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1
Escribano, Luis, Alberto Orfao, Jesús Villarrubia, Beatriz Díaz-Agustín, Carlos Cerveró, Agustín Rios, José L. Velasco, Juana Ciudad, José L. Navarro und Jesús F. San Miguel. „Immunophenotypic Characterization of Human Bone Marrow Mast Cells. A Flow Cytometric Study of Normal and Pathological Bone Marrow Samples“. Analytical Cellular Pathology 16, Nr. 3 (1998): 151–59. http://dx.doi.org/10.1155/1998/341340.
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The goal of the present paper was to define the immunophenotype of bone marrow mast cells (BMMC) from healthy controls and patients with hematologic malignancies (HM) based on the use of multiple stainings with monoclonal antibodies analyzed by flow cytometry. Our results show that BMMC from both groups of individuals display a similar but heterogenous immunophenotype. The overall numbers of BMMC are higher in the HM group of individuals (p= 0.08). Three patterns of antigen expression were detected: (1) markers constantly positive in all cases analyzed (CD9, CD29, CD33, CD43, CD44, CD49d, CD49e, CD51, CD71, CD117, and FcεRI), (2) antigens that were constantly negative (CD1a, CD2, CD3, CD5, CD6, CD11a, CD14, CD15, CD16, CD19, CD20, CD21, CD23, CD25, CD30, CD34, CD38, CD41a, CD42b, CD65, CD66b, HLA-DR, and CD138), and (3) markers that were positive in a variable proportion of cases – CD11b (50%), CD11c (77%), CD13 (40%), CD18 (20%), CD22 (68%), CD35 (27%), CD40 (67%), CD54 (88%) and CD61 (40%). In addition, BMMC from all cases explored were CD45+, and this antigen was expressed at an intensity similar to that of mature granulocytes.In summary, our results show that BMMC from both healthy controls and HM patients display a relatively heterogenous immunophenotype. Interestingly, we have observed clear differences between the immunophenotype of BMMC and MC from other tissues. This could be due either to the heterogeneity of human MC according to their tissue localization or to the sensitivity of the method used for antigen detection.
2
Shirure, Venktesh S., Tiantian Liu, Luis F. Delgadillo, Chaz M. Cuckler, David F. J. Tees, Fabian Benencia, Douglas J. Goetz und Monica M. Burdick. „CD44 variant isoforms expressed by breast cancer cells are functional E-selectin ligands under flow conditions“. American Journal of Physiology-Cell Physiology 308, Nr. 1 (01.01.2015): C68—C78. http://dx.doi.org/10.1152/ajpcell.00094.2014.
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Adhesion of circulating tumor cells to vascular endothelium is mediated by specialized molecules that are functional under shear forces exerted by hematogenous flow. Endothelial E-selectin binding to glycoforms of CD44 mediates shear-resistant cell adhesion in numerous physiological and pathological conditions. However, this pathway is poorly understood in breast cancer and is the focus of the present investigation. All breast cancer cell lines used in this study strongly expressed CD44. In particular, BT-20 cells expressed CD44s and multiple CD44v isoforms, whereas MDA-MB-231 cells predominantly expressed CD44s but weakly expressed CD44v isoforms. CD44 expressed by BT-20, but not MDA-MB-231, cells possessed E-selectin ligand activity as detected by Western blotting and antigen capture assays. Importantly, CD44 expressed by intact BT-20 cells were functional E-selectin ligands, regulating cell rolling and adhesion under physiological flow conditions, as found by shRNA-targeted silencing of CD44. Antigen capture assays strongly suggest greater shear-resistant E-selectin ligand activity of BT-20 cell CD44v isoforms than CD44s. Surprisingly, CD44 was not recognized by the HECA-452 MAb, which detects sialofucosylated epitopes traditionally expressed by selectin ligands, suggesting that BT-20 cells express a novel glycoform of CD44v as an E-selectin ligand. The activity of this glycoform was predominantly attributed to N-linked glycans. Furthermore, expression of CD44v as an E-selectin ligand correlated with high levels of fucosyltransferase-3 and -6 and epithelial, rather than mesenchymal, cell phenotype. Together, these data demonstrate that expression of CD44 as a functional E-selectin ligand may be important in breast cancer metastasis.
3
HERMIDA-GÓMEZ, TAMARA, ISAAC FUENTES-BOQUETE, MARIA JOSÉ GIMENO-LONGAS, EMMA MUIÑOS-LÓPEZ, SILVIA DÍAZ-PRADO, FRANCISCO JAVIER de TORO und FRANCISCO JAVIER BLANCO. „Quantification of Cells Expressing Mesenchymal Stem Cell Markers in Healthy and Osteoarthritic Synovial Membranes“. Journal of Rheumatology 38, Nr. 2 (15.11.2010): 339–49. http://dx.doi.org/10.3899/jrheum.100614.
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Objective.To quantify cells expressing mesenchymal stem cell (MSC) markers in synovial membranes from human osteoarthritic (OA) and healthy joints.Methods.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.Results.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.Conclusion.Synovial membranes from patients with OA contain more cells positive for CD44, CD90, and CD105 antigens than those from joints with undamaged cartilage.
4
Sallusto, F., und A. Lanzavecchia. „Efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte/macrophage colony-stimulating factor plus interleukin 4 and downregulated by tumor necrosis factor alpha.“ Journal of Experimental Medicine 179, Nr. 4 (01.04.1994): 1109–18. http://dx.doi.org/10.1084/jem.179.4.1109.
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Using granulocyte/macrophage colony-stimulating factor (GM-CSF) and interleukin 4 we have established dendritic cell (DC) lines from blood mononuclear cells that maintain the antigen capturing and processing capacity characteristic of immature dendritic cells in vivo. These cells have typical dendritic morphology, express high levels of major histocompatibility complex (MHC) class I and class II molecules, CD1, Fc gamma RII, CD40, B7, CD44, and ICAM-1, and lack CD14. Cultured DCs are highly stimulatory in mixed leukocyte reaction (MLR) and are also capable of triggering cord blood naive T cells. Most strikingly, these DCs are as efficient as antigen-specific B cells in presenting tetanus toxoid (TT) to specific T cell clones. Their efficiency of antigen presentation can be further enhanced by specific antibodies via FcR-mediated antigen uptake. Incubation of these cultured DCs with tumor necrosis factor alpha (TNF-alpha) or soluble CD40 ligand (CD40L) for 24 h results in an increased surface expression of MHC class I and class II molecules, B7, and ICAM-1 and in the appearance of the CD44 exon 9 splice variant (CD44-v9); by contrast, Fc gamma RII is markedly and sometimes completely downregulated. The functional consequences of the short contact with TNF-alpha are in increased T cell stimulatory capacity in MLR, but a 10-fold decrease in presentation of soluble TT and a 100-fold decrease in presentation of TT-immunoglobulin G complexes.
5
Kansas, GS, MJ Muirhead und MO Dailey. „Expression of the CD11/CD18, leukocyte adhesion molecule 1, and CD44 adhesion molecules during normal myeloid and erythroid differentiation in humans“. Blood 76, Nr. 12 (15.12.1990): 2483–92. http://dx.doi.org/10.1182/blood.v76.12.2483.2483.
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Abstract We have used three-color flow cytometry to investigate the pattern of expression of the CD11/CD18, CD44, and leukocyte adhesion molecule 1 (LAM-1) adhesion molecules during myeloid and erythroid differentiation in humans. The earliest myeloid cells, identified as CD33loCD15-, were exclusively CD44hi but contained both leukocyte function-associated antigen 1 (LFA-1hi) and LFA-1lo cells, as well as LAM-1+ and LAM-1- cells. This CD33loCD15- myeloid subpopulation expressed only low levels of CD11c and failed to express CD11b, CD14, or any lymphoid (CD3, CD16, CD19) antigens or glycophorin. Commitment to monocyte differentiation, suggested by the presence of an LFA-1hi CD11c+ subset within the CD33loCD15- subpopulation, was clearly signaled by upregulation of CD33; these monocyte-lineage committed cells were exclusively CD33hi, CD44hi, CD11ahi, CD11c+, and exhibited a broad range of intensity of CD15 expression. Later stages of monopoiesis were identified by acquisition of CD11b, and subsequently of CD14. Myeloid cells committed to granulopoiesis remained LFA-1lo, and underwent a sharp upregulation of CD15 along with downregulation of both CD33 and CD44. Successive stages of granulocyte development were marked by expression of CD11b and, subsequently, of CD16. The earliest cells capable of erythroid differentiation were CD44hi, LFA-1lo, and LAM-1+. Both LFA-1 and LAM-1 were lost before the onset of glycophorin (glyco) expression, whereas CD44 expression remained high on glyco+ cells, which also expressed CD45. CD44 expression was intermediate on glyco+ CD71+ cells, and low on glyco+ CD45- CD71- cells, similar to normal, circulating erythrocytes. Our results allow us to phenotypically define discrete stages in the normal development of monocytes, neutrophils, and erythrocytes. The expression of LFA-1, LAM-1, and high levels of CD44 on the most primitive hematopoietic cells detectable by flow cytometry suggests that at least some of these molecules are critically involved in leukocyte adhesion during development.
