmol/L) assisted proteomics approach. We applied a strategy involving aptamer-facilitated biomarker discovery. Biotin-tagged AraHH001 was incubated with lysates of mTECs and the aptamer-proteins were then conjugated with streptavidin magnetic beads. Finally, the bound proteins were separated by sodiumdodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with silver staining. We identified troponin T via matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry, the molecular target of aptamer AraHH001, and its presence was confirmed by measuring mRNA, protein levels, western blot, immunostaining, a gel shift assay of AraHH001 with troponin T. We first report here on the discovery of troponin T on mTECs, a promising and interesting diagnostic tool in the development of antiangiogenic therapy techniques the involves the targeting of the tumor vasculature. PMID:24810801

  17. Differential expression of prognostic proteomic markers in primary tumour, venous tumour thrombus and metastatic renal cell cancer tissue and correlation with patient outcome.

    PubMed

    Laird, Alexander; O'Mahony, Fiach C; Nanda, Jyoti; Riddick, Antony C P; O'Donnell, Marie; Harrison, David J; Stewart, Grant D

    2013-01-01

    Renal cell carcinoma (RCC) is the most deadly of urological malignancies. Metastatic disease affects one third of patients at diagnosis with a further third developing metastatic disease after extirpative surgery. Heterogeneity in the clinical course ensures predicting metastasis is notoriously difficult, despite the routine use of prognostic clinico-pathological parameters in risk stratification. With greater understanding of pathways involved in disease pathogenesis, a number of biomarkers have been shown to have prognostic significance, including Ki67, p53, vascular endothelial growth factor receptor 1 (VEGFR1) and ligand D (VEGFD), SNAIL and SLUG. Previous pathway analysis has been from study of the primary tumour, with little attention to the metastatic tumours which are the focus of targeted molecular therapies. As such, in this study a tissue microarray from 177 patients with primary renal tumour, renal vein tumour thrombus and/or RCC metastasis has been created and used with Automated Quantitative Analysis (AQUA) of immunofluorescence to study the prognostic significance of these markers in locally advanced and metastatic disease. Furthermore, this has allowed assessment of differential protein expression between the primary tumours, renal vein tumour thrombi and metastases. The results demonstrate that clinico-pathological parameters remain the most significant predictors of cancer specific survival; however, high VEGFR1 or VEGFD can predict poor cancer specific survival on univariate analysis for locally advanced and metastatic disease. There was significantly greater expression of Ki67, p53, VEGFR1, SLUG and SNAIL in the metastases compared with the primary tumours and renal vein tumour thrombi. With the exception of p53, these differences in protein expression have not been shown previously in RCC. This confirms the importance of proliferation, angiogenesis and epithelial to mesenchymal transition in the pathogenesis and metastasis of RCC. Importantly

  18. The expansion of adult stem/progenitor cells and their marker expression fluctuations are linked with pituitary plastic adaptation during gestation and lactancy.

    PubMed

    Vaca, Alicia Maldré; Guido, Carolina Beatriz; Sosa, Liliana Del Valle; Nicola, Juan Pablo; Mukdsi, Jorge; Petiti, Juan Pablo; Torres, Alicia Ines

    2016-08-01

    Extensive evidence has revealed variations in the number of hormone-producing cells in the pituitary gland, which occur under physiological conditions such as gestation and lactancy. It has been proposed that new hormone-producing cells differentiate from stem cells. However, exactly how and when this takes place is not clear. In this work, we used immunoelectron microscopy to identify adult pituitary stem/progenitor cells (SC/P) localized in the marginal zone (MZ), and additionally, we detected GFRa2-, Sox2-, and Sox9-positive cells in the adenoparenchyma (AP) by fluorescence microscopy. Then, we evaluated fluctuations of SC/P mRNA and protein level markers in MZ and AP during gestation and lactancy. An upregulation in stemness markers was shown at term of gestation (AT) in MZ, whereas there were more progenitor cell markers in the middle of gestation and active lactancy. Concerning committed cell markers, we detected a rise in AP at beginning of lactancy (d1L). We performed a BrdU uptake analysis in MZ and AP cells. The highest level of BrdU uptake was observed in MZ AT cells, whereas in AP this was detected in d1L, followed by a decrease in both the MZ and AP. Finally, we detected double immunostaining for BrdU-GFRa2 in MZ AT cells and BrdU-Sox9 in the AP d1L cells. Taken together, we hypothesize that the expansion of the SC/P niche took place mainly in MZ from pituitary rats in AT and d1L. These results suggest that the SC niche actively participates in pituitary plasticity during these reproductive states, contributing to the origin of hormone cell populations.

  19. The Neuropeptide Y Y1 Receptor: A Diagnostic Marker? Expression in MCF-7 Breast Cancer Cells Is Down-Regulated by Antiestrogens In Vitro and in Xenografts

    PubMed Central

    Memminger, Martin; Keller, Max; Lopuch, Miroslaw; Pop, Nathalie; Bernhardt, Günther; von Angerer, Erwin; Buschauer, Armin

    2012-01-01

    The neuropeptide Y (NPY) Y1 receptor (Y1R) has been suggested as a tumor marker for in vivo imaging and as a therapeutic target. In view of the assumed link between estrogen receptor (ER) and Y1R in mammary carcinoma and with respect to the development of new diagnostic tools, we investigated the Y1R protein expression in human MCF-7 cell variants differing in ER content and sensitivity against antiestrogens. ER and Y1R expression were quantified by radioligand binding using [3H]-17β-estradiol and the Y1R selective antagonist [3H]-UR-MK114, respectively. The latter was used for cellular binding studies and for autoradiography of MCF-7 xenografts. The fluorescent ligands Cy5-pNPY (universal Y1R, Y2R and Y5R agonist) and UR-MK22 (selective Y1R antagonist), as well as the selective antagonists BIBP3226 (Y1R), BIIE0246 (Y2R) and CGP71683 (Y5R) were used to identify the NPY receptor subtype(s) by confocal microscopy. Y1R functionality was determined by mobilization of intracellular Ca2+. Sensitivity of MCF-7 cells against antiestrogen 4-hydroxytamoxifen correlated directly with the ER content. The exclusive expression of Y1Rs was confirmed by confocal microscopy. The Y1R protein was up-regulated (100%) by 17β-estradiol (EC50 20 pM) and the predominant role of ERα was demonstrated by using the ERα-selective agonist “propylpyrazole triol”. 17β-Estradiol-induced over-expression of functional Y1R protein was reverted by the antiestrogen fulvestrant (IC50 5 nM) in vitro. Furthermore, tamoxifen treatment of nude mice resulted in an almost total loss of Y1Rs in MCF-7 xenografts. In conclusion, the value of the Y1R as a target for therapy and imaging in breast cancer patients may be compromised due to Y1R down-regulation induced by hormonal (antiestrogen) treatment. PMID:23236424

  20. Neuroendocrine marker expression in thyroid epithelial tumors.

    PubMed

    Satoh, F; Umemura, S; Yasuda, M; Osamura, R Y

    2001-01-01

    Tissue sections from 50 cases with thyroid tumors, composed of 11 follicular adenomas, 10 follicular carcinomas, 14 papillary carcinomas, 10 anaplastic carcinomas, and 5 medullary carcinomas, were immunohistochemically analyzed for representative neuroendocrine markers. Immunoexpression ratios of these neuroendocrine markers were as follows: Follicular adenomas, neuron-specific enolase (NSE)63.6%, synaptophysin (SynP) 45.5%, Leu7 27.3%, NCAM 45.5%, chromogranin A (CgA) 0%, SNAP25 0%; follicular carcinomas, NSE 90.0%, SynP 80.0%, Leu7 80.0%, NCAM 0%, CgA 0%, SNAP25 0%; papillary carcinomas, NSE 85.7%, SynP 78.6%, Leu7 100%, NCAM 7.0%, CgA 0%, SNAP25.0%; anaplastic carcinomas, NSE 10.0%, SynP 0%, Leu7 0%, NCAM 0%, CgA 0%, SNAP25 0%; medullary carcinomas, NSE 100%, SynP100%, Leu7 80.0%, NCAM 40.0%, CgA 100%, SNAP25 100%. The two follicular carcinomas, which were morphologically characterized by "insular" (or "alveolar") arrangements, showed distinct immunoexpression of NSE and SynP at the same time. By in situ hybridization (ISH), expression of mRNA for NSE was confirmed in cases with marked immunoexpression of NSE. Although no endocrine granules were found, our results suggested that a specific type of follicular carcinoma, i.e., insular variant, may be immaturely neuroendocrine-differentiated.

  1. Expression of unusual immunohistochemical markers in mucinous breast carcinoma.

    PubMed

    de Andrade Natal, Rodrigo; Derchain, Sophie F; Pavanello, Marina; Paiva, Geisilene R; Sarian, Luis O; Vassallo, José

    2017-04-01

    Mucinous breast carcinoma is characterized by the production of variable amounts of mucin. Some studies have addressed immunohistochemical characterization of mucinous breast carcinoma using a limited set of antibodies. However, the purpose of the present study was to investigate a larger panel of markers not widely used in daily practice and to determine their pathological implications. Forty patients diagnosed with mucinous breast carcinoma were enrolled. An immunohistochemical study was performed on whole sections of paraffin embedded tissue, using antibodies for the following markers: estrogen receptor alpha and beta, progesterone receptor, androgen receptor, HER2, EGFR, Ki-67, E-cadherin, β-catenin, p53, chromogranin, synaptophysin, GCDFP15, mammaglobin, and CDX2. The pure mucinous type was more prevalent in older patients and more frequently expressed GCDFP15. Capella type B presented more frequently with a high Ki-67 index and neuroendocrine differentiation. Although there was a lower frequency of vascular invasion and lymph node metastases in the pure type, the difference was not statistically significant. No case expressed CDX2 (a marker for gastrointestinal tumors), while 85% of the cases expressed at least one of the two typical breast markers (GCDFP15 and mammaglobin), suggesting that these markers may be reliably used for differential diagnosis. Expression of estrogen receptor beta was related to the presence of mucin cell producing lymph node metastasis, with potential prognostic and predictive value. our findings support the immunohistochemical homogeneity of mucinous breast carcinomas because only minor differences were found when subgrouping them into Capella types A and B or into types pure and mixed. Copyright © 2017 Elsevier GmbH. All rights reserved.

  2. Effect of insoluble fibre on intestinal morphology and mRNA expression pattern of inflammatory, cell cycle and growth marker genes in a piglet model.