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Kansas, GS, MJ Muirhead und MO Dailey. „Expression of the CD11/CD18, leukocyte adhesion molecule 1, and CD44 adhesion molecules during normal myeloid and erythroid differentiation in humans“. Blood 76, Nr. 12 (15.12.1990): 2483–92. http://dx.doi.org/10.1182/blood.v76.12.2483.bloodjournal76122483.
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We have used three-color flow cytometry to investigate the pattern of expression of the CD11/CD18, CD44, and leukocyte adhesion molecule 1 (LAM-1) adhesion molecules during myeloid and erythroid differentiation in humans. The earliest myeloid cells, identified as CD33loCD15-, were exclusively CD44hi but contained both leukocyte function-associated antigen 1 (LFA-1hi) and LFA-1lo cells, as well as LAM-1+ and LAM-1- cells. This CD33loCD15- myeloid subpopulation expressed only low levels of CD11c and failed to express CD11b, CD14, or any lymphoid (CD3, CD16, CD19) antigens or glycophorin. Commitment to monocyte differentiation, suggested by the presence of an LFA-1hi CD11c+ subset within the CD33loCD15- subpopulation, was clearly signaled by upregulation of CD33; these monocyte-lineage committed cells were exclusively CD33hi, CD44hi, CD11ahi, CD11c+, and exhibited a broad range of intensity of CD15 expression. Later stages of monopoiesis were identified by acquisition of CD11b, and subsequently of CD14. Myeloid cells committed to granulopoiesis remained LFA-1lo, and underwent a sharp upregulation of CD15 along with downregulation of both CD33 and CD44. Successive stages of granulocyte development were marked by expression of CD11b and, subsequently, of CD16. The earliest cells capable of erythroid differentiation were CD44hi, LFA-1lo, and LAM-1+. Both LFA-1 and LAM-1 were lost before the onset of glycophorin (glyco) expression, whereas CD44 expression remained high on glyco+ cells, which also expressed CD45. CD44 expression was intermediate on glyco+ CD71+ cells, and low on glyco+ CD45- CD71- cells, similar to normal, circulating erythrocytes. Our results allow us to phenotypically define discrete stages in the normal development of monocytes, neutrophils, and erythrocytes. The expression of LFA-1, LAM-1, and high levels of CD44 on the most primitive hematopoietic cells detectable by flow cytometry suggests that at least some of these molecules are critically involved in leukocyte adhesion during development.
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Parsons, SF, J. Jones, DJ Anstee, PA Judson, B. Gardner, E. Wiener, J. Poole, N. Illum und SN Wickramasinghe. „A novel form of congenital dyserythropoietic anemia associated with deficiency of erythroid CD44 and a unique blood group phenotype [In(a-b- ), Co(a-b-)]“. Blood 83, Nr. 3 (01.02.1994): 860–68. http://dx.doi.org/10.1182/blood.v83.3.860.860.
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Abstract We have used a panel of well-characterized monoclonal antibodies (MoAbs) to examine the blood cells of a patient with a novel form of congenital dyserythropoietic anemia (CDA) characterized by intra- erythroblastic and intra-erythrocytic membranous inclusions. Twelve antibodies defining three nonoverlapping epitope groups on the extracellular domain of CD44 all failed to react with the red blood cells (RBCs) of the patient. A rabbit antibody to the cytoplasmic domain of CD44 from normal RBCs failed to react with the patient's RBC ghosts. In contrast, the patient's lymphocytes, granulocytes, and monocytes showed apparently normal CD44 expression. Bone marrow preparations stained with CD44 antibodies and visualized with 125I antimouse Ig (F(ab')2) followed by autoradiography showed positive staining of lymphocytes and myeloid cells but not of most orthotolidine- positive erythroblasts. The patient's RBCs also gave weaker than normal reactions with MoAbs of anti-LWab specificity while MoAbs to glycophorins A, B, and C, Rh polypeptides, CD47, CD55, CD58, CD59, acetylcholinesterase, and Lutheran and Kell glycoproteins all gave normal reactions. Agglutination tests with human blood grouping sera demonstrated that the RBCs of the patient have the unique phenotype In(a-b-), Co(a-b-) and that they also lack the high incidence RBC antigen AnWj. The phenotype In(a-b-) would be expected because these antigens are known to be expressed on CD44. There is also some evidence associating the AnWj antigen with CD44. However, the CO blood group locus is on chromosome 7p whereas that for CD44 is on chromosome 11p. Quantitative binding assays using 125I-labeled Fab fragments of CD44 antibodies did not show any evidence for reduced levels of CD44 on RBCs from the parents of the patient or from her unaffected sister. The parents and sister had the common Colton blood group phenotype [Co(a+b- )]. Neither deficiency of CD44 nor absence of Colton antigens are general features of CDA because erythrocytes from patients with CDA I, CDA II, CDA III, and two other unclassified CDAs had normal expression of CD44 and normal Colton blood group phenotypes. Further analysis of the defect(s) present in the patient's erythroid cells may provide useful information regarding membrane assembly and the regulation of differentiation in normal erythroid cells.
8
Parsons, SF, J. Jones, DJ Anstee, PA Judson, B. Gardner, E. Wiener, J. Poole, N. Illum und SN Wickramasinghe. „A novel form of congenital dyserythropoietic anemia associated with deficiency of erythroid CD44 and a unique blood group phenotype [In(a-b- ), Co(a-b-)]“. Blood 83, Nr. 3 (01.02.1994): 860–68. http://dx.doi.org/10.1182/blood.v83.3.860.bloodjournal833860.
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We have used a panel of well-characterized monoclonal antibodies (MoAbs) to examine the blood cells of a patient with a novel form of congenital dyserythropoietic anemia (CDA) characterized by intra- erythroblastic and intra-erythrocytic membranous inclusions. Twelve antibodies defining three nonoverlapping epitope groups on the extracellular domain of CD44 all failed to react with the red blood cells (RBCs) of the patient. A rabbit antibody to the cytoplasmic domain of CD44 from normal RBCs failed to react with the patient's RBC ghosts. In contrast, the patient's lymphocytes, granulocytes, and monocytes showed apparently normal CD44 expression. Bone marrow preparations stained with CD44 antibodies and visualized with 125I antimouse Ig (F(ab')2) followed by autoradiography showed positive staining of lymphocytes and myeloid cells but not of most orthotolidine- positive erythroblasts. The patient's RBCs also gave weaker than normal reactions with MoAbs of anti-LWab specificity while MoAbs to glycophorins A, B, and C, Rh polypeptides, CD47, CD55, CD58, CD59, acetylcholinesterase, and Lutheran and Kell glycoproteins all gave normal reactions. Agglutination tests with human blood grouping sera demonstrated that the RBCs of the patient have the unique phenotype In(a-b-), Co(a-b-) and that they also lack the high incidence RBC antigen AnWj. The phenotype In(a-b-) would be expected because these antigens are known to be expressed on CD44. There is also some evidence associating the AnWj antigen with CD44. However, the CO blood group locus is on chromosome 7p whereas that for CD44 is on chromosome 11p. Quantitative binding assays using 125I-labeled Fab fragments of CD44 antibodies did not show any evidence for reduced levels of CD44 on RBCs from the parents of the patient or from her unaffected sister. The parents and sister had the common Colton blood group phenotype [Co(a+b- )]. Neither deficiency of CD44 nor absence of Colton antigens are general features of CDA because erythrocytes from patients with CDA I, CDA II, CDA III, and two other unclassified CDAs had normal expression of CD44 and normal Colton blood group phenotypes. Further analysis of the defect(s) present in the patient's erythroid cells may provide useful information regarding membrane assembly and the regulation of differentiation in normal erythroid cells.
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Mancuso, Patrizia, Ines Martin Padura, Giuliana Gregato, Paola Marighetti, Angelica Calleri, Chiara Corsini, Giancarlo Pruneri, Visnu Lohsiriwat, Jean Yves Petit und Francesco Bertolini. „CD45-CD34+ Endothelial Progenitor Cells (EPCs) from Human Adipose Tissue Promote Tumor Growth and Metastases“. Blood 118, Nr. 21 (18.11.2011): 2208. http://dx.doi.org/10.1182/blood.v118.21.2208.2208.