    PubMed

    Schedle, Karl; Pfaffl, Michael W; Plitzner, Christian; Meyer, Heinrich H D; Windisch, Wilhelm

    2008-12-01

    The effects of insoluble dietary fibre differing in lignin content on intestinal morphology and mRNA expression was tested in an animal model of 48 weaned piglets. Engaged fibre sources were wheat bran (rich in cellulose and hemicellulose) and pollen from Chinese Masson pine (Pinus massoniana) (rich in lignin), respectively. The fibre sources were added to a basal diet as follows: no addition (control), 3.0% wheat bran, 1.27% pine pollen, and 2.55% pine pollen. The 12 animals of each feeding group were fed four experimental diets ad libitum for 37 days and were then slaughtered for retrieving tissue samples from stomach, jejunum, ileum, colon and mesenterial lymph nodes. Both fibre sources increased villus height of mucosa in jejunum (+10% on average) and ileum (+16% on average). Results of mRNA expression rates of inflammatory, cell cycle and growth marker genes (NFkappaB, TNFalpha, TGFbeta, Caspase3, CDK4, IGF1) were specific to fibre source and tissue: wheat bran induced an up-regulation of NFkappaB in stomach and jejunum, as well as TNFalpha and TGFbeta, and Caspase3 in jejunum. Pine pollen induced down regulation of NFkappaB, TNFalpha, TGFbeta, Caspase3, CDK4 and IGF1 in the colon as well as up-regulation of NFkappaB and TGFbeta in mesenterial lymph nodes. Finally, an overall data comparison based on a hierarchical cluster analysis showed a close relation between gene regulation in different gut sections and organs, as well as between small intestine morphology and zootechnical performance.

  3. Marker-specific sorting of rare cells using dielectrophoresis

    PubMed Central

    Hu, Xiaoyuan; Bessette, Paul H.; Qian, Jiangrong; Meinhart, Carl D.; Daugherty, Patrick S.; Soh, Hyongsok T.

    2005-01-01

    Current techniques in high-speed cell sorting are limited by the inherent coupling among three competing parameters of performance: throughput, purity, and rare cell recovery. Microfluidics provides an alternate strategy to decouple these parameters through the use of arrayed devices that operate in parallel. To efficiently isolate rare cells from complex mixtures, an electrokinetic sorting methodology was developed that exploits dielectrophoresis (DEP) in microfluidic channels. In this approach, the dielectrophoretic amplitude response of rare target cells is modulated by labeling cells with particles that differ in polarization response. Cell mixtures were interrogated in the DEP-activated cell sorter in a continuous-flow manner, wherein the electric fields were engineered to achieve efficient separation between the dielectrophoretically labeled and unlabeled cells. To demonstrate the efficiency of marker-specific cell separation, DEP-activated cell sorting (DACS) was applied for affinity-based enrichment of rare bacteria expressing a specific surface marker from an excess of nontarget bacteria that do not express this marker. Rare target cells were enriched by >200-fold in a single round of sorting at a single-channel throughput of 10,000 cells per second. DACS offers the potential for automated, surface marker-specific cell sorting in a disposable format that is capable of simultaneously achieving high throughput, purity, and rare cell recovery. PMID:16236724

  4. Cancer stem cell marker glycosylation: Nature, function and significance.

    PubMed

    Mallard, Brody W; Tiralongo, Joe

    2017-08-01

    Glycans are essential for the maintenance of normal biological function, with alterations in glycan expression being a hallmark of cancer. Cancer stem cells (CSCs) are a subset of cells within a tumour capable of self-renewal, cellular differentiation and resistances to conventional therapies. As is the case with stem cells, marker proteins present on the cell surface are frequently used to identify and enrich CSCs, with the expression of these markers statistical correlating with the likelihood of cancer recurrence and overall patient survival. As such CSC markers are of high clinical relevance. The majority of markers currently used to identify CSC populations are glycoproteins, and although the diverse biological roles for many of these markers are known, the nature and function of the glycan moiety on these glycoproteins remains to be fully elucidated. This mini-review summarises our current knowledge regarding the types and extent of CSC marker glycosylation, and the various roles that these glycans play in CSC biology, including in mediating cell adhesion, metastasis, evading apoptosis, tear shear resistance, tumour growth, maintaining pluripotency, self-renewal, trafficking, maintaining stability, maintaining enzymatic activity and aiding epithelial mesenchymal transitioning. Given that CSCs markers have multiple diverse biological functions, and are potentially of significant diagnostic and therapeutic benefit the search for new markers that are uniquely expressed on CSCs is vital to selectively target/identify this subset of cancer cells. As such we have also outlined how high-throughput lectin microarrays can be used to successfully profile the glycosylation status of CSC and to identify glyco-markers unique to CSCs.

  5. Novel Identity and Functional Markers for Human Corneal Endothelial Cells

    PubMed Central

    Bartakova, Alena; Alvarez-Delfin, Karen; Weisman, Alejandra D.; Salero, Enrique; Raffa, Gabriella A.; Merkhofer, Richard M.; Kunzevitzky, Noelia J.; Goldberg, Jeffrey L.

    2016-01-01

    Purpose Human corneal endothelial cell (HCEC) density decreases with age, surgical complications, or disease, leading to vision impairment. Such endothelial dysfunction is an indication for corneal transplantation, although there is a worldwide shortage of transplant-grade tissue. To overcome the current poor donor availability, here we isolate, expand, and characterize HCECs in vitro as a step toward cell therapy. Methods Human corneal endothelial cells were isolated from cadaveric corneas and expanded in vitro. Cell identity was evaluated based on morphology and immunocytochemistry, and gene expression analysis and flow cytometry were used to identify novel HCEC-specific markers. The functional ability of HCEC to form barriers was assessed by transendothelial electrical resistance (TEER) assays. Results Cultured HCECs demonstrated canonical morphology for up to four passages and later underwent endothelial-to-mesenchymal transition (EnMT). Quality of donor tissue influenced cell measures in culture including proliferation rate. Cultured HCECs expressed identity markers, and microarray analysis revealed novel endothelial-specific markers that were validated by flow cytometry. Finally, canonical HCECs expressed higher levels of CD56, which correlated with higher TEER than fibroblastic HCECs. Conclusions In vitro expansion of HCECs from cadaveric donor corneas yields functional cells identifiable by morphology and a panel of novel markers. Markers described correlated with function in culture, suggesting a basis for cell therapy for corneal endothelial dysfunction. PMID:27196322

  6. Porcine EPCs downregulate stem cell markers and upregulate endothelial maturation markers during in vitro cultivation.

    PubMed

    Avci-Adali, Meltem; Nolte, Andrea; Simon, Perikles; Ziemer, Gerhard; Wendel, Hans P

    2009-10-01

    In recent years, interest in endothelial progenitor cells (EPCs) in the field of tissue engineering and regenerative medicine has increased tremendously. However, each clinical stem cell application requires prior validation through animal experiments. This study investigates the isolation and characterization of porcine EPCs from peripheral blood and the change of their cell surface marker expression during in vitro cultivation. RT-PCR demonstrated that the EPCs express stem cell markers CD34 and CD133, which decrease with in vitro cultivation time. Throughout the cultivation process EPCs did not express monocytic (CD14) or haematopoietic marker (CD45). Surprisingly, the CD31 and VE-cadherin expression in EPCs was significantly higher than in endothelial cells (ECs). In contrast, the VEGFR2 and E-selectin expression was significantly lower than in ECs, but increased during the expansion process. This study clarifies the characteristic properties of porcine EPCs during cell culture and may help to improve the impact of EPC-based therapies in porcine animal studies.

  7. MCM4 and MCM7, potential novel proliferation markers, significantly correlated with Ki-67, Bmi1, and cyclin E expression in esophageal adenocarcinoma, squamous cell carcinoma, and precancerous lesions.

    PubMed

    Choy, Bonnie; LaLonde, Amy; Que, Jianwen; Wu, Tongtong; Zhou, Zhongren

    2016-11-01

    Minichromosomal maintenance (MCM) proteins are participants of DNA replication and may represent more accurate markers in determining the proliferative fraction within a tumor than proliferative marker Ki-67. Our study investigated the correlation between MCM4 and MCM7 expression and Ki-67, Bmi1, and cyclin E expression in esophageal adenocarcinoma, squamous cell carcinoma, and precancerous lesions. MCM4 and MCM7 expression had similar distribution as Ki-67 and Bmi1 expression in esophageal carcinoma and precancerous lesions. The mean percentage of MCM4, MCM7, and Ki-67 expression increased from squamous epithelium (5.5%, 7.3%, and 5.9%, respectively), to columnar cell metaplasia (11.2, 13.5%, and 3.4%), Barrett's esophagus (27.7%, 35.3%, and 8.3%), low-grade dysplasia (42.6%, 52.2%, and 12.9%), high-grade dysplasia (63.2%, 77.7%, and 29.6%), adenocarcinoma (61.3%, 75.5%, and 24.5%), and squamous cell carcinoma (74.1, 85.4%, and 36.3%). The percentages of MCM4 and MCM7 expression were significantly higher than Ki-67 expression. Using univariate analysis we found a high percentage of MCM4 expression (>70%) to be significantly associated with lymph node metastasis and shorter survival in the adenocarcinoma group. We also demonstrated the percentage of MCM4 and MCM7 expression to be significantly correlated with Ki-67, Bmi1, and cyclin E expression in esophageal carcinoma and precancerous lesions. MCM4 and MCM7 may serve as more sensitive proliferative markers for the evaluation of esophageal lesions. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  8. High expression of stathmin 1 is a strong prognostic marker in oral squamous cell carcinoma patients treated by docetaxel-containing regimens.

    PubMed

    Harada, Koji; Ferdous, Tarannum; Harada, Toyoko; Ueyama, Yoshiya

    2017-02-01

    Stathmin 1 is an oncoprotein that regulates cell cycle by modulating microtubule dynamics and can cause uncontrolled cell proliferation in mutated state. The present study examined stathmin 1 expression in 49 patients with oral squamous cell carcinoma (OSCC) treated with docetaxel (Doc)-containing regimens by immunohistochemical staining. Moreover, we investigated the relationship between stathmin 1 expression and clinicopathological features, as well as the prognosis of above patients. Stathmin 1 could be detected in the cytoplasm of tumor cells in OSCC tissues though its expression level was variable. There was no correlation between stathmin 1 expression and patient gender, or age in OSCC. However, stathmin 1 expression of tumor cell was significantly correlated with T classification (P = 0.0017), N classification (P = 0.0171), stage (P < 0.0001), therapeutic efficacy (P < 0.0001), and patient outcome (P = 0.0387). In addition, high expression of stathmin 1 in tumor cells was associated with shorter overall survival (OS, P = 0.0017). Multivariate analysis also revealed that high expression of stathmin 1 was a predictor of reduced survival (P = 0.0241). These findings suggest that patients with OSCC tumors showing high expression of stathmin 1 might have poor therapeutic effects and worse clinical outcomes in OSCC treated with Doc-containing regimen.

  9. High expression of the stem cell marker nestin is an adverse prognostic factor in WHO grade II-III astrocytomas and oligoastrocytomas

    PubMed Central

    Hatanpaa, Kimmo J.; Foong, Chan; Raisanen, Jack M.; Oliver, Dwight; Hiemenz, Matthew C.; Burns, Dennis K.; White, Charles L.; Whitworth, L. Anthony; Mickey, Bruce; Stegner, Martha; Habib, Amyn A.; Fink, Karen; Maher, Elizabeth A.; Bachoo, Robert M.