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Abstract Abstract 2208 A catalytic role has been proposed in neoplastic angiogenesis and cancer progression for bone marrow-derived endothelial progenitor cells (EPCs). However, in preclinical and clinical studies the quantitative role of marrow-derived EPCs in cancer vascularization was found to be extremely variable. Adipose tissue represents an attractive source of autologous adult stem cells due to its abundance and surgical accessibility. Lipotransfer aspirates (LAs) from patients undergoing breast reconstruction after breast cancer surgery were analyzed by six colors flow cytometry and tissue culture. After collagenase digestion, cells were stained with the nuclear binding antigen Syto16 and 7-AAD and with CD34, CD45, CD133, CD31, CD140b, CD105, CD90, CD44, CD13, CD144, CD10, CD29, CD109, CD117, CD146,CD16, CD11c, CD14, CD38, CXCR4, VEGFR-1, VEGFR-2, VEGFR-3, Tie-2. The absolute count of CD45-CD34+ cells was obtained using reference beads in Trucount tubes (BD, Mountain View, CA). LAs were found to contain a large amount of CD45-CD34+ cells fulfilling the most recent criteria for EPC identification. These CD45-CD34+ cells included two subpopulations: CD45-CD34++ CD13+ CD140b+ CD44+ CD90++ cells and CD45-CD34+ CD31+CD105+ cells. We found in the adipose tissue about 263 fold more CD45-CD34+ EPCs/mL when compared to the bone marrow. In particular, the median of CD45-CD34+CD31- cells/mL was 181,046 (range 35,970–465,357), and the median of CD45-CD34+CD31+ cells/mL was 76,946 (range13,982-191,287). When compared to marrow-derived CD34+ cells, purified CD45-CD34+ adipose cells expressed similar levels of stemness-related genes such as NANOG, SOX2, Lin28 and significantly increased levels of angiogenesis-related genes such as CD144, VEGFR2, ALK-1. In vitro, CD45-CD34+ cells generated mature endothelial cells and capillary tubes as well as mature mesenchymal cells. When coinjected with triple negative human breast cancer MDA-MB-436 and HCC1937 cells in the mammary fat of a murine model of human breast cancer, purified CD45-CD34+ cells significantly increased tumor growth, and immunohistochemistry studies demonstrated the presence of human CD31+, CD34+, CD105+ endothelial cells lining the vessels of orthotopic breast cancers growing in mice co-injected with human adipose tissue-derived CD45-CD34+ cells. Moreover, in a mouse model of breast cancer metastatization we found an increased number of lung and axillary lymph node metastases when purified CD34+ WAT cells were injected into the third mammary fat pad after the primary tumor resection. In conclusion our data demonstrate that the phenotype of adipose derived EPCs is consistent with that reported for both bone marrow and circulating EPCs, but their frequency in adipose tissue is more than 250 fold higher. Further studies are ongoing to clarify what cell populations residing in the adipose tissue can be used safely for breast reconstruction and what are at risk for supporting the growth of otherwise quiescent cancer cells still resident after surgery. Disclosures: No relevant conflicts of interest to declare.
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Rappa, G., F. Anzanello und A. Lorico. „CD24 expression and breast cancer stem cell phenotype“. Journal of Clinical Oncology 27, Nr. 15_suppl (20.05.2009): 11106. http://dx.doi.org/10.1200/jco.2009.27.15_suppl.11106.
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11106 Background: Several studies suggest the existence of breast cancer-initiating cells (BCIC), responsible for tumor development and progression. Initial reports that only the CD44+CD24−/low subpopulation contains BCIC have been challenged by subsequent studies. We examined the relationship between CD24 and biological properties of breast cancer cells. Methods: MA-11 breast carcinoma cells, originating from bone marrow micrometastases, are CD44+ and have an heterogeneous expression of CD24 (214,000/cell; range 0–1,120,000). We have previously reported that upon in vitro culture as mammospheres under stem cell-like conditions, MA-11 cells acquired increased tumorigenicity and a CD44+CD24−/low phenotype. We have now investigated the relationship between CD24 expression and tumorigenicity in the MA-11 model. Results: Upon passage of MA-11 mammospheres in adherent culture, cells rapidly re-expressed CD 24. The rapid increase in CD24 was consistent with antigen up-regulation, not selection of CD24−/low cells. Exposure of adherent MA-11 cells to imatinib for 72h resulted in a reversible decrease in CD24 from 214,000 to 15,800/cell. CD44+CD24−/low cells, sorted by flow cytometry, generated CD44+CD24high, and CD44+CD24highgenerated CD44+CD24−/low. Immediately after sorting, >90% CD44+CD24−/low cells were in G0/G1. After 24–48 h in culture, cell cycle distribution, growth rate and invasiveness of the sorted cell populations were equivalent. Upon injection and s.c. growth, CD24 expression of CD44+CD24−/low populations and clones increased from 10,000 to 220,000/cell. Similarly, CD44+CD24−/low clones derived from human MCF-7 breast carcinoma cells formed tumors containing >99% CD44+CD24high cells. The average number of CD24 per cell was equivalent for tumors formed upon injection of CD44+CD24−/low, CD44+CD24+, mammosphere-derived cells or parental adherent MA-11 cells. The tumorigenic potentials of sorted CD44+CD24−/low, CD44+CD24−/lowsub-populations and clones in nu/nu mice were equivalent. Conclusions: CD44+CD24−/low breast cancer cells are not associated with increased tumorigenicity; the high CD24 level of mouse xenografts derived from both CD44+CD24−/low and CD44+CD24hi breast cancer cells suggests an important role for CD24 in tumor growth. No significant financial relationships to disclose.
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Schuurhuis, Gerrit J., Rolf Wouters, Angèle Kelder, Arjo P. Rutten, Alexander N. Snel, Monique Terwijn, Paul Noordhuis, Sonja Zweegman und Gert J. Ossenkoppele. „Specificity of Markers of Leukemia Initiating Cells with a New Multiparameter Flow Cytometry Based Appraoch; Impact for Prognostic and Therapeutic Applications“. Blood 116, Nr. 21 (19.11.2010): 1834. http://dx.doi.org/10.1182/blood.v116.21.1834.1834.
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Abstract Abstract 1834 Leukemia stem cells (LSC) are proposed to underly relapse of AML. In order to develop methods to specifically detect LSCs and to design LSC specific therapies, discrimination between LSC and normal hematopoietic stem cells (HSC) is a prerequisite. HSC and LSC may be either CD34-positive (CD34+CD38-) or CD34-negative. The latter may be contained in stem cell compartments defined by functional parameters: Hoechst 33342 efflux (side population, SP) and ALDH activity. To discriminate between LSCs and HSCs aberrant cell surface markers have been described. Additional flowcytometric parameters (scatter, and level of expression of CD34/CD45) allow further discrimination (Terwijn Blood 2009; 114:165). We applied this new approach to compare the specificity of expression of the LSC markers CD123, CLL-1, CD96, CD44, CD47, and CD33. We studied LSCs in diagnosis AML, HSCs in normal bone marrow (NBM) and regenerating BM (RBM) and HSCs present at diagnosis in the AML BM itself. The Table shows the results for the CD34+CD38-cells. Large differences were found for LSCs: CD44>CD33>CD47>CD123≥CLL-1 and CD96 (column 2; median and ranges shown in all columns). Expression on HSCs in NBM (column 4) shows the lack of LSC specificity of several of these markers: CD44>CD47>CD33>CD123>CD96 and CLL-1. A similar result was found for RBM for the three markers studied: CD33>CD123>CLL-1 (column 5). Expression in HSCs present in the AML BM itself was: CD33>CD123>CLL-1 and CD96 (column 3). CD44 and CD47 could not be properly analyzed due to extensive overlap of LSC and HSC (column 3). In order to determine whether expression on HSC would negatively impact diagnostic and targeted therapy, we defined an in vitro “therapeutic window” (TW=expr. in LSC/expr. in HSC) in columns 6–8: each individual value for LSC for a particular marker was divided by the median value of HSC in NBM (column 7), RBM (column 8) or divided by each corresponding HSC value in the AML BM itself (column 6). Overall, TW was slightly higher for CLL-1 than for CD96, CD123 and CD33, irrespective HSC source (CD33 had a high TW in diagnosis BM). The TW for CD44 and CD47 was low (column 7) and not analyzable in the AML BM itself. Experiments were repeated for SP cells: TW for CLL-1 (for the 3 HSC sources, resp) were 2.1, 3.8, and 3.8; for CD123: 2.0, 1.5, and 2.5 and for CD33: 1.9, 1.3, and 1.6. CLL-1 was thus slightly superior for SPs. The overall conclusions from this project are that, due to a high specificity, CD123, CLL-1, CD96 and CD33, but not CD44 and CD47 are suitable for diagnostic purposes. Whether or not a similar conclusion can be drawn for therapeutic applications remains to be investigated. Large differences exist in expression of the different antigens for most individual AML cases; for therapeutic applications, for each AML case, the optimal antigen or, most likely, combinations of antigens will be needed. The most important conclusion is that, by using our approach, it is now possible to discriminate between concomitantly present LSCs and HSCs. This approach may have high impact for both prognostic purposes (e.g. prediction of relapse) and prospective isolation of LSC and HSC from the same AML BM to design new therapeutic modalities. It may further serve as a surrogate marker to monitor effectiveness of a treatment and to design antibody/ligand combination therapies in a personalized setting. Disclosures: No relevant conflicts of interest to declare.
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Marlina, Marlina, Rizki Rahmadian, Armenia Armenia, Wahyu Widowati, Rizal Rizal, Hanna Sari Widya Kusuma, Satrio Haryo Benowo Wibowo, Wahyu Setia Widodo und Ika Adhani Sholihah. „Isolation, Characterization, Proliferation and Differentiation of Synovial Membrane-derived Mesenchymal Stem Cells (SM-MSCs) from Osteoarthritis Patients“. Molecular and Cellular Biomedical Sciences 4, Nr. 2 (01.07.2020): 76. http://dx.doi.org/10.21705/mcbs.v4i2.100.