    2015-01-01

    Infiltrating astrocytomas and oligoastrocytomas of low to anaplastic grade (WHO grades II and III), in spite of being associated with a wide range of clinical outcomes, can be difficult to subclassify and grade by the current histopathologic criteria. Unlike oligodendrogliomas and anaplastic oligodendrogliomas that can be identified by the 1p/19q codeletion and the more malignant glioblastomas (WHO grade IV astrocytomas) that can be diagnosed solely based on objective features on routine hematoxylin and eosin sections, no such objective criteria exist for the subclassification of grade II-III astrocytomas and oligoastrocytomas (A+OA II-III). In this study, we evaluated the prognostic and predictive value of the stem cell marker nestin in adult A+OA II-III (n=50) using immunohistochemistry and computer-assisted analysis on tissue microarrays. In addition, the correlation between nestin mRNA level and total survival was analyzed in the NCI Rembrandt database. The results showed that high nestin expression is a strong adverse prognostic factor for total survival (p=0.0004). The strength of the correlation was comparable to but independent of the isocitrate dehydrogenase 1/2 (IDH 1/2) mutation status. Histopathological grading and subclassification did not correlate significantly with outcome, although the interpretation of this finding is limited by the fact that grade III tumors were treated more aggressively than grade II tumors. These results suggest that nestin level and IDH 1/2 mutation status are strong prognostic features in A+OA II-III and possibly more helpful for treatment planning than routine histopathological variables such as oligodendroglial component (astrocytoma vs. oligoastrocytoma) and WHO grade (grade II vs. III). PMID:24519516

  10. High expression of the stem cell marker nestin is an adverse prognostic factor in WHO grade II-III astrocytomas and oligoastrocytomas.

    PubMed

    Hatanpaa, Kimmo J; Hu, Tianshen; Vemireddy, Vamsidhara; Foong, Chan; Raisanen, Jack M; Oliver, Dwight; Hiemenz, Matthew C; Burns, Dennis K; White, Charles L; Whitworth, L Anthony; Mickey, Bruce; Stegner, Martha; Habib, Amyn A; Fink, Karen; Maher, Elizabeth A; Bachoo, Robert M

    2014-03-01

    Infiltrating astrocytomas and oligoastrocytomas of low to anaplastic grade (WHO grades II and III), in spite of being associated with a wide range of clinical outcomes, can be difficult to subclassify and grade by the current histopathologic criteria. Unlike oligodendrogliomas and anaplastic oligodendrogliomas that can be identified by the 1p/19q codeletion and the more malignant glioblastomas (WHO grade IV astrocytomas) that can be diagnosed solely based on objective features on routine hematoxylin and eosin sections, no such objective criteria exist for the subclassification of grade II-III astrocytomas and oligoastrocytomas (A+OA II-III). In this study, we evaluated the prognostic and predictive value of the stem cell marker nestin in adult A+OA II-III (n = 50) using immunohistochemistry and computer-assisted analysis on tissue microarrays. In addition, the correlation between nestin mRNA level and total survival was analyzed in the NCI Rembrandt database. The results showed that high nestin expression is a strong adverse prognostic factor for total survival (p = 0.0004). The strength of the correlation was comparable to but independent of the isocitrate dehydrogenase 1/2 (IDH 1/2) mutation status. Histopathological grading and subclassification did not correlate significantly with outcome, although the interpretation of this finding is limited by the fact that grade III tumors were treated more aggressively than grade II tumors. These results suggest that nestin level and IDH 1/2 mutation status are strong prognostic features in A+OA II-III and possibly more helpful for treatment planning than routine histopathological variables such as oligodendroglial component (astrocytoma vs. oligoastrocytoma) and WHO grade (grade II vs. III).

  11. TGF-β1 pathway affects the protein expression of many signaling pathways, markers of liver cancer stem cells, cytokeratins, and TERT in liver cancer HepG2 cells.

    PubMed

    Wang, Xin-Hong; Liu, Ming-Na; Sun, Xun; Xu, Chun-Huan; Liu, Jing; Chen, Jing; Xu, Rui-Ling; Li, Bao-Xin

    2016-03-01

    Liver cancer is one of the most common human malignancies, and transforming growth factor-beta (TGF-β) pathway plays a key role in its pathogenesis. To study the relationship between TGF-β pathway and the related protein expression of many signaling pathway, markers of stem cells, CK family, and others, liver cancer HepG2 cells were transfected with siRNA directed against TGF-β1 or were treated with exogenous TGF-β1. Then, these protein levels were measured by Western blotting. After siRNA transfection, TGF-β1 protein level was decreased, indicating that the siRNA against it was effective. In exogenous TGF-β1 group, the expression of smad4, smad2/3, and β-catenin proteins was increased, whereas that of p-smad2/3, CD133, cleaved Notch1, and epithelial cell adhesion molecule (EpCAM) proteins at 48 h was decreased. The expression of CK8 and CK18 proteins was increased at 24 h and was decreased at 48 and 96 h. In TGF-β1-silenced group, the expression of smad2/3, β-catenin, cleaved-notch1, and CK18 proteins was decreased, while that of smad4, p-smad2/3, CD133, EpCAM, and CK8 proteins was increased. TERT protein expression was slightly increased in exogenous TGF-β1 group at 48 h and in TGF-β1-silenced group at 96 h. TGF-β1 did not affect the protein expression of CK19 and HIF-1. Thus, TGF-β1 pathway plays an important role in cell regulation of liver cancer through the modulation of these proteins. These data will contribute to the understanding of the pathogenesis of liver cancer and the role of TGF-β pathway in this process.

  12. Increased expression of putative cancer stem cell markers in the bone marrow of prostate cancer patients is associated with bone metastasis progression.

    PubMed

    Ricci, Estelle; Mattei, Eve; Dumontet, Charles; Eaton, Colby L; Hamdy, Freddy; van der Pluije, Gabri; Cecchini, Marco; Thalmann, George; Clezardin, Philippe; Colombel, Marc

    2013-12-01

    The number of cells positive for the α-6 and α-2 integrin subunits and the c-Met receptor in primary tumors and bone biopsies from prostate cancer patients has been correlated with metastasis and disease progression. The objective of this study was to quantify disseminated tumour cells present in bone marrow in prostate cancer patients using specific markers and determine their correlation with metastasis and survival. Patients were included at different stage of prostate cancer disease, from localised to metastatic castration-resistant prostate cancer. Healthy men were used as a control group. Bone marrow samples were collected and nucleated cells separated. These were stained for CD45, α-2, α-6 integrin subunits and c-Met and samples were processed for analysis and quantification of CD45-/α2+/α6+/c-met + cells using flow cytometry. Clinical and pathological parameters were assessed and survival measured. Statistical analyses were made of associations between disease specific parameters, bone marrow flow cytometry data, prostate-specific antigen (PSA) progression free survival and bone metastases progression free survival. For all markers, the presence of more than 0.1% positive cells in bone marrow aspirates was significantly associated with the risk of biochemical progression, the risk of developing metastasis and death from prostate cancer. Quantification of cells carrying putative stem cell markers in bone marrow is a potential indicator of disease progression. Functional studies on isolated cells are needed to show more specifically their property for metastatic spread in prostate cancer. © 2013 Wiley Periodicals, Inc.

  13. Impact of the putative cancer stem cell markers and growth factor receptor expression on the sensitivity of ovarian cancer cells to treatment with various forms of small molecule tyrosine kinase inhibitors and cytotoxic drugs

    PubMed Central

    Puvanenthiran, Soozana; Essapen, Sharadah; Seddon, Alan M.; Modjtahedi, Helmout

    2016-01-01

    Increased expression and activation of human epidermal growth factor receptor (EGFR) and HER-2 have been reported in numerous cancers. The aim of this study was to determine the sensitivity of a large panel of human ovarian cancer cell lines (OCCLs) to treatment with various forms of small molecule tyrosine kinase inhibitors (TKIs) and cytotoxic drugs. The aim was to see if there was any association between the protein expression of various biomarkers including three putative ovarian cancer stem cell (CSC) markers (CD24, CD44, CD117/c-Kit), P-glycoprotein (P-gp), and HER family members and response to treatment with these agents. The sensitivity of 10 ovarian tumour cell lines to the treatment with various forms of HER TKIs (gefitinib, erlotinib, lapatinib, sapitinib, afatinib, canertinib, neratinib), as well as other TKIs (dasatinib, imatinib, NVP-AEW541, crizotinib) and cytotoxic agents (paclitaxel, cisplatin and doxorubicin), as single agents or in combination, was determined by SRB assay. The effect on these agents on the cell cycle distribution, and downstream signaling molecules and tumour migration were determined using flow cytometry, western blotting, and the IncuCyte Clear View cell migration assay respectively. Of the HER inhibitors, the irreversible pan-TKIs (canertinib, neratinib and afatinib) were the most effective TKIs for inhibiting the growth of all ovarian cancer cells, and for blocking the phosphorylation of EGFR, HER-2, AKT and MAPK in SKOV3 cells. Interestingly, while the majority of cancer cells were highly sensitive to treatment with dasatinib, they were relatively resistant to treatment with imatinib (i.e., IC50 >10 μM). Of the cytotoxic agents, paclitaxel was the most effective for inhibiting the growth of OCCLs, and of various combinations of these drugs, only treatment with a combination of NVP-AEW541 and paclitaxel produced a synergistic or additive anti-proliferative effect in all three cell lines examined (i.e., SKOV3, Caov3, ES2

  14. HPV status, cancer stem cell marker expression, hypoxia gene signatures and tumour volume identify good prognosis subgroups in patients with HNSCC after primary radiochemotherapy: A multicentre retrospective study of the German Cancer Consortium Radiation Oncology Group (DKTK-ROG).

    PubMed

    Linge, Annett; Lohaus, Fabian; Löck, Steffen; Nowak, Alexander; Gudziol, Volker; Valentini, Chiara; von Neubeck, Cläre; Jütz, Martin; Tinhofer, Inge; Budach, Volker; Sak, Ali; Stuschke, Martin; Balermpas, Panagiotis; Rödel, Claus; Grosu, Anca-Ligia; Abdollahi, Amir; Debus, Jürgen; Ganswindt, Ute; Belka, Claus; Pigorsch, Steffi; Combs, Stephanie E; Mönnich, David; Zips, Daniel; Buchholz, Frank; Aust, Daniela E; Baretton, Gustavo B; Thames, Howard D; Dubrovska, Anna; Alsner, Jan; Overgaard, Jens; Krause, Mechthild; Baumann, Michael

    2016-12-01

    To investigate the impact of the tumour volume, HPV status, cancer stem cell (CSC) marker expression and hypoxia gene signatures, as potential markers of radiobiological mechanisms of radioresistance, in a contemporary cohort of patients with locally advanced head and neck squamous cell carcinoma (HNSCC), who received primary radiochemotherapy (RCTx). For 158 patients with locally advanced HNSCC of the oral cavity, oropharynx or hypopharynx who were treated at six DKTK partner sites, the impact of tumour volume, HPV DNA, p16 overexpression, p53 expression, CSC marker expression and hypoxia-associated gene signatures on outcome of primary RCTx was retrospectively analyzed. The primary endpoint of this study was loco-regional control (LRC). Univariate Cox regression revealed a significant impact of tumour volume, p16 overexpression, and SLC3A2 and CD44 protein expression on LRC. The tumour hypoxia classification showed a significant impact only for small tumours. In multivariate analyses an independent correlation of tumour volume, SLC3A2 expression, and the 15-gene hypoxia signature with LRC was identified (CD44 protein n/a because of no event in the CD44-negative group). Logistic modelling showed that inclusion of CD44 protein expression and p16 overexpression significantly improved the performance to predict LRC at 2years compared to the model with tumour volume alone. Tumour volume, HPV status, CSC marker expression and hypoxia gene signatures are potential prognostic biomarkers for patients with locally advanced HNSCC, who were treated by primary RCTx. The study also supports that the individual tumour volumes should generally be included in biomarker studies and that panels of biomarkers are superior to individual parameters. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  15. Expression of putative markers of pluripotency in equine embryonic and adult tissues.

    PubMed

    Esteves, Cristina L; Sharma, Ruchi; Dawson, Lucy; Taylor, Sarah E; Pearson, Gemma; Keen, John A; McDonald, Kieran; Aurich, Christine; Donadeu, F Xavier

    2014-12-01

    Expression of several putative markers of pluripotency (OCT4, SOX2, NANOG, LIN28A, REX1, DNMT3B and TERT) was examined in a range of equine tissues, including early embryos, induced pluripotent stem cells (iPSCs), testis, adipose- and bone marrow-derived mesenchymal stromal cells (MSCs), and keratinocytes. Transcript levels of all markers were highest in embryos and iPSCs and, except for SOX2, were very low or undetectable in keratinocytes. Mean expression levels of all markers were lower in testis than in embryos or iPSCs and, except for DNMT3B, were higher in testis than in MSCs. Expression of OCT4, NANOG and DNMT3B, but not the other markers, was detected in MSCs. Of all markers analysed, only LIN28A, REX1 and TERT were associated exclusively with pluripotent cells in the horse.

  16. Expression of endoplasmic reticulum stress markers GRP78 and CHOP induced by oxidative stress in blue light-mediated damage of A2E-containing retinal pigment epithelium cells.

    PubMed

    Feng, Jingyang; Chen, Xunjie; Sun, Xiangjun; Wang, Fenghua; Sun, Xiaodong

    2014-01-01

    Age-related lipofuscin N-retinylidene-N-retinylethanolamine (A2E) accumulated in human retinal pigment epithelium (RPE) cells confers susceptibility to blue light-mediated damage, which represents one pathogenesis of age-related macular degeneration. This study investigated the expression of 2 best-characterized endoplasmic reticulum (ER) stress markers, glucose-related protein 78 (GRP78) and C/EBP homologous protein (CHOP), as well as their regulation by oxidative stress after blue light-mediated damage of A2E-containing RPE cells. ARPE-19 cells were incubated with A2E (10, 25, 50 μM) for 2 h and exposed to blue light for 20 min. A2E distributions in RPE cells were assessed via laser scanning confocal microscope and liquid chromatography-mass spectrometry. Cell viability was measured by a Cell Titer 96 Aqueous One Solution cell proliferation assay. The quantity of intracellular reactive oxygen species (ROS) was detected by dihydroethidium fluorescence using flow cytometry. Expressions of GRP78 and CHOP were measured at both mRNA and protein levels. To examine the role of oxidative stress in regulating GRP78 and CHOP expression, RPE cells were pretreated with the antioxidant N-acetylcysteine (NAC) for 2 h. RNA interference of GRP78 performed by short hairpin RNA was used to evaluate the effect of GRP78 in blue light-mediated damage of RPE cells. After blue light exposure, A2E-treated RPE cells showed a gradual decrease in cell viability and a particular increase in ROS levels. Meanwhile, the expressions of GRP78 and CHOP in A2E-treated RPE cells were significantly increased at different time points after illumination. Pretreatment with NAC attenuated the expression of 2 ER stress markers, especially CHOP in A2E and blue light-treated RPE cells. Silencing of GRP78 by RNA interference upregulated CHOP and caspase-12 expression as well as aggravated the blue light-mediated damage of A2E-laden RPE cells. RPE cells exhibited ROS accumulation and subsequent elevation of

  17. Hypoxia markers are expressed in interneurons exposed to recurrent seizures.

    PubMed

    Gualtieri, Fabio; Marinelli, Carla; Longo, Daniela; Pugnaghi, Matteo; Nichelli, Paolo F; Meletti, Stefano; Biagini, Giuseppe

    2013-03-01

    An early but transient decrease in oxygen availability occurs during experimentally induced seizures. Using pimonidazole, which probes hypoxic insults, we found that by increasing the duration of pilocarpine-induced status epilepticus (SE) from 30 to 120 min, counts of pimonidazole-immunoreactive neurons also increased (P < 0.01, 120 vs 60 and 30 min). All the animals exposed to SE were immunopositive to pimonidazole, but a different scenario emerged during epileptogenesis when a decrease in pimonidazole-immunostained cells occurred from 7 to 14 days, so that only 1 out of 4 rats presented with pimonidazole-immunopositive cells. Pimonidazole-immunoreactive cells robustly reappeared at 21 days post-SE induction when all animals (7 out of 7) had developed spontaneous recurrent seizures. Specific neuronal markers revealed that immunopositivity to pimonidazole was present in cells identified by neuropeptide Y (NPY) or somatostatin antibodies. At variance, neurons immunopositive to parvalbumin or cholecystokinin were not immunopositive to pimonidazole. Pimonidazole-immunopositive neurons expressed remarkable immunoreactivity to hypoxia-inducible factor 1α (HIF-1α). Interestingly, surgical samples obtained from pharmacoresistant patients showed neurons co-labeled by HIF-1α and NPY antibodies. These interneurons, along with parvalbumin-positive interneurons that were negative to HIF-1α, showed immunopositivity to markers of cell damage, such as high-mobility group box 1 in the cytoplasm and cleaved caspase-3 in the nucleus. These findings suggest that interneurons are continuously endangered in rodent and human epileptogenic tissue. The presence of hypoxia and cell damage markers in NPY interneurons of rats and patients presenting with recurrent seizures indicates a mechanism of selective vulnerability in a specific neuronal subpopulation.

  18. Hodgkin's lymphoma cells are efficiently engrafted and tumor marker CD30 is expressed with constitutive nuclear factor-kappaB activity in unconditioned NOD/SCID/gammac(null) mice.

    PubMed

    Dewan, Md Zahidunnabi; Watanabe, Mariko; Ahmed, Sunjida; Terashima, Kazuo; Horiuchi, Sankichi; Sata, Tetsutaro; Honda, Mitsuo; Ito, Mamoru; Watanabe, Toshiki; Horie, Ryouichi; Yamamoto, Naoki

    2005-08-01

    As there are very few reproducible animal models without conditioning available for the study of human B-cell-type Hodgkin's lymphoma (HL), we investigated the ability of HL cells to induce tumors using novel NOD/SCID/gammac(null) (NOG) mice. Four human Epstein-Barr virus-negative cell lines (KM-H2 and L428 originated from B cells, L540 and HDLM2 originated from T cells) were inoculated either subcutaneously in the postauricular region or intravenously in the tail of unmanipulated NOG mice. All cell lines successfully engrafted and produced tumors with infiltration of cells in various organs of all mice. Tumor cells had classical histomorphology as well as expression patterns of the tumor marker CD30, which is a cell surface antigen expressed on HL. Tumor progression in mice inoculated with B-cell-type, but not T-cell-type, HL cells correlated with an elevation in serum human interleukin-6 levels. Tumor cells from the mice also retained strong nuclear factor (NF)-kappaB DNA binding activity, and the induced NF-kappaB components were indistinguishable from those cultured in vitro. The reproducible growth behavior and preservation of characteristic features of both B-cell-type and T-cell-type HL in the mice suggest that this new xenotransplant model can provide a unique opportunity to understand and investigate the mechanism of pathogenesis and malignant cell growth, and to develop novel anticancer therapies.

  19. Circulating soluble triggering receptor expressed on myeloid cells-1 (sTREM-1) as diagnostic and prognostic marker in neonatal sepsis.

    PubMed

    Adly, Amira A M; Ismail, Eman A; Andrawes, Nevine G; El-Saadany, Marwa A

    2014-02-01

    Triggering receptor expressed on myeloid cells-1 (TREM-1) is an important receptor involved in the innate inflammatory response and sepsis. We assessed soluble TREM-1 (sTREM-1) in 112 septic neonates (63 culture-positive and 49 culture-negative) and 40 healthy controls as a potential early diagnostic and prognostic marker for neonatal sepsis (NS). Studied neonates were evaluated for early- or late-onset sepsis using clinical and laboratory indicators upon admission. sTREM-1 was measured on initial sepsis evaluation and at 48h after antibiotic therapy. For ethical reasons, cord blood samples were collected from control neonates and only samples from neonates that proved to be healthy by clinical examination and laboratory analysis were further analyzed for sTREM-1. Baseline sTREM-1 levels were significantly elevated in culture-proven (1461.1±523pg/mL) and culture-negative sepsis (1194±485pg/mL) compared to controls (162.2±61pg/mL) with no significant difference between both septic groups. Culture-positive or negative septic preterm neonates had significantly higher sTREM-1 compared to full term neonates. sTREM-1 was significantly higher in neonates with early sepsis than late sepsis and was associated with high mortality. sTREM-1 was significantly decreased 48h after antibiotic therapy compared to baseline or levels in neonates with persistently positive cultures. sTREM-1 was positively correlated to white blood cells (WBCs), absolute neutrophil count, immature/total neutrophil (I/T) ratio, C-reactive protein (hs-CRP) and sepsis score while negatively correlated to gestational age and weight. hs-CRP and sepsis score were independently related to sTREM-1 in multiregression analysis. sTREM-1 cutoff value of 310pg/mL could be diagnostic for NS with 100% sensitivity and specificity (AUC, 1.0 and 95% confidence interval [CI], 0.696-1.015) while the cutoff value 1100pg/mL was predictive of survival with 100% sensitivity and 97% specificity (AUC, 0.978 and 95% CI, 0

  20. CRLF2 over-expression is a poor prognostic marker in children with high risk T-cell acute lymphoblastic leukemia

    PubMed Central

    Palmi, Chiara; Savino, Angela M.; Silvestri, Daniela; Bronzini, Ilaria; Cario, Gunnar; Paganin, Maddalena; Buldini, Barbara; Galbiati, Marta; Muckenthaler, Martina U.; Bugarin, Cristina; Mina, Pamela Della; Nagel, Stefan; Barisone, Elena; Casale, Fiorina; Locatelli, Franco; Nigro, Luca Lo; Micalizzi, Concetta; Parasole, Rosanna; Pession, Andrea; Putti, Maria C.; Santoro, Nicola; Testi, Anna M.; Ziino, Ottavio; Kulozik, Andreas E.; Zimmermann, Martin; Schrappe, Martin; Villa, Antonello; Gaipa, Giuseppe; Basso, Giuseppe; Biondi, Andrea; Valsecchi, Maria G.; Stanulla, Martin; Conter, Valentino; te Kronnie, Geertruy; Cazzaniga, Giovanni

    2016-01-01

    Pediatric T-ALL patients have a worse outcome compared to BCP-ALL patients and they could benefit from new prognostic marker identification. Alteration of CRLF2 gene, a hallmark correlated with poor outcome in BCP-ALL, has not been reported in T-ALL. We analyzed CRLF2 expression in 212 T-ALL pediatric patients enrolled in AIEOP-BFM ALL2000 study in Italian and German centers. Seventeen out of 120 (14.2%) Italian patients presented CRLF2 mRNA expression 5 times higher than the median (CRLF2-high); they had a significantly inferior event-free survival (41.2%±11.9 vs. 68.9%±4.6, p=0.006) and overall survival (47.1%±12.1 vs. 73.8%±4.3, p=0.009) and an increased cumulative incidence of relapse/resistance (52.9%±12.1 vs. 26.2%±4.3, p=0.007) compared to CRLF2-low patients. The prognostic value of CRLF2 over-expression was validated in the German cohort. Of note, CRLF2 over-expression was associated with poor prognosis in the high risk (HR) subgroup where CRLF2-high patients were more frequently allocated. Interestingly, although in T-ALL CRLF2 protein was localized mainly in the cytoplasm, in CRLF2-high blasts we found a trend towards a stronger TSLP-induced pSTAT5 response, sensitive to the JAK inhibitor Ruxolitinib. In conclusion, CRLF2 over-expression is a poor prognostic marker identifying a subset of HR T-ALL patients that could benefit from alternative therapy, potentially targeting the CRLF2 pathway. PMID:27449287

  1. Forkhead box protein P3 (Foxp3) expression serves as an early chronic inflammation marker of squamous cell differentiation and aggressive pathology of urothelial carcinomas in neurological patients.