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Background: Mesenchymal stem cells (MSCs) are the cells which has high renewal capacity and and are capable for differentiating into some types of cells. MSCs can be obtained from several tissues including bone marrow, synovial membrane, blood, adipose tissue and periosteum. The proliferation and self-repair ability of MSCs are the advantages to use as stem cells-based therapy of various diseases. The aim of this study was to determine the differentiation, characterization and priliferation of synovial membrane-derived MSCs (SM-MSCs).Materials and Methods: The cells proliferation capacity was determined by cell counting using trypan blue, characterization of MSCs (cluster of differentiation (CD)90, CD11b, CD73, CD34, CD19, CD45, CD105 and human leukocyte antigen-DR isotype (HLA-DR)) using flow cytometry analysis, and differentiation capability into three lineage cells was determined with red alcian blue, oil red O and alizarin staining.Results: The type culture of SM-MSCs was adherent and showed positive CD44, CD105, CD73, CD90 and negative of CD19, HLA-DR, CD11b, CD45, CD34 surface marker. Based on the result, SM-MSCs P3 showed differentiation potency into adipogenic, chondrogenic, and osteogenic lineage cells. The population doubling time of SM-MSCs has increased from P3 to P8. The population doubling time of SM-MSCs P3 was 1.69 days and SM-MSCs P8 was 3.64 days.Conclusion: The results indicated that SM-MCSCs from osteoarthritis patients are able to differentiate into osteocytes, chondrocytes, adipocytes and highly express of CD105, CD73, CD90, CD44 and negative for CD34, CD45, CD14, CD19.Keywords: synovial membrane, mesenchymal stromal cells, adipocyte, chondrocyte, osteocyte
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Florian, Stefan, Karoline Sonneck, Alexander W. Hauswirth, Maria-Theresa Krauth, Wolfgang R. Sperr und Peter Valent. „Phenotyping of Neoplastic (CD34+/CD38−/CD123+) Stem Cells in Myeloid Malignancies Reveals Expression of Multiple Molecular Targets.“ Blood 106, Nr. 11 (16.11.2005): 1381. http://dx.doi.org/10.1182/blood.v106.11.1381.1381.
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Abstract Recent data suggest that myeloid neoplasms are organized hierarchically in terms of self renewal and maturation of early progenitor cells, similar to normal myelopoiesis. In acute myeloid leukemia (AML), the NOD/SCID mouse-repopulating leukemic stem cells usually co-express CD123 with CD34, but lack CD38. So far, however, little is known about expression of other markers and targets on these progenitors. In the present study, expression of target antigens on CD34+/CD38− cells was analyzed by multicolor flow cytometry in patients with AML (n=18), myelodysplastic syndromes (MDS, n=6), chronic myeloid leukemia (CML, n=8), systemic mastocytosis (SM, n=9), and normal bone marrow (n=5). The IL-3Ra chain (CD123) was found to be expressed on CD34+/CD38− cells in a majority of all patients in all disease-categories. Independent of the type of disease, the vast majority of these stem cells also co-expressed aminopeptidase-N (CD13) and the target receptor CD44 in all patients. CD34+/CD38− progenitor cells expressed variable amounts of the Mylotarg® receptor CD33, KIT (CD117), HLA-DR, and AC133 (CD133). With regard to AC133, two distinct subpopulations of progenitor cells were detected in many cases, namely a CD133+ and a clearly CD133- cell-fraction. In patients with AML, the levels of CD33 varied from patient to patient with a broad range of reactivity, whereas in most patients with MDS, CML, and SM, CD33 was found to be consistently expressed on most progenitors. In most patients, neoplastic stem cells did not express substantial amounts of the GM-CSF receptor alpha chain (CD116), Thy-1 (CD90), E-NPP3 (CD203c), MDR-1 (CD243), or PAR-2. In the normal bone marrow, CD34+/CD38− cells co-expressed CD13, CD44 and CD45, but did not express CD33, CD116, or CD123. In conclusion, neoplastic stem cells in various myeloid neoplasms appear to express a similar phenotype including target receptors such as CD13, CD33, and CD44. These antigens may thus be attractive targets of therapy in AML. However, since many of these targets are not expressed on all stem cells in all patients, the elimination of the entire clone may require combinations of targeted antibodies or use of additional drugs. In other cases (CD13, CD44, CD45), the target antigen is also expressed on normal stem cells, so that targeted therapy is likely to be an ablative maneuver and thus would require a combined stem cell transplantation approach.
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Vicente, A., A. Varas, R. S. Acedón, E. Jiminez, J. J. Mulqoz und A. G. Zapata. „Appearance and Maturation of T-Cell Subsets During Rat Thymus Ontogeny“. Developmental Immunology 5, Nr. 4 (1998): 319–31. http://dx.doi.org/10.1155/1998/24239.
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In previous papers, we have described the ontogenetical development of thymic stromal-cell components (epithelium, macrophages, dendritic cells) of Wistar rats. Here, we correlate those results with the maturation of rat T-cell precursors along the fetal and postnatal life. First T-cell precursors, which colonize the thymus anlage around days 13-14 of gestation, largely express CD45, CD43, CD53, and Thy 1 cell markers, and in a lesser proportion the OX22 antigen. Rat CD3-CD4-CD8-thymocytes present in the earliest stages of gestation could be subdivided in three major cell subpopulations according to the CD44 and CD25 expression: CD44-/+CD25-→ CD44+CD25+→ CD44+CD25-On fetal days 17-18, a certain proportion of CD4-CD8-cells weakly,express the TcRβchain, in correlation with the appearance of the first immature CD4-CD8+thymocytes. This cell subpopulation, in progress to the CD4+CD8+stage, upregulates CD8αbefore the CD8βchain, expresses the CD53 antigen, and exhibits a high proliferative rate. First mature thymocytes arising from the DP (CD4+CD8+) cells appear on fetal days 20-21. Then, the CD4+:CD8+cell ratio is ≤1 changing to adult values (2-3) just after birth. Also, the percentage of VβTcR repertoire covered in adult thymus is reached during the postnatal period, being lower during the fetal life. Finally, in correlation with the beginning of thymocyte emigration to the periphery a new wave of T-cell maturation apparently occurs in the perinatal rat thymus.
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Zhang, Bao Yue, Hui Xue Song, Tan Chen und Zhan Hui Wang. „A Microfluidic Platform for Multi-Antigen Immunofluorescence Assays“. Applied Mechanics and Materials 108 (Oktober 2011): 200–205. http://dx.doi.org/10.4028/www.scientific.net/amm.108.200.
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Traditional cell-based assays such as cell immunoassay that utilizes plastic (chamber slides, dishes, microtiter plates), Magnetic bead, enzyme-linked immunsorbent assays (ELISA) [1], FACS cell sorting is labor intensive, time consuming, and requires a large numbers of cells or reagents. In this report, a microfluidic device integrated with cell culture, washing, fixation, and antigen-antibody reaction is presented for high-throughput immunoassay. Using this microfluidic device, each assay can be performed on a small number of cells and nanolitre or picolitre of reagents, this is particularly beneficial for rare or expensive cell types such as stem cells, or flow sorted cell populations. The capability of the microfluidic device was demonstrated for seeding human umbilical cord blood mesenchymal stem cells (UC-MSCs) in chambers and detecting the expression of surface markers (CD34, CD44, CD45, CD73, CD105, HLA-DR) by immunofluorescence assay.
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Collado, Antonia, Antonia de Andres, Encarnación Cañadas, Francisco Ruiz-Cabello, Ovidio Gomez, Susana Pedrinaci und Federico Garrido. „Characterization of CD44 Antigen during Lymphoid Ontogeny“. Immunobiology 183, Nr. 1-2 (September 1991): 1–11. http://dx.doi.org/10.1016/s0171-2985(11)80181-6.
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Camp, R. L., T. A. Kraus, M. L. Birkeland und E. Puré. „High levels of CD44 expression distinguish virgin from antigen-primed B cells.“ Journal of Experimental Medicine 173, Nr. 3 (01.03.1991): 763–66. http://dx.doi.org/10.1084/jem.173.3.763.
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The in vitro polyclonal stimulation of B cells through their surface immunoglobulin (Ig) induces substantial increases in CD44 protein levels within 24 hours, whereas other stimuli (e.g., lipopolysaccharide, phorbol 12,13 dibutyrate, and interleukin 4) fail to significantly upregulate CD44. The marked increase in CD44 protein expression on anti-Ig-treated B lymphocytes correlates with an increase in CD44-specific mRNA. Cell sorting experiments with B cells isolated from trinitrophenyl-keyhole limpet hemocyanin-immunized mice demonstrate that both short-term antigen-specific, IgG-secreting cells and long-term antigen-primed B cells are exclusively CD44high. We speculate that the rapid and sustained increase in CD44 expression mediated by surface Ig stimulation may alter the homing properties of antigen-primed B cells.
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Griffioen, Arjan W., Marieke J. H. Coenen, Cora A. Damen, Sandra M. M. Hellwig, David H.J. van Weering, Wim Vooys, Geert H. Blijham und Gerard Groenewegen. „CD44 Is Involved in Tumor Angiogenesis; an Activation Antigen on Human Endothelial Cells“. Blood 90, Nr. 3 (01.08.1997): 1150–59. http://dx.doi.org/10.1182/blood.v90.3.1150.
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Abstract CD44 is described to be an activation molecule in a number of different cell types. We investigated the role of CD44 on human endothelial cells (EC) and in tumor angiogenesis. Using flow cytometry we showed that EC from the vasculature of human solid tumors display an enhanced expression of CD44 as compared to EC from normal tissue. This finding was confirmed by immunohistochemical studies on frozen tissue sections. Because tumors are dependent on angiogenesis, the role of angiogenic stimuli in the enhanced CD44 expression was investigated. We found that basic fibroblast growth factor (bFGF ) and vascular endothelial growth factor were able to efficiently upregulate CD44 expression on cultured human EC. The upregulation reached maximal levels after treatment for 3 days with 10 ng/mL bFGF. The physiological impact of this upregulation was shown by the enhanced binding of EC to hyaluronate after pretreatment with bFGF. In a next set of studies that were designed to unravel the regulation of CD44 expression on EC we concluded that CD44 is an activation antigen on human EC since (1) human umbilical vein derived endothelial cells, which in vivo do not express CD44, begin to express CD44 when plated and cultured, (2) CD44 expression is enhanced after subculture of confluent cultures, (3) CD44 is predominantly expressed on the BrdU incorporating subset of cultured EC. The specific expression of CD44 on activated and tumor EC prompted us to study the usefulness of CD44 as an endothelial target for therapy with immunotoxins. In vitro experiments showed that EC are efficiently killed after targeting immunotoxin to CD44.
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Griffioen, Arjan W., Marieke J. H. Coenen, Cora A. Damen, Sandra M. M. Hellwig, David H.J. van Weering, Wim Vooys, Geert H. Blijham und Gerard Groenewegen. „CD44 Is Involved in Tumor Angiogenesis; an Activation Antigen on Human Endothelial Cells“. Blood 90, Nr. 3 (01.08.1997): 1150–59. http://dx.doi.org/10.1182/blood.v90.3.1150.1150_1150_1159.
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CD44 is described to be an activation molecule in a number of different cell types. We investigated the role of CD44 on human endothelial cells (EC) and in tumor angiogenesis. Using flow cytometry we showed that EC from the vasculature of human solid tumors display an enhanced expression of CD44 as compared to EC from normal tissue. This finding was confirmed by immunohistochemical studies on frozen tissue sections. Because tumors are dependent on angiogenesis, the role of angiogenic stimuli in the enhanced CD44 expression was investigated. We found that basic fibroblast growth factor (bFGF ) and vascular endothelial growth factor were able to efficiently upregulate CD44 expression on cultured human EC. The upregulation reached maximal levels after treatment for 3 days with 10 ng/mL bFGF. The physiological impact of this upregulation was shown by the enhanced binding of EC to hyaluronate after pretreatment with bFGF. In a next set of studies that were designed to unravel the regulation of CD44 expression on EC we concluded that CD44 is an activation antigen on human EC since (1) human umbilical vein derived endothelial cells, which in vivo do not express CD44, begin to express CD44 when plated and cultured, (2) CD44 expression is enhanced after subculture of confluent cultures, (3) CD44 is predominantly expressed on the BrdU incorporating subset of cultured EC. The specific expression of CD44 on activated and tumor EC prompted us to study the usefulness of CD44 as an endothelial target for therapy with immunotoxins. In vitro experiments showed that EC are efficiently killed after targeting immunotoxin to CD44.
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Weiss, Johannes M., Jonathan Sleeman, Andreas C. Renkl, Henning Dittmar, Christian C. Termeer, Sabine Taxis, Norma Howells et al. „An Essential Role for CD44 Variant Isoforms in Epidermal Langerhans Cell and Blood Dendritic Cell Function“. Journal of Cell Biology 137, Nr. 5 (02.06.1997): 1137–47. http://dx.doi.org/10.1083/jcb.137.5.1137.
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Upon antigen contact, epidermal Langerhans cells (LC) and dendritic cells (DC) leave peripheral organs and home to lymph nodes via the afferent lymphatic vessels and then assemble in the paracortical T cell zone and present antigen to T lymphocytes. Since splice variants of CD44 promote metastasis of certain tumors to lymph nodes, we explored the expression of CD44 proteins on migrating LC and DC. We show that upon antigen contact, LC and DC upregulate pan CD44 epitopes and epitopes encoded by variant exons v4, v5, v6, and v9. Antibodies against CD44 epitopes inhibit the emigration of LC from the epidermis, prevent binding of activated LC and DC to the T cell zones of lymph nodes, and severely inhibit their capacity to induce a delayed type hypersensitivity reaction to a skin hapten in vivo. Our results demonstrate that CD44 splice variant expression is obligatory for the migration and function of LC and DC.
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Milde, Kerstin F., Rodolfo Alejandro, Daniel H. Mintz und Ricardo L. Pastori. „Molecular cloning of the canine CD44 antigen cDNA“. Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression 1218, Nr. 1 (Mai 1994): 112–14. http://dx.doi.org/10.1016/0167-4781(94)90111-2.
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Dzudzilo, Madara, Regīna Kleina, Ingrīda Čēma, Anita Dabuzinskiene und Šimons Svirskis. „Expression and Localisation of CD44 Antigen as a Prognostic Factor of Oral Leukoplakia“. Proceedings of the Latvian Academy of Sciences. Section B. Natural, Exact, and Applied Sciences. 75, Nr. 2 (01.04.2021): 68–74. http://dx.doi.org/10.2478/prolas-2021-0012.
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Abstract It is essential to identify markers that could indicate the presence of early molecular changes in premalignant tissues like oral leukoplakia (OL). CD44 adhesion molecule is not only a stem cell marker, but also determines cell proliferation and migration in malignant processes. The aim of our study was to assess the amount and pattern of CD44 antigen expression by epithelial and mononuclear cells in the lamina propria under OL and their role in premalignant lesions. The current study included 102 cases of OL and ten biopsies from healthy oral mucosa. Immunohistochemical CD44 antigen expression was determined in 34 cases by a standard EnVision imaging system in three points of OL: both edges and centre. Statistical analysis was done using GraphPad Prism software version 8.4.0. In OL, statistically significant overexpression of membranous CD44 was demonstrated compared to healthy mucosa (p < 0.0001). The intra-cytoplasmatic CD44 expression of epithelium together with characteristic nuclear changes may be used as a predictive factor for potential malignant transformation of non-homogenous leukoplakia. CD44 expression in mononuclear cells under the basal membrane in OL (p < 0.05) possibly influences the process of premalignant lesion transformation into intraepithelial cancer. Further study of CD44 antigen expression in intra-cytoplasmatic structures is required for better explanation of the role of this glycoprotein.
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Cheung, Sarah K. C., Po-Kai Chuang, Han-Wen Huang, Wendy W. Hwang-Verslues, Candy Hsin-Hua Cho, Wen-Bin Yang, Chia-Ning Shen et al. „Stage-specific embryonic antigen-3 (SSEA-3) and β3GalT5 are cancer specific and significant markers for breast cancer stem cells“. Proceedings of the National Academy of Sciences 113, Nr. 4 (17.12.2015): 960–65. http://dx.doi.org/10.1073/pnas.1522602113.
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The discovery of cancer stem cells (CSCs), which are responsible for self-renewal and tumor growth in heterogeneous cancer tissues, has stimulated interests in developing new cancer therapies and early diagnosis. However, the markers currently used for isolation of CSCs are often not selective enough to enrich CSCs for the study of this special cell population. Here we show that the breast CSCs isolated with CD44+CD24-/loSSEA-3+ or ESAhiPROCRhiSSEA-3+ markers had higher tumorigenicity than those with conventional markers in vitro and in vivo. As few as 10 cells with CD44+CD24-/loSSEA-3+ formed tumor in mice, compared with more than 100 cells with CD44+CD24-/lo. Suppression of SSEA-3 expression by knockdown of the gene encoding β-1,3-galactosyltransferase 5 (β3GalT5) in the globo-series pathway, led to apoptosis in cancer cells specifically but had no effect on normal cells. This finding is further supported by the analysis of SSEA-3 and the two related globo-series epitopes SSEA4 and globo-H in stem cells (embryonic stem cells and induced pluripotent stem cells) and various normal and cancer cells, and by the antibody approach to target the globo-series glycans and the late-stage clinical trials of a breast cancer vaccine.
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Sperr, WR, HC Bankl, G. Mundigler, G. Klappacher, K. Grossschmidt, H. Agis, P. Simon, P. Laufer, M. Imhof und T. Radaszkiewicz. „The human cardiac mast cell: localization, isolation, phenotype, and functional characterization“. Blood 84, Nr. 11 (01.12.1994): 3876–84. http://dx.doi.org/10.1182/blood.v84.11.3876.bloodjournal84113876.