    PubMed

    Phé, Véronique; Rouprêt, Morgan; Cussenot, Olivier; Chartier-Kastler, Emmanuel; Gamé, Xavier; Compérat, Eva

    2015-04-01

    To establish whether the expression of forkhead box protein P3 (Foxp3) provides specific diagnostic information about neurological patients with urothelial carcinoma of the bladder (UCB). UCB tissue samples from neurological patients were retrieved and compared with control samples. The expression of Foxp3 was analysed via immunohistochemistry of microarray tissue sections. The correlation between Foxp3 expression, histological parameters and tumour stage was assessed. Overall, 20 UCB tissue samples and 20 others without UCB from neurological patients, and 46 UCB tissue samples from non-neurological patients were analysed. The distribution of pT of UCB in the neurological patients was as follows: one low-grade pTa (5%), three high-grade pTa (15%), three pT1(15%), one pT2(5%), seven pT3(35%) and five pT4(25%). Squamous cell differentiation was seen in nine UCB samples (45%). Foxp3 expression was detected in tumour tissues, including one pTa high grade, one pT1, one pT2, five pT3 and five pT4 tumours. Foxp3 was expressed in 11/13 muscle-invasive tumours. All tumours displaying squamous cell differentiation expressed Foxp3. Foxp3 was not expressed in the pT3 tumours that displayed sarcomatoid and micropapillary properties. Among the bladder samples without UCB from neurological patients, no expression of Foxp3 was observed. Among the UCB samples from the non-neurological patients, only seven displayed squamous cell differentiation. All tumours that displayed squamous cell differentiation expressed Foxp3, including one pTa high grade, four pT3 and two pT4 tumours. Other tumours displaying urothelial differentiation did not express Foxp3. The expression of Foxp3 correlated to squamous cell differentiation in neurological (P = 0.004) and non-neurological UCB tissue (P < 0.001). In neurological, but not non-neurological UCB tissue, the expression of Foxp3 correlated with the muscle-invasive stage (P = 0.022). Elevated expression of Foxp3 appears to be a characteristic of

  2. Loss of p16 expression is associated with the stem cell characteristics of surface markers and therapeutic resistance in estrogen receptor-negative breast cancer.

    PubMed

    Arima, Yoshimi; Hayashi, Naoki; Hayashi, Hidemi; Sasaki, Mikako; Kai, Kazuharu; Sugihara, Eiji; Abe, Eriko; Yoshida, Atsushi; Mikami, Shuji; Nakamura, Seigo; Saya, Hideyuki

    2012-06-01

    Triple-negative breast cancer [TNBC, which is negative for the estrogen receptor (ER), progesterone receptor, and human epidermal growth factor receptor 2] is a high-risk form of the disease without a specific therapy. DNA microarray and immunohistochemical analyses have shown that most TNBCs fall within the basal-like histological subset of breast cancers, which frequently exhibit inactivation of the retinoblastoma tumor suppressor (Rb) and upregulation of the cyclin-dependent kinase inhibitor p16(INK4a) (p16). However, downregulation of p16 expression has been observed in some basal-like breast cancer cell lines, suggesting that such cells can be divided into two groups according to Rb and p16 status. We now show that cells that are CD44(+) and CD24(-) , a phenotype associated with stem-like breast cancer cells, are more abundant in ER(-) /p16(-) breast cancer cell lines than in ER(-) /p16(+) lines. It was also found that p16 expression was downregulated in mammospheres from an ER-negative breast cancer cell line. Depletion of p16 by RNA interference in ER-negative breast cancer cells increased the percentage of CD44(+) /CD24(-) cells and increased the expression of mRNA of the ES-like genes Nanog, Oct4, and Sox2 through an Rb-independent pathway. Furthermore, such depletion of p16 reduced chemosensitivity. The loss of p16 expression may thus reduce the response of ER-negative breast cancer cells to chemotherapy by conferring cancer stem cell-like properties. Consistent with this conclusion, immunohistochemical analysis of the clinical samples suggests that low p16 expression in TNBC is associated with resistance to preoperative chemotherapy. Copyright © 2011 UICC.

  3. Pancreatic cancer stem cell markers and exosomes - the incentive push

    PubMed Central

    Heiler, Sarah; Wang, Zhe; Zöller, Margot

    2016-01-01

    Pancreatic cancer (PaCa) has the highest death rate and incidence is increasing. Poor prognosis is due to late diagnosis and early metastatic spread, which is ascribed to a minor population of so called cancer stem cells (CSC) within the mass of the primary tumor. CSC are defined by biological features, which they share with adult stem cells like longevity, rare cell division, the capacity for self renewal, differentiation, drug resistance and the requirement for a niche. CSC can also be identified by sets of markers, which for pancreatic CSC (Pa-CSC) include CD44v6, c-Met, Tspan8, alpha6beta4, CXCR4, CD133, EpCAM and claudin7. The functional relevance of CSC markers is still disputed. We hypothesize that Pa-CSC markers play a decisive role in tumor progression. This is fostered by the location in glycolipid-enriched membrane domains, which function as signaling platform and support connectivity of the individual Pa-CSC markers. Outside-in signaling supports apoptosis resistance, stem cell gene expression and tumor suppressor gene repression as well as miRNA transcription and silencing. Pa-CSC markers also contribute to motility and invasiveness. By ligand binding host cells are triggered towards creating a milieu supporting Pa-CSC maintenance. Furthermore, CSC markers contribute to the generation, loading and delivery of exosomes, whereby CSC gain the capacity for a cell-cell contact independent crosstalk with the host and neighboring non-CSC. This allows Pa-CSC exosomes (TEX) to reprogram neighboring non-CSC towards epithelial mesenchymal transition and to stimulate host cells towards preparing a niche for metastasizing tumor cells. Finally, TEX communicate with the matrix to support tumor cell motility, invasion and homing. We will discuss the possibility that CSC markers are the initial trigger for these processes and what is the special contribution of CSC-TEX. PMID:27468191

  4. Identification of novel dendritic cell subset markers in human blood.

    PubMed

    Schütz, Fabian; Hackstein, Holger

    2014-01-10

    Human dendritic cells (DC) are key regulators of innate and adaptive immunity that can be divided in at least three major subpopulations: plasmacytoid DC (pDC), myeloid type 1 DC (mDC1) and myeloid type 2 DC (mDC2) exhibiting different functions. However, research, diagnostic and cell therapeutic studies on human DC subsets are limited because only few DC subset markers have been identified so far. Especially mDC2 representing the rarest blood DC subset are difficult to be separated from mDC1 and pDC due to a paucity of mDC2 markers. We have combined multiparameter flow cytometry analysis of human blood DC subsets with systematic expression analysis of 332 surface antigens in magnetic bead-enriched blood DC samples. The initial analysis revealed eight novel putative DC subset markers CD26, CD85a, CD109, CD172a, CD200, CD200R, CD275 and CD301 that were subsequently tested in bulk peripheral blood mononuclear cell (PBMC) samples from healthy blood donors. Secondary analysis of PBMC samples confirmed three novel DC subset markers CD26 (dipeptidyl peptidase IV), CD85a (Leukocyte immunoglobulin-like receptor B3) and CD275 (inducible costimulator ligand). CD85a is specifically expressed in mDC1 and CD26 and CD275 represent novel mDC2 markers. These markers will facilitate human DC subset discrimination and additionally provide insight into potentially novel DC subset-specific functions.

  5. Androgen deprivation and stem cell markers in prostate cancers

    PubMed Central

    Tang, Yao; Hamburger, Anne W; Wang, Linbo; Khan, Mohammad Afnan; Hussain, Arif

    2010-01-01

    In our previous studies using human LNCaP xenografts and TRAMP (transgenic adenocarcinoma of mouse prostate) mice, androgen deprivation therapy (ADT) resulted in a temporary cessation of prostate cancer (PCa) growth, but then tumors grew faster with more malignant behaviour. To understand whether cancer stem cells might play a role in PCa progression in these animal models, we investigated the expressions of stem cell-related markers in tumors at different time points after ADT. In both animal models, enhanced expressions of stem cell markers were observed in tumors of castrated mice, as compared to non-castrated controls. This increased cell population that expressed stem cell markers is designated as stem-like cells (SLC) in this article. We also observed that the SLC peaked at relatively early time points after ADT, before tumors resumed their growth. These results suggest that the SLC population may play a role in tumor re-growth and disease progression, and that targeting the SLC at their peak-expression time point may prevent tumor recurrence following ADT. PMID:20126580

  6. Statistical classification of multivariate flow cytometry data analyzed by manual gating: stem, progenitor, and epithelial marker expression in nonsmall cell lung cancer and normal lung.

    PubMed

    Normolle, Daniel P; Donnenberg, Vera S; Donnenberg, Albert D

    2013-01-01

    The use of supervised classification to extract markers from primary flow cytometry data is an emerging field that has made significant progress, spurred by the growing complexity of multidimensional flow cytometry. Whether the markers are extracted without supervision or by conventional gate and region methods, the number of candidate variables identified is typically larger than the number of specimens (p > n) and many variables are highly intercorrelated. Thus, comparison across groups or treatments to determine which markers are significant is challenging. Here, we utilized a data set in which 86 variables were created by conventional manual analysis of individual listmode data files, and compared the application of five multivariate classification methods to discern subtle differences between the stem/progenitor content of 35 nonsmall cell lung cancer and adjacent normal lung specimens. The methods compared include elastic-net, lasso, random forest, diagonal linear discriminant analysis, and best single variable (best-1). We described a broadly applicable methodology consisting of: 1) variable transformation and standardization; 2) visualization and assessment of correlation between variables; 3) selection of significant variables and modeling; and 4) characterization of the quality and stability of the model. The analysis yielded both validating results (tumors are aneuploid and have higher light scatter properties than normal lung), as well as leads that require followup: Cytokeratin+ CD133+ progenitors are present in normal lung but reduced in lung cancer; diploid (or pseudo-diploid) CD117+CD44+ cells are more prevalent in tumor. We anticipate that the methods described here will be broadly applicable to a variety of multidimensional cytometry problems.