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We have isolated and characterized the human cardiac mast cell (CMC) and compared this novel mast cell (MC type with MC obtained from uterus, skin, and lung. Heart tissue was obtained from 14 patients with cardiomyopathy (CMP, heart transplantation). CMC were isolated by enzymatic digestion using collagenase, pronase-E, hyaluronidase, and DNAse. Substantial amounts of CMC (0.5% to 1.5% of isolated cells) were found in the atrial appendages but not in ventricular digests or other sites of the heart (< 0.1%). In situ staining of atrial tissue revealed the presence of CMC in the myocardium (2.16 +/- 0.7 MC/mm2), endocardium (2.24 +/- 0.9 MC/mm2), and epicardium. As assessed by combined toluidine blue/immunofluorescence staining with monoclonal antibodies (MoAbs), isolated CMC expressed surface IgE, the receptor for stem cell factor (c-kit receptor/CD117), the p24 antigen (CD9), the Pgp-1 homing receptor (CD44), the pan leukocyte antigen (CD45), and the ICAM-1 antigen (CD54). CMC were not recognized by MoAbs to lymphocyte function associated antigen 2 (LFA-2; CD2), T-cell receptor (TcR; CD3), T4 antigen (CD4), LFA-1 alpha-chain (CD11a), C3biR alpha-chain (CD11b), CR4 alpha-chain (CD11c), LPS-R related Ag (CD14), 3-FAL/x-hapten (CD15), Fc gamma RIII (CD16), lactosylceramid (CDw17), the B-cell antigen CD19, or CR1 (CD35). In situ expression of leukocyte antigens on CMC was demonstrable by indirect immunoperoxidase staining technique and double-labeling immunohistochemistry. Almost all CMC (90%) reacted with MoAbs against tryptase and chymase and thus were MCTC. Cardiac mast cells were also stained by the heparin-binding dye Berberine sulfate and expressed measurable amounts of histamine (4.6 +/- 1.4 pg per cell). Cross linking of either IgE receptor or SCF receptor (c-kit) on CMC resulted in histamine secretion (non-specific release: < 6% of total histamine, alpha IgE induced: 12% to 52%; SCF-induced release: 9% to 18%), whereas neither substance P (a skin MC agonist) nor the basophil agonist FMLP showed an effect on CMC. Together, the CMC is an MCTC primarily located in the appendage of the atrium. This novel type of MC exhibits surface membrane antigen and functional properties similar to those of lung and uterus MC.
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van der Voort, Robbert, Robert M. J. Keehnen, Esther A. Beuling, Marcel Spaargaren und Steven T. Pals. „Regulation of Cytokine Signaling by B Cell Antigen Receptor and Cd40-Controlled Expression of Heparan Sulfate Proteoglycans“. Journal of Experimental Medicine 192, Nr. 8 (09.10.2000): 1115–24. http://dx.doi.org/10.1084/jem.192.8.1115.
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Recently, biochemical, cell biological, and genetic studies have converged to reveal that integral membrane heparan sulfate proteoglycans (HSPGs) are critical regulators of growth and differentiation of epithelial and connective tissues. As a large number of cytokines involved in lymphoid tissue homeostasis or inflammation contain potential HS-binding domains, HSPGs presumably also play important roles in the regulation of the immune response. In this report, we explored the expression, regulation, and function of HSPGs on B lymphocytes. We demonstrate that activation of the B cell antigen receptor (BCR) and/or CD40 induces a strong transient expression of HSPGs on human tonsillar B cells. By means of these HSPGs, the activated B cells can bind hepatocyte growth factor (HGF), a cytokine that regulates integrin-mediated B cell adhesion and migration. This interaction with HGF is highly selective since the HSPGs did not bind the chemokine stromal cell–derived factor (SDF)-1α, even though the affinities of HGF and SDF-1α for heparin are similar. On the activated B cells, we observed induction of a specific HSPG isoform of CD44 (CD44-HS), but not of other HSPGs such as syndecans or glypican-1. Interestingly, the expression of CD44-HS on B cells strongly promotes HGF-induced signaling, resulting in an HS-dependent enhanced phosphorylation of Met, the receptor tyrosine kinase for HGF, as well as downstream signaling molecules including Grb2-associated binder 1 (Gab1) and Akt/protein kinase B (PKB). Our results demonstrate that the BCR and CD40 control the expression of HSPGs, specifically CD44-HS. These HSPGs act as functional coreceptors that selectively promote cytokine signaling in B cells, suggesting a dynamic role for HSPGs in antigen-specific B cell differentiation.
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Oelschlaegel, Uta, Kirsten Poppe-Thiede, Kristina Hoelig, Martin Bornhaeuser, Gerhard Ehninger und Frank Kroschinsky. „Differences in the Expression of Adhesion Molecules and Mobilization Efficacy in Healthy Stem Cell Donors and Patients with Hematologic Malignancies.“ Blood 104, Nr. 11 (16.11.2004): 2879. http://dx.doi.org/10.1182/blood.v104.11.2879.2879.
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Abstract Stem cell mobilization is achieved by short term administration of G-CSF in healthy donors and G-CSF +/− chemotherapy in patients with malignant diseases. It is known that the crosstalk between adhesion molecules, bone marrow microenvironment, and cytokines facilitates the multi step process of stem cell mobilization from bone marrow to peripheral blood. But still, the biological basis of the large variability in mobilization efficacy remains unclear. The aim of the present study was to evaluate if poor and good mobilizers - healthy donors as well as patients with hematologic malignancies - show differences in the expression of adhesion molecules (VLA-4, L-selectin, LFA-1, PECAM-1, CD44), the chemokine receptor CXCR4 and the G-CSF receptor measured by flow cytometry on CD34 positive cells. Therefore, we investigated 200 aphereses from 132 healthy PBSC donors and 68 patients with a 4-color staining: CD34/CD45 combined with two different adhesion molecules in each sample. The quantitative (mean fluorescence intensity) and qualitative (%) antigen expression was assessed. Donors/patients were divided into three groups according to the peripheral CD34-positive cell count at the day of apheresis: ≤ 30/μl (poor mobilizer; 36 donors, 30 pts.), 31–100/μl (standard mobilizer; 56 donors, 23 pts.), > 100/μl (good mobilizer; 40 donors, 15 pts.). The quantitative antigen expression was significantly higher for LFA-1 in poor vs. good mobilizing donors (GeoMean: 22 vs. 17; p=0.007) and patients (26 vs. 18; p<0.033) and for PECAM-1 (419 vs. 240; p<0.019; only in patients). In contrast, L-selectin showed a significantly lower expression in poor vs. good mobilizing donors (55 vs. 101; p=0.006). Considering the percentage of antigen positivity, LFA-1 was expressed in a significantly higher proportion of CD34 positive cells in poor mobilizing donors (66% vs. 53%; p=0.002) as well as patients (82% vs. 51%; p<0.011). In contrast, CXCR4 and L-selectin expression was significantly lower in samples of poor mobilizing patients (48 % vs. 62%, p<0.001; 90% vs. 95%, p=0.025). VLA-4, PECAM-1, and CD44 were expressed in 100% of CD34 positive cells independent of mobilization capacity. Comparing healthy donors and patients, the qualitative and quantitative adhesion molecule expression is much lower in donors vs. patients for almost all tested antigens and independent of mobilization efficiency. In contrast, the chemokine receptor CXCR4 has a significantly higher expression in CD34+ cells of healthy donors on the one hand, suggesting its downregulation during recovery from myeloablative chemotherapy in patients on the other hand. In summary, our analyses suggest that a higher LFA-1 expression is correlated with a reduced mobilization efficacy in healthy donors as well as patients with hematologic malignancies. The expression of the other tested antigen seems to be regulated differently in donors and patients.
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Croft, M., D. D. Duncan und S. L. Swain. „Response of naive antigen-specific CD4+ T cells in vitro: characteristics and antigen-presenting cell requirements.“ Journal of Experimental Medicine 176, Nr. 5 (01.11.1992): 1431–37. http://dx.doi.org/10.1084/jem.176.5.1431.
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Because of the low frequency of T cells for any particular soluble protein antigen in unprimed animals, the requirements for naive T cell responses in specific antigens have not been clearly delineated and they have been difficult to study in vitro. We have taken advantage of mice transgenic for the V beta 3/V alpha 11 T cell receptor (TCR), which can recognize a peptide of cytochrome c presented by IEk. 85-90% of CD4+ T cells in these mice express the transgenic TCR, and we show that almost all such V beta 3/V alpha 11 receptor-positive cells have a phenotype characteristic of naive T cells, including expression of high levels of CD45RB, high levels of L-selectin (Mel-14), low levels of CD44 (Pgp-1), and secretion of interleukin 2 (IL-2) as the major cytokine. Naive T cells, separated on the basis of CD45RB high expression, gave vigorous responses (proliferation and IL-2 secretion) to peptide antigen presented in vitro by a mixed antigen-presenting cell population. At least 50% of the T cell population appeared to respond, as assessed by blast transformation, entry into G1, and expression of increased levels of CD44 by 24 h. Significant contributions to the response by contaminating memory CD4+ cells were ruled out by demonstrating that the majority of the CD45RB low, L-selectin low, CD44 high cells did not express the V beta 3/V alpha 11 TCR and responded poorly to antigen. We find that proliferation and IL-2 secretion of the naive CD4 cells is minimal when resting B cells present peptide antigen, and that both splenic and bone marrow-derived macrophages are weak stimulators. Naive T cells did respond well to high numbers of activated B cells. However, dendritic cells were the most potent stimulators of proliferation and IL-2 secretion at low cell numbers, and were far superior inducers of IL-2 at higher numbers. These studies establish that naive CD4 T cells can respond vigorously to soluble antigen and indicate that maximal stimulation can be achieved by presentation of antigen on dendritic cells. This model should prove very useful in further investigations of activation requirements and functional characteristics of naive helper T cells.
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Min, Yoo-Hong, Ji Yeon Kim, Hoi Kyung Jeung, Seung Tae Lee und June-Won Cheong. „Ligation of CD44 by A3D8 Monoclonal Antibody Induces Terminal Differentiation of THP-1 Leukemia Cells by Promoting Cross-Talk between Extracellular Signal-Regulated Kinase and Phosphoinositide-3 Kinase/Akt Signaling Pathway.“ Blood 104, Nr. 11 (16.11.2004): 4460. http://dx.doi.org/10.1182/blood.v104.11.4460.4460.
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Abstract Ligation of CD44 with anti-CD44 monoclonal antibody A3D8 induces terminal differentiation of human leukemia cells. However, the underlying molecular mechanisms remain largely unknown. In this study, we examined the importance of MEK/ERK, p38 MAPK, and phosphatidylinositol 3-kinase (PI3-K)/Akt pathway in A3D8-induced monocytic differentiation of THP-1 leukemia cells. THP-1 cells displayed cytologic changes typical of mature monocytes and an increased expression of monocyte-specific antigen CD14 (49% ± 4%) and of myeloid-specific antigen CD11b (68% ± 6%) after 3 days of A3D8 treatment. The level of CD15 was also increased. The increase in the expression of these differentiation antigens was dose- and time-dependent. We found that CD44 ligation with A3D8 led to rapid and sustained activation of the essential kinases in the extracellular signal-regulated kinase pathway such as phospho-Raf-1, phospho-MEK1/2, and phospho-ERK1/2. However, the total levels of these kinases were not affected during the course of differentiation. Ser473 Akt phosphorylation was also observed shortly after the cells were exposed to A3D8 and sustained thereafter. In contrast, the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and C-Jun N-terminal kinase were not increased by CD44 ligation. Pretreatment of cells with 20mM of MEK1 inhibitor PD98059 abrogated the A3D8-induced activation of MEK1/2 and ERK1/2 and potently inhibited the THP-1 differentiation. However, a p38 MAPK inhibitor SB203580 did not abolished the CD44 ligation-induced terminal differentiation of THP-1 cells. Pretreatment of cells with PI3-K inhibitor LY294002 resulted in a nearly complete inhibition of A3D8-induced terminal differentiation. Overexpression of dominant-negative Akt also reduced the A3D8-induced THP-1 differentiation, suggesting that A3D8-induced THP-1 differentiation is coupled to PI3-K/Akt activation. Surprisingly, the pharmacological inhibition of PI3-K blocked not only A3D8-induced Akt activation but also A3D8-induced activation of Raf, MEK and ERK pathway. By contrast, pretreatment of cells with PD98059 did not inhibit the A3D8-induced Akt activation, suggesting that PI3-K/Akt is the upstream of Raf/MEK/ERK pathway in A3D8-induced terminal differentiation of THP-1 leukemia cells. Taken together, our findings demonstrated that the cross-talk between the activation of A3D8-inducible PI3-K/Akt pathway and the Raf/MEK/ERK pathway in THP-1 cell exists, and plays a critical role during CD44 ligation-induced THP-1 differentiation.
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Jaggupilli, Appalaraju, und Eyad Elkord. „Significance of CD44 and CD24 as Cancer Stem Cell Markers: An Enduring Ambiguity“. Clinical and Developmental Immunology 2012 (2012): 1–11. http://dx.doi.org/10.1155/2012/708036.
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Cancer stem cell population is a subset of cells capable of dictating invasion, metastasis, heterogeneity, and therapeutic resistance in tumours. Eradication of this rare population is a new insight in cancer treatment. However, prospective identification, characterization, and isolation of these CSCs have been a major challenge. Many studies were performed on surface markers for potential identification and isolation of CSCs. Lack of universal expression of surface markers limits their usage and no best combination of markers has yet been confirmed to identify CSCs capable of initiating and metastasizing tumours. CD44, a hyaluronic acid receptor, is one of the most commonly studied surface markers, which is expressed by almost every tumour cell. CD24, a heat stable antigen, is another surface marker expressed in many tumour types. However, their expression and prognostic value in isolating CSCs are still an enduring ambiguity. In this critical review, we assess the role of CD44 and CD24 in tumour initiation, development, and metastasis. We mainly focus on analysing the significance of CD44 and CD24 as CSC surface markers in combination or with other putative markers in different types of cancer.
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Utama, Bobby Indra. „ISOLATION OF AMNIOTIC FLUID MESENCHYMAL STEM CELLS (AF-MSCs) OBTAINED FROM CAESAREAN SECTIONS“. JOURNAL OBGIN EMAS 2, Nr. 1 (28.11.2019): 1–9. http://dx.doi.org/10.25077/aogj.2.1.1-9.2018.
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Amniotic fluid is a liquid that fills the amniotic cavity which has defense and nutritional functions in fetal development. Human aterm amniotic fluid can be an ideal alternative as a source of mesenchymal stem cells, originating from the neonate. Preclinical studies of second and third trimester amnion fluid cells confirmed the number of potential donors from this wasted material. In several studies, AF-MSCs express mesenchymal markers such as CD90, CD73 (SH3, SH), CD105 (SH2), CD29, CD166, CD49e, CD58 and CD44 (MHC class I). These cells also express HLA-ABC antigens, CD 34, CD 45 which are hematopoietic markers, and endothelial CD31 markers. There is no expression of CD10, CD11b, CD14, CD34, CD117, EMA and HLA-DR, DP, DQ antigens. Most of AF-MSCs have pluripotent properties which are characterized by the discovery of octamer binding protein 3/4 (Oct-3/4), transcription factors Nanog (Nanog), and stage-specific embryonic antigen 4 (SSEA-4) on RT-PCR examination. From this study, 8 million cells was isolated. These cells will be used for research on pelvic organ prolapse therapy by using AF-MSCs. AF-MSCs isolation totally takes 6 weeks. From 1 flask, 2 million of stem cells was obtained.
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Ng, David P., und Brent Wood. „Unsupervised Discovery of Early Markers of Erythroid Maturation in Human Donor Marrow“. Blood 124, Nr. 21 (06.12.2014): 4304. http://dx.doi.org/10.1182/blood.v124.21.4304.4304.
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Abstract Antigenic expression during erythroid maturation is not well understood. Despite the large number of surface antigens currently described for leukocytes, only a few (e.g. CD36, CD45, CD71, CD117, and CD235a) have been well characterized on erythroid cells. Here, we apply novel bioinformatic tools to select antigens with high potential for identifying successive stages of erythroid maturation in an unsupervised and unbiased manner from a high dimensional dataset. In brief, flow cytometry was performed on 3 normal donor bone marrows using Becton-Dickinson lyoplates containing a total of 275 unique antibodies and 8 gating reagents (CD15, CD19, CD34, CD38, CD45, CD71, CD117, and CD123). Each of the 275 independent data files from the same donor was aligned using the gating reagents and a weighted nearest neighbor algorithm in order to synthesize a flow data set with 283 antigens and 50,000 events. We applied a modified SPADE algorithm to generate a maturational path for cells of the erythroid lineage starting with CD34+/CD38- progenitors through the mature erythrocyte stage. A non-parametric Kruskal–Wallis test was used to identify and rank antigens that show differential expression along the erythroid maturational sequence. To discern antigens having the greatest discrimination for early vs. late erythroid maturation, we identified 15 antigens common to the top 25 most differentially expressed antigens from each donor sample. As expected, our method correctly identified the three erythroid gating reagents (CD45, CD71 and CD117) as well as previously a previously described erythroid associated antigen (CD36). A few of the identified antigens have been described are less common but also known to be differentially expressed on erythroid cells (e.g. integrin members CD29, CD44, and CD49d), however several additional novel antigens were also identified with strong differential expression including CD46, CD58, CD81, CD98, CD99, CD164, CD220 and CD321 (Figure 1). Of note, CD49d, CD98 and CD164 are of particular interest as they appear to be gradually lost during maturation from the early normoblast through the mature erythrocyte stages. This is in contrast with the other antigens that show high expression on pronormoblasts—with rapid decline during the early normoblast stage to the level of erythrocytes. We examined selected antigens from the synthetic data of all 3 samples using manual gating, and interestingly CD99 retained expression among the erythroid cells to late stage normoblasts in 2 of the 3 samples, suggesting some element of phenotypic variability among normal individuals for this antigen. Additionally, several antigens appear useful in distinguishing erythroid lineage cells from non-erythroid precursors including beta-2 microglobulin, CD50, and HLA-ABC which all showed ubiquitous expression in non-erythroid precursors with low to absent expression among cells of the erythroid lineage. In summary, using a novel unbiased method for large-scale antigen discovery, we have identified multiple novel antigens that are differentially down regulated with progressive erythroid maturation and appear useful in further delineating erythroid progenitor maturation. Additional work is underway to correlate these antigens with our morphologic understanding of erythroid maturation. Further work in characterizing these changes in myeloid stem cell disorders is on going and may be of diagnostic utility in the diagnosis of myelodysplastic syndromes. Figure 1 [Red=High Expression, Blue=Low Expression] Figure 1. [Red=High Expression, Blue=Low Expression] Disclosures No relevant conflicts of interest to declare.