  7. Shades of gray: The delineation of marker expression within the adult rodent subventricular zone.

    PubMed

    Mamber, Carlyn; Kozareva, Danka A; Kamphuis, Willem; Hol, Elly M

    2013-12-01

    The research field of adult neurogenesis is rapidly expanding with more and more information becoming available on the identity of the cells located within the subventricular zone (SVZ). Much of our understanding is based on rodent studies. The SVZ is comprised of several different cell types including B1 astrocytes, transit amplifying progenitor cells (C cells), and neuroblasts (A cells). B1 astrocytes are the quiescent neural stem cells that continue to divide throughout a lifespan. They give rise to a progenitor cell, termed a C cell, which in turn, generates neuroblasts destined for the olfactory bulb. There is much controversy over how to distinguish various SVZ cell types. This review summarizes the known markers for rodent SVZ cell types, with particular attention paid towards B1 astrocytes and C cells. Unfortunately, there is no perfect stem cell marker. B1 astrocytes, C cells, and neuroblasts gain and lose marker expression patterns throughout their lineage progression. These expression patterns often overlap at the transition from one cell type to another. The SVZ cell lineage must be seen as a continuum, rather than a static and inert system. This view will aid in understanding the mechanisms underlying marker expression and cellular behavior in the SVZ. Copyright © 2013 Elsevier Ltd. All rights reserved.

  8. Human lymphocyte markers defined by antibodies derived from somatic cell hybrids. II. A hybridoma secreting antibody against an antigen expressed by human B and null lymphocytes.

    PubMed

    Beckman, I G; Bradley, J; Brooks, D A; Kupa, A; McNamara, P J; Thomas, M E; Zola, H

    1980-06-01

    A hybridoma (FMC4) has been derived which secretes antibody showing selective reaction with human B lymphocytes, monocytes and some null lymphocytes. Few, if any, T lymphocytes in normal blood are stained, although stimulation of lymphocytes with PHA leads to an increase in the proportion of cells reacting with the hybridoma antibody. The antibody reacts with B and null lymphoblastoid cell lines but not with T cell lines. B chronic lymphocytic leukaemia (CLL) cells but not T-CLLs are stained and null-type acute lymphoblastic leukaemia (ALL) cells but not T-type ALL also react. Normal blood myeloid cells do not react with FMC4 supernatant whilst some myeloid leukaemias do. The expression of the antigen reacting with FMC4 supernatant suggests that FMC4 may secrete an antibody against the human equivalent of the Ia antigen.

  9. Human lymphocyte markers defined by antibodies derived from somatic cell hybrids. II. A hybridoma secreting antibody against an antigen expressed by human B and null lymphocytes.

    PubMed Central

    Beckman, I G; Bradley, J; Brooks, D A; Kupa, A; McNamara, P J; Thomas, M E; Zola, H

    1980-01-01

    A hybridoma (FMC4) has been derived which secretes antibody showing selective reaction with human B lymphocytes, monocytes and some null lymphocytes. Few, if any, T lymphocytes in normal blood are stained, although stimulation of lymphocytes with PHA leads to an increase in the proportion of cells reacting with the hybridoma antibody. The antibody reacts with B and null lymphoblastoid cell lines but not with T cell lines. B chronic lymphocytic leukaemia (CLL) cells but not T-CLLs are stained and null-type acute lymphoblastic leukaemia (ALL) cells but not T-type ALL also react. Normal blood myeloid cells do not react with FMC4 supernatant whilst some myeloid leukaemias do. The expression of the antigen reacting with FMC4 supernatant suggests that FMC4 may secrete an antibody against the human equivalent of the Ia antigen. PMID:6968260

  10. HINTW, a W-chromosome HINT gene in chick, is expressed ubiquitously and is a robust female cell marker applicable in intraspecific chimera studies.

    PubMed

    Nagai, Hiroki; Sezaki, Maiko; Bertocchini, Federica; Fukuda, Kimiko; Sheng, Guojun

    2014-05-01

    Grafting and transplantation experiments in embryology require proper distinction between host and donor tissues. For the avian model this has traditionally been achieved by using two closely related species (e.g., chick and quail) followed by species-specific antibody staining. Here, we show that an in situ hybridization probe against the HINTW gene is a robust and reliable marker for female-derived chicken cells. At all pre-circulation stages tested, all cells in female embryos, independently confirmed by PCR analysis, were strongly positive for HINTW, whereas all male embryos were negative. This probe is broadly applicable in intra-specific chick/chick chimera studies, and as a proof of principle, we utilized this probe to detect female cells in three experimental settings: (1) to mark female donor cells in a node transplantation assay; (2) to distinguish female cells in male/female twins generated by the Cornish pasty culture; and (3) to detect female half of the embryo in artificially generated bilateral gynandromorphs. A rapid, PCR based pre-screening step increases the efficiency of obtaining desired donor/host sex combination from 25% to 100%. For most avian chimera studies, this female-specific in situ probe is a low cost alternative to the commonly used QCPN antibody and to ubiquitous-GFP chicken strains which are not widely available to the research community. © 2014 Wiley Periodicals, Inc.

  11. A novel selectable marker based on Aspergillus niger arginase expression.

    PubMed

    Dave, Kashyap; Ahuja, Manmeet; Jayashri, T N; Sirola, Rekha Bisht; Punekar, Narayan S

    2012-06-10

    Selectable markers are valuable tools in transforming asexual fungi like Aspergillus niger. An arginase (agaA) expression vector and a suitable arginase-disrupted host would define a novel nutritional marker/selection for transformation. The development of such a marker was successfully achieved in two steps. The single genomic copy of A. niger arginase gene was disrupted by homologous integration of the bar marker. The agaA disruptant was subsequently complemented by transforming it with agaA expression vectors. Both citA and trpC promoters were able to drive the expression of arginase cDNA. Such agaA+ transformants displayed arginase expression pattern distinct from that of the parent strain. The results are also consistent with a single catabolic route for arginine in this fungus. A simple yet novel arginine-based selection for filamentous fungal transformation is thus described.

  12. Electron microscopic analysis of Drosophila midline glia during embryogenesis and larval development using beta-galactosidase expression as endogenous cell marker.

    PubMed

    Stollewerk, A; Klămbt, C; Cantera, R

    1996-10-15

    To thoroughly study developmental problems it is often desirable to identify specific cells at the resolution of the electron microscope (TEM). Specific antibodies, and immunogold and other antibody labelling techniques can be successfully used with the TEM. But for these techniques to be successful there must be substantial adjustments for each antibody and tissue analyzed. To develop a more generally applicable labelling method we took advantage of the enhancer trap technique in Drosophila. Enhancer trap fly strains show cell- and/or tissue-specific beta-galactosidase expression which can be visualized by a simple X-gal staining procedure. To combine the power of the enhancer trap approach with electron microscopy, we have improved the fixation and staining conditions, which allow detection of X-gal crystals (by TEM) and thus provide precise information on ultrastructural morphology. We have tested our technique using the well-known midline glial cells and examined these cells between late embryonic and pupal developmental stages. The four embryonic midline glial cells found in each neuromere reside ventrally and dorsally to the midline of the neuropile and are closely associated with unpaired neurons, major commissures, and other types of glial cells. During larval and pupal life dramatic cell growth and endomitotic nuclear replication occur in midline glial cells. By the end of larval life, the giant midline glial cells fragment to give rise to a variable number of small midline glial cells. Here we show that the combination of transmission electron microscopy with cytochemical detection of beta-galactosidase expression represents a promising and valuable tool for the study of the morphology and development of specific cell types.

  13. Co-expression of the Follicle Stimulating Hormone Receptor and Stem Cell Markers: A Novel Approach to Target Ovarian Cancer Stem Cells

    DTIC Science & Technology

    2012-09-01

    Award Number: 11 1 0623 TITLE: Co-expression of the Follicle Stimulating Hormone Receptor and Stem...Annual 3. DATES COVERED 1 Sep 2011 – 31 Aug 2012 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER Coexpression of the Follicle Stimulating Hormone ...Unlimited 13. SUPPLEMENTARY NOTES 14. ABSTRACT The purpose of this project is to determine whether the Follicle-stimulating Hormone Receptor (FSHR) is co

  14. Aberrant expression of epithelial and neuroendocrine markers in alveolar rhabdomyosarcoma: a potentially serious diagnostic pitfall.

    PubMed

    Bahrami, Armita; Gown, Allen M; Baird, Geoffrey S; Hicks, M John; Folpe, Andrew L

    2008-07-01

    Alveolar rhabdomyosarcoma may be extremely difficult to distinguish from other primitive round cell neoplasms without ancillary immunohistochemistry and/or genetic study. Particularly in adults and in the head and neck locations, the differential diagnosis of alveolar rhabdomyosarcoma includes small cell carcinoma and neuroepithelial tumors, such as esthesioneuroblastoma. We have recently seen cases of genetically confirmed alveolar rhabdomyosarcoma, which were misdiagnosed owing to expression of cytokeratins and neuroendocrine markers. We studied a large group of well-characterized alveolar rhabdomyosarcomas for expression of such markers. Forty-four alveolar rhabdomyosarcomas (18 genetically confirmed) were retrieved from our archives and immunostained for wide-spectrum cytokeratin (OSCAR), low molecular weight cytokeratin (Cam5.2), synaptophysin, chromogranin A, and CD56 using commercially available antibodies. Cases were scored as 'negative', 'rare' (<5% positive cells), '1+' (5-25%), '2+' (26-50%) and '3+' (>51%). The tumors occurred in 23 males and 21 females at a mean age of 18 years (range, <1-64 years), and involved many sites. Fifty percent of cases (22 of 44) expressed wide-spectrum cytokeratin, and scored almost equally as rare, 1+, and 2+, but rarely 3+. Cam5.2 was positive in 52% (14 of 27). Forty-three percent of cases (16 of 37) expressed at least one of the specific neuroendocrine markers, 32% (12 of 37) expressed synaptophysin, 22% (eight of 36) expressed chromogranin A, and 11% expressed both. Expression of synaptophysin and chromogranin A was typically confined to rare cells but could be more widespread. Thirty-two percent of cases (12 of 37) expressed the wide-spectrum cytokeratin and at least one of the neuroendocrine markers, and 8% (three of 36) expressed cytokeratin and both neuroendocrine markers. CD56 expression was nearly ubiquitous. Aberrant expression of epithelial and neuroendocrine markers is relatively common in alveolar

  15. Cinnamaldehyde, Carvacrol and Organic Acids Affect Gene Expression of Selected Oxidative Stress and Inflammation Markers in IPEC-J2 Cells Exposed to Salmonella typhimurium.

    PubMed

    Burt, Sara A; Adolfse, Simone J M; Ahad, Dina S A; Tersteeg-Zijderveld, Monique H G; Jongerius-Gortemaker, Betty G M; Post, Jan A; Brüggemann, Holger; Santos, Regiane R

    2016-12-01

    Essential oils and organic acids are used as feed additives to improve health status and reduce colonization with pathogens. Although bactericidal in vitro, concentrations achieved in the animal gut are probably not lethal to pathogens. The aim of this study was to investigate the effects of cinnamaldehyde, carvacrol and cinnamic, lactic and propionic acids on the ability of Salmonella typhimurium ATCC 14028 (ST) to invade intestinal epithelial cells (IPEC-J2) and on the expression levels of immune related genes in the cells. The minimum inhibitory concentration (MIC) and non-inhibitory concentration (NIC) were determined and influence on the invasion capacity of ST was investigated. The structure of fimbriae and flagella was analysed by electron microscopy, and expression levels of HSP70, IkBa, IL-8 and IL-10 in the IPEC-J2 cells were carried out by q-PCR. Cinnamaldehyde, carvacrol and cinnamic and propionic acids inhibited ST invasion but not cell viability, bacterial viability and motility or the development of flagella. Propionic acid and cinnamaldehyde in combination with cinnamic acid caused structural impairment of fimbriae. Cinnamaldehyde up-regulated expression of HSP70 irrespective of the presence of organic acids or ST; exposure to carvacrol induced HSP70 only in the presence of propionic acid and ST. © 2016 The Authors. Phytotherapy Research published by John Wiley & Sons Ltd.

  16. Expression of KISS1 and KISS1R (GPR54) may be used as favorable prognostic markers for patients with non-small cell lung cancer.

    PubMed

    Sun, Yan-Bin; Xu, Shun

    2013-08-01

    Lung cancer is the most commonly diagnosed cancer worldwide. Loss of KISS1 expression has been associated with progression and poor prognosis of various cancers, however, the precise role of KISS1 expression in non-small cell lung cancer (NSCLC) is not well defined. KISS1 receptor (KISS1R, also named GPR54) coupled to KISS1, has been shown to play a pivotal role in suppressing cancer metastasis. In this study, 56 NSCLC specimens were divided into stage IIIB (locally advanced) and stage IV (metastatic). The mRNA and protein levels of KISS1 and KISS1R in cancer tissues were found to be lower compared to that in normal tissues using RT-PCR and western blot analysis, respectively. In addition, the expression of both KISS1 and KISS1R in stage IV NSCLC was lower compared to that in stage IIIB stage NSCLC. The cumulative survival rate of the patients with KISS1 or KISS1R expression was significantly higher compared to that without expression. KISS1 or KISS1R expression in NSCLC can be used to indicate favorable prognosis for disease outcome. Metastin, the product of the KISS1 gene, was lower in the serum of patients with stage IV NSCLC compared to that in stage IIIB NSCLC.

  17. c-Met Expression Is a Marker of Poor Prognosis in Patients With Locally Advanced Head and Neck Squamous Cell Carcinoma Treated With Chemoradiation

    SciTech Connect

    Baschnagel, Andrew M.; Williams, Lindsay; Hanna, Alaa; Chen, Peter Y.; Krauss, Daniel J.; Pruetz, Barbara L.; Akervall, Jan; Wilson, George D.

    2014-03-01

    Purpose: To examine the prognostic significance of c-Met expression in relation to p16 and epidermal growth factor receptor (EGFR) in patients with locally advanced head and neck squamous cell carcinoma (HNSCC) treated with definitive concurrent chemoradiation. Methods and Materials: Archival tissue from 107 HNSCC patients treated with chemoradiation was retrieved, and a tissue microarray was assembled. Immunohistochemical staining of c-Met, p16, and EGFR was performed. c-Met expression was correlated with p16, EGFR, clinical characteristics, and clinical endpoints including locoregional control (LRC), distant metastasis (DM), disease-free survival (DFS), and overall survival (OS). Results: Fifty-one percent of patients were positive for p16, and 53% were positive for EGFR. Both p16-negative (P≤.001) and EGFR-positive (P=.019) status predicted for worse DFS. Ninety-three percent of patients stained positive for c-Met. Patients were divided into low (0, 1, or 2+ intensity) or high (3+ intensity) c-Met expression. On univariate analysis, high c-Met expression predicted for worse LRC (hazard ratio [HR] 2.27; 95% CI, 1.08-4.77; P=.031), DM (HR 4.41; 95% CI, 1.56-12.45; P=.005), DFS (HR 3.00; 95% CI, 1.68-5.38; P<.001), and OS (HR 4.35; 95% CI, 2.13-8.88; P<.001). On multivariate analysis, after adjustment for site, T stage, smoking history, and EGFR status, only high c-Met expression (P=.011) and negative p16 status (P=.003) predicted for worse DFS. High c-Met expression was predictive of worse DFS in both EGFR-positive (P=.032) and -negative (P=.008) patients. In the p16-negative patients, those with high c-Met expression had worse DFS (P=.036) than did those with low c-Met expression. c-Met expression was not associated with any outcome in the p16-positive patients. Conclusions: c-Met is expressed in the majority of locally advanced HNSCC cases, and high c-Met expression predicts for worse clinical outcomes. High c-Met expression predicted for worse DFS in p16

  18. PTEN expression is a prognostic marker for patients with non-small cell lung cancer: a systematic review and meta-analysis of the literature

    PubMed Central

    Xiao, Jian; Hu, Cheng-Ping; He, Bi-Xiu; Chen, Xi; Lu, Xiao-Xiao; Xie, Ming-Xuan; Li, Wei; He, Shu-Ya; You, Shao-Jin; Chen, Qiong

    2016-01-01

    Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a known tumor suppressor in non-small cell lung cancer (NSCLC). By performing a systematic review and meta-analysis of the literature, we determined the prognostic value of decreased PTEN expression in patients with NSCLC. We comprehensively and systematically searched through multiple online databases up to May 22, 2016 for NSCLC studies reporting on PTEN expression and patient survival outcome. Several criteria, including the Newcastle-Ottawa Quality Assessment Scale (NOS), were used to discriminate between studies. In total, 23 eligible studies with a total of 2,505 NSCLC patients were included in our meta-analysis. Our results demonstrated that decreased expression of PTEN correlated with poor overall survival in NSCLC patients and was indicative of a poor prognosis for disease-free survival and progression-free survival in patients with NSCLC. PMID:27506936

  19. Identification of cancer stem cell markers in human malignant mesothelioma cells

    SciTech Connect

    Ghani, Farhana Ishrat; Yamazaki, Hiroto; Iwata, Satoshi; Okamoto, Toshihiro; Aoe, Keisuke; Okabe, Kazunori; Mimura, Yusuke; Fujimoto, Nobukazu; Kishimoto, Takumi; Yamada, Taketo; Xu, C. Wilson; Morimoto, Chikao

    2011-01-14

    Research highlights: {yields} We performed serial transplantation of surgical samples and established new cell lines of malignant mesothelioma. {yields} SP cell and expressions of CD9/CD24/CD26 were often observed in mesothelioma cell lines. {yields} SP and CD24{sup +} cells proliferated by asymmetric cell division-like manner. CD9{sup +} and CD24{sup +} cells have higher potential to generate spheroid colony. {yields} The marker-positive cells have clear tendency to generate larger tumors in mice. -- Abstract: Malignant mesothelioma (MM) is an aggressive and therapy-resistant neoplasm arising from the pleural mesothelial cells and usually associated with long-term asbestos exposure. Recent studies suggest that tumors contain cancer stem cells (CSCs) and their stem cell characteristics are thought to confer therapy-resistance. However, whether MM cell has any stem cell characteristics is not known. To understand the molecular basis of MM, we first performed serial transplantation of surgical samples into NOD/SCID mice and established new cell lines. Next, we performed marker analysis of the MM cell lines and found that many of them contain SP cells and expressed several putative CSC markers such as CD9, CD24, and CD26. Interestingly, expression of CD26 closely correlated with that of CD24 in some cases. Sorting and culture assay revealed that SP and CD24{sup +} cells proliferated by asymmetric cell division-like manner. In addition, CD9{sup +} and CD24{sup +} cells have higher potential to generate spheroid colony than negative cells in the stem cell medium. Moreover, these marker-positive cells have clear tendency to generate larger tumors in mouse transplantation assay. Taken together, our data suggest that SP, CD9, CD24, and CD26 are CSC markers of MM and could be used as novel therapeutic targets.

  20. Prognostic significance of nestin expression in patients with resected non-small cell lung cancer treated with platinum-based adjuvant chemotherapy; relationship between nestin expression and epithelial to mesenchymal transition related markers

    PubMed Central

    Ryuge, Shinichiro; Sato, Yuichi; Nagashio, Ryo; Hiyoshi, Yasuhiro; Katono, Ken; Igawa, Satoshi; Nakashima, Hiroyasu; Shiomi, Kazu; Ichinoe, Masaaki; Murakumo, Yoshiki; Saegusa, Makoto; Satoh, Yukitoshi; Masuda, Noriyuki

    2017-01-01

    Introduction Although adjuvant platinum-based chemotherapy (AC) has been shown to improve survival of patients with completely resected stage II and stage IIIA non-small cell lung cancer (NSCLC), its effect is limited. Nestin is a class VI intermediate filament protein expressed in neural stem cells and several cancer cells including NSCLC. In the present study, we aimed to determine its prognostic significance concerning survival in NSCLC patients receiving AC. Methods Nestin expression in cancer cells was immunohistochemically studied in 90 patients with completely resected stage II and stage IIIA NSCLC treated with AC and its association with clinicopathologic parameters, including ABCG2, E-cadherin, and vimentin expression, was evaluated. Kaplan-Meier survival analysis and Cox proportional hazards models were used to estimate the effect of nestin expression on survival. Results Nestin expression was observed in 28 of the 90 (31.1%) NSCLCs. Clinicopathologically, nestin expression was associated with loss of E-cadherin expression (P = 0.006) and vimentin positive expression (P < 0.001). In survival analysis, nestin expression was significantly associated with a poorer prognosis (P = 0.028). Multivariable analysis confirmed that nestin expression is an independent prognostic indicator in NSCLC patients receiving AC (HR = 2.56; 95% CI, 1.23–5.30, P = 0.01). Conclusion The present study reveals that nestin expression is a prognostic indicator of a poorer survival probability in NSCLC patients receiving AC, although its prognostic significance still requires confirmation with larger patient populations. PMID:28358810

  1. Mammalian target of rapamycin inhibitors, temsirolimus and torin 1, attenuate stemness-associated properties and expression of mesenchymal markers promoted by phorbol-myristate-acetate and oncostatin-M in glioblastoma cells.