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Brown, T. A., T. Bouchard, T. St John, E. Wayner und W. G. Carter. „Human keratinocytes express a new CD44 core protein (CD44E) as a heparan-sulfate intrinsic membrane proteoglycan with additional exons.“ Journal of Cell Biology 113, Nr. 1 (01.04.1991): 207–21. http://dx.doi.org/10.1083/jcb.113.1.207.
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We previously identified a 90-kD (GP90), collagen-binding, membrane glycoprotein, termed extracellular matrix receptor III (ECMR III), that is homologous to the lymphocyte homing receptor and CD44 antigen (Gallatin, W. M., E. A. Wayner, P. A. Hoffman, T. St. John, E. C. Butcher, and W. G. Carter. 1989. Proc. Natl. Acad. Sci. USA. 86:4654-4658). CD44 is abundantly expressed in many epithelial tissues, and is localized predominantly to filopodia in cultured keratinocytes. Here we establish CD44 as a polymorphic family of related membrane proteoglycans and glycoproteins possessing extensive diversity in both glycosylation and core protein sequence. Human neonatal foreskin keratinocytes (HFKs) and QG56 lung squamous carcinoma cells express an alternatively spliced form of the CD44 core protein (termed CD44E) that contains an additional 132 amino acids in the carbohydrate attachment region of the extracellular domain. HFKs, HT1080 fibrosarcoma and QG56 cells, as well as many other human cells, contain varying ratios of GP90 and structurally related, higher molecular mass forms of CD44 that express the following characteristics: (a) each form reacted with anti-CD44 (mAbs) P1G12, P3H9, and P3H5. Each of these mAbs recognized a distinct, nonoverlapping epitope present on each CD44 form. (b) Differences in mass were due primarily to variation in carbohydrate moieties, including sulfated aspargine-linked glycopeptides (GP), chondroitin sulfate (CS), and heparan sulfate (HS) glycosaminoglycans, as well as O-linked mucin and polylactosamine structure(s). The major polymorphic forms were designated HT1080 GP90 and CS180, QG56 GP230, and HFK HS/CS250, based on dominant carbohydrate moieties and relative mass. (c) The polymorphic forms use CD44 and CD44E core proteins, each containing a unique set of potential attachment sites for O- and N-glycosides and glycosaminoglycans. (d) Immunofluorescence microscopy, differential extraction with Triton-X-114 detergent, and incorporation into liposomes indicated that all the forms were membrane bound glycoconjugates. These results define CD44 as a structurally diverse, but immunologically related, set of intrinsic membrane macromolecules, and suggests that these structurally varied forms might be expected to manifest multiple functions.
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Lai, Xiaoyu, He Huang, Li Huang und Fenfang Zeng. „Isolation of Human Bone Marrow Mesenchymal Stem Cells by a Novel Monoclonal Antibody, ZUC3.“ Blood 108, Nr. 11 (16.11.2006): 2562. http://dx.doi.org/10.1182/blood.v108.11.2562.2562.
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Abstract Objective: Due to absence of a single definitive marker of mesenchymal stem cells (MSCs) and low incidence in human bone marrow, the primary culture of MSCs, conventionally isolated with its characteristic of adherent, were considered to be heterogeneous containing of several subpopulations, which had currently limited our understanding of their biology and therapeutic applications. In our previous study, a novel murine monoclonal antibody (McAb) ZUC3 was produced by hybridoma technology, which was specifically reactive with human MSCs, while showed negative cross-reactivity when screened against a variety of human tissues. Now, ZUC3 antigen positive MSCs population would be further identified by magnetic-activated cell sorting (MACS). Methods: Bone marrow were taken from the iliac crest of normal healthy adult volunteers, and mononuclear cells were separated by density gradient centrifugation, then separated into positively- and negatively-labelled fractions with McAb ZUC3 by immunomagnetic activated cell sorting. The purity of positive cells was analyzed by flow cytometry, then ZUC3 antigen positive and negative cells were plated respectively in human MSCs medium consisting of 10% FBS, LG-DMEM. Characteristics of ZUC3 antigen positive cells phenotype was analyzed by flow cytometry, and proliferation and multiple differentiation potential of the cells was observed in vitro. Results: Flow cytometric analysis showed that ZUC3 antigen expression by cultured MSCs and mononuclear cells derived from bone marrow were 91.31±2.92%, 0.96±0.28% respectively, and western blotting showed the molecular mass of antigen was about 33KD. The purity of the recovered fractions for ZUC3 by MACS was 76.82±6.32%. The positive cells have adhered to culture flask in vitro, and the quantity of adhered cells that had fibroblast-like morphology increased and proliferated during primary expansion period, while the negative cells were observed as round shape cells without any proliferation. It was demonstrated that ZUC3 antigen positive cells continued growth with spindle-shape, extending beyond 30 population doublings in long-term culture. Analyzed by flow cytometry, the culture-expanded positive cells were uniformly positive for CD29, CD44, CD105, CD106, and lack typical hematopoietic antigens such as CD14, CD34, CD45, HLA-DR, which demonstrated that ZUC3 postive cells sorted from bone marrow mononuclear cells by McAb were MSCs. With proper medium, the ZUC3 antigen positive cells could be successfully induced to differentiate into adipocytes, osteoblasts, and neuro-like cells which were positive of neuron markers such as nestin, NSE and NF-M. Conclusion: ZUC3 McAb was a specific surface marker against human MSCs for cell sorting. The ZUC3 antigen positive cells separated from bone marrow mononuclear cells had potential capacity of high proliferation and multiple differentiation.
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Cizkova, Katerina, Jakub Malohlava und Zdenek Tauber. „Cell Membrane Nanostructure is Altered by Heat-Induced Antigen Retrieval: A Possible Consequence for Immunocytochemical Detection of Membranous Antigens“. Microscopy and Microanalysis 26, Nr. 1 (14.11.2019): 139–47. http://dx.doi.org/10.1017/s1431927619015113.
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AbstractHeat-induced antigen retrieval (HIAR) treatment improves the antigen immunodetection in formalin-fixed, paraffin-embedded tissue samples and it can also improve the detection of intracellular antigens in alcohol-fixed cytological samples, although it could deleteriously impact immunodetection, particularly that of membranous antigens. We examined the differences in cell surface topography on MCF7 cells fixed in methanol/acetone (M/A) or 4% paraformaldehyde (4% PFA), as well as the changes caused by HIAR treatment at three different temperatures (60, 90, and 120°C), using atomic force microscopy. Furthermore, the consequences for immunostaining of five membranous antigens [epidermal growth factor receptor (EGFR), E-cadherin, CD9, CD24, and CD44] were examined. Our results illustrate that while there was no one single optimal immunostaining condition for the tested antibodies, the surface topography could be an important factor in successful staining. Generally, the best conditions for successful immunostaining were M/A fixation with no HIAR treatment, whereas in 4% PFA-fixed cells, HIAR treatment at 120°C was optimal. These conditions showed similarity in cell surface skewness. A correlation factor between successful immunocytochemical staining and the skewness parameter was 0.8000. Our results indicate that the presence of valleys, depressions, scratches, and pits on the cell surface is unfavorable for the successful immunodetection of cell surface antigens.
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Camp, R. L., A. Scheynius, C. Johansson und E. Puré. „CD44 is necessary for optimal contact allergic responses but is not required for normal leukocyte extravasation.“ Journal of Experimental Medicine 178, Nr. 2 (01.08.1993): 497–507. http://dx.doi.org/10.1084/jem.178.2.497.
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The in vivo administration of certain monoclonal antibodies (mAbs) against the adhesion receptor, CD44, into normal mice induces both a modulation of CD44 from the surface of peripheral lymphocytes, and a concomitant increase in the amount of soluble CD44 in the serum. CD44-negative lymphocytes isolated from anti-CD44-treated mice exhibit normal homing patterns upon adoptive transfer, and are capable of reexpressing CD44 upon activation. The treatment of haptensensitized mice with anti-CD