    PubMed

    Chandrika, Goparaju; Natesh, Kumar; Ranade, Deepak; Chugh, Ashish; Shastry, Padma

    2017-03-01

    The phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin signaling pathway is crucial for tumor survival, proliferation, and progression, making it an attractive target for therapeutic intervention. In glioblastoma, activated mammalian target of rapamycin promotes invasive phenotype and correlates with poor patient survival. A wide range of mammalian target of rapamycin inhibitors are currently being evaluated for cytotoxicity and anti-proliferative activity in various tumor types but are not explored sufficiently for controlling tumor invasion and recurrence. We recently reported that mammalian target of rapamycin inhibitors-rapamycin, temsirolimus, torin 1, and PP242-suppressed invasion and migration promoted by tumor necrosis factor-alpha and phorbol-myristate-acetate in glioblastoma cells. As aggressive invasion and migration of tumors are associated with mesenchymal and stem-like cell properties, this study aimed to examine the effect of mammalian target of rapamycin inhibitors on these features in glioblastoma cells. We demonstrate that temsirolimus and torin 1 effectively reduced the constitutive as well as phorbol-myristate-acetate/oncostatin-M-induced expression of mesenchymal markers (fibronectin, vimentin, and YKL40) and neural stem cell markers (Sox2, Oct4, nestin, and mushashi1). The inhibitors significantly abrogated the neurosphere-forming capacity induced by phorbol-myristate-acetate and oncostatin-M. Furthermore, we demonstrate that the drugs dephosphorylated signal transducer and activator transcription factor 3, a major regulator of mesenchymal and neural stem cell markers implicating the role of signal transducer and activator transcription factor 3 in the inhibitory action of these drugs. The findings demonstrate the potential of mammalian target of rapamycin inhibitors as "stemness-inhibiting drugs" and a promising therapeutic approach to target glioma stem cells.

  2. Embryonic stem cells: from markers to market.

    PubMed

    Deb, Kaushik Dilip; Jayaprakash, Anitha Devi; Sharma, Vijay; Totey, Satish

    2008-02-01

    ABSTRACT Embryonic stem cells are considered the mother of all kinds of tissues and cells and it is envisioned as the holy grail of regenerative medicine. However, their use in cell replacement therapies (CRT) has so far been limited and their potentials are yet to be fully realized. The use of human embryonic stem cells (hESC) involves many safety issues pertaining to culture conditions and epigenetic changes. The role and importance of an epigenomic signature in derivation and maintenance of hESC are discussed. We provide a list of important epigenetic markers, which should be studied for evaluation of safety in hESC-based cell replacement therapies. These genes also need to be screened to determine an epigenetic signature for pluripotency in the hESCs. Finally a comprehensive list of all known stemness signature genes and the marker genes for different germ line lineages are presented. This review aims at summing up most of the intriguing molecules that can play a role in the maintenance of pluripotency and can help in determining hESC differentiation to various lineages. Extensive understanding of these markers will eventually help the researchers to transform the hESC research from bench to the bedside. The use of hESCs in CRTs is still in its infancy; much effort is warranted to turn them into the much dreamed about magic wand of regenerative medicine.

  3. Bone marker gene expression in calvarial bones: different bone microenvironments.

    PubMed

    Al-Amer, Osama

    2017-12-01

    In calvarial mice, mesenchymal stem cells (MSCs) differentiate into osteoprogenitor cells and then differentiate into osteoblasts that differentiate into osteocytes, which become embedded within the bone matrix. In this case, the cells participating in bone formation include MSCs, osteoprogenitor cells, osteoblasts and osteocytes. The calvariae of C57BL/KaLwRijHsD mice consist of the following five bones: two frontal bones, two parietal bones and one interparietal bone. This study aimed to analyse some bone marker genes and bone related genes to determine whether these calvarial bones have different bone microenvironments. C57BL/KaLwRijHsD calvariae were carefully excised from five male mice that were 4-6 weeks of age. Frontal, parietal, and interparietal bones were dissected to determine the bone microenvironment in calvariae. Haematoxylin and eosin staining was used to determine the morphology of different calvarial bones under microscopy. TaqMan was used to analyse the relative expression of Runx2, OC, OSX, RANK, RANKL, OPG, N-cadherin, E-cadherin, FGF2 and FGFR1 genes in different parts of the calvariae. Histological analysis demonstrated different bone marrow (BM) areas between the different parts of the calvariae. The data show that parietal bones have the smallest BM area compared to frontal and interparietal bones. TaqMan data show a significant increase in the expression level of Runx2, OC, OSX, RANKL, OPG, FGF2 and FGFR1 genes in the parietal bones compared with the frontal and interparietal bones of calvariae. This study provides evidence that different calvarial bones, frontal, parietal and interparietal, contain different bone microenvironments.

  4. Expression of microRNAs in squamous cell carcinoma of human head and neck and the esophagus: miR-205 and miR-21 are specific markers for HNSCC and ESCC.

    PubMed

    Kimura, Sotai; Naganuma, Seiji; Susuki, Dai; Hirono, Yasuo; Yamaguchi, Akio; Fujieda, Shigeharu; Sano, Kazuo; Itoh, Hiroshi

    2010-06-01

    MicroRNAs (miRNAs) are non-coding small RNAs that regulate cell proliferation and functions by interfering with the translation of target mRNAs. Altered expression of miRNA is known to induce various human malignancies. We examined the expression of miRNAs in squamous cell carcinoma of human head and neck (HNSCC) and esophagus (ESCC), compared to that in normal squamous epithelia as well as malignancies of other organs. Microarray analysis showed up-regulation of miR-21, miR-16 and miR-30a-5p in HNSCC and ESCC cell lines compared to normal squamous epithelial cell lines, and consistent high expression of miR-205 and let-7a in both normal and malignant squamous epithelial cell lines. Validation study using real-time quantitative RT-PCR in formalin-fixed paraffin-embedded cancer tissues and paired normal epithelia obtained by Laser-captured microdissection revealed that miR-205 showed highest expression in both malignant and benign squamous epithelia, although it was less expressed in cell lines and tissues other than squamous epithelia. MiR-21, which is an oncogenic miRNA in various malignancies, was also up-regulated in HNSCC and ESCC compared to paired normal squamous epithelia. These results suggest that miR-205 might be a specific marker miRNA of both normal and malignant squamous epithelia, while miR-21 might be a putative oncogenic miRNA in HNSCC and ESCC.

  5. Expression of the SST receptor 2 in uveal melanoma is not a prognostic marker.

    PubMed

    Kouch-el Filali, Mariam; Kilic, Emine; Melis, Marleen; de Klein, Annelies; de Jong, Marion; Luyten, Gregorius P M

    2008-11-01

    Uveal melanoma (UM) cells and neurohormone-producing cells both originate from the neural crest. Somatostatin receptors subtype 2 (SSTR2) are over-expressed in several tumors, often from neuroendocrine origin, and synthetic antagonists like octreotide and octreotate are being used as diagnostic or therapeutic agents. We investigated the SSTR2 expression in UM, and determined whether this expression was related to prognosis of the disease. UM cell lines and fresh primary UM samples were tested for SSTR2 expression by autoradiography (AR) using 125I-Tyr3-octreotate. Furthermore, UM cell lines were analyzed for SSTR2 mRNA expression with quantitative real-time RT-PCR. Using AR, cell-surface SSTR2 expression was demonstrated in two UM metastatic cell lines, but no expression was detected in three cell lines derived from primary UM. However, all primary and metastatic UM cell lines showed mRNA expression levels for SSTR2 using quantitative real-time RT-PCR. Only three of 14 primary UM demonstrated moderate SSTR2 expression, and this expression was not significantly associated with tumor-free survival or any tested prognostic factor. Based on the rare and low expression of SSTR2 found in primary UM specimens and in UM cell lines, we conclude that SSTR2 is not widely expressed in UM. Furthermore, SSTR2 expression was not associated with tumor-free survival and prognostic factors. Therefore SSTR2 is not suited as prognostic marker or therapeutic target in UM.

  6. [Expression of Myocardial Specificity Markers MEF-2C and Cx43 in Rat Bone Marrow-derived Mesenchymal Stem Cells Induced by Electrical Stimulation In Vitro].

    PubMed

    Tang, Min; Yang, Gang; Jiang, Jian; He, Xueling; Li, Huiming; Zhang, Mengying; Wu, Wenchao; Liu, Xiaojing; Li, Liang

    2015-06-01

    Bone marrow-derived mesenchymal stem cells (BMSCs) for repairing damaged heart tissue are a new kind of important treatment options because of their potential to differentiate into cardiomyocytes. We in this experiment investigated the effect of different electrical stimulation time on the expression of myocardial specificity gene and protein in rat bone marrow mesenchymal stem cells (rBMSCs) in vitro. The rBMSCs of second or third generation were randomly divided into three groups, i.e, electrical stimulation (ES) group, 5-Azacytidine (5-Aza) group and the control group. The rBMSCs in the ES groups with complete medium were exposed to 2 V, 2 Hz, 5 ms electrical stimulation for 0. 5 h, 2 h, 4 h, and 6 h respectively every day for 10 days. Those in the 5-Aza group were induced by 5-Aza (10 μmol/L) for 24 h, and then cultured with complete medium for 10 days. Those in the control group were only cultured with complete medium, without any treatment, for 10 days. The rBMSCs' morphological feature in each group was observed with inverted phase microscope. The mRNA expression of myocyte-specific enhancer factor 2C (MEF-2C) and connexin 43 (Cx43) were examined with Real-Time quantitative PCR and the protein expression of MEF-2C, Cx43 were detected with Western Blot method. The results showed that the mRNA expression level of the MEF-2C, Cx43 and the protein expression level of MEF-2C, Cx43 were significantly higher in the ES group and 5-Aza group than those in the relative control group (P < 0.05). It suggests that electrical stimulation could play a part of role in the induction of the rBMSCs to differentiate into the cariomyocyte-like cells in vitro and the effectiveness of the electrical stimulation with 2 h/d had the best in our experiment. But the mechanism how electrical stimulation promotes the differentiation of rBMSC into cardiomyocyte is still unclear.

  7. Expression of early and late cellular damage markers by ARPE-19 cells following prolonged treatment with UV-A radiation.

    PubMed

    Tringali, Giuseppe; Sampaolese, Beatrice; Clementi, Maria Elisabetta

    2016-10-01

    Pathological alterations to the retinal pigment epithelium underlie several eye diseases, which lead to visual impairment and even blindness. Exposure to ultraviolet (UV) radiation is associated with some skin and ocular pathologies; UV radiation may induce DNA breakdown and cause cellular damage through the production of reactive oxygen species (ROS), thus leading to programmed cell death. The present study aimed to investigate the production of ROS and the gene expression levels of anti‑ and proapoptotic proteins [B‑cell lymphoma 2 (Bcl‑2), Bcl‑2‑associated X protein (Bax) and caspase‑3] in human retinal pigment epithelial cells (ARPE‑19) treated with UV‑A for 5 h consecutively. The results demonstrated that prolonged exposure to UV‑A induced: i) Cell death, the decrease in cell viability was time‑dependent and reached statistical significance after 3 h; ii) a significant and substantial increase in ROS levels that remained constant for the duration of the experiment, the levels were significantly increased after 1 h of exposure; iii) an activation of apoptotic genes (Bax and caspase‑3) after 1 h of treatment, which was accompanied by a decrease in the anti‑apoptotic gene Bcl‑2; and iv) a loss of apoptotic signals and a rapid decrease in cellular viability after 3 h of consecutive treatment. These processes may trigger necrosis, which was observed in the cells following treatment with UV‑A for 5 consecutive hours. In conclusion, the present study is the first, to the best of our knowledge, to provide in vitro evidence regarding the sequence of events that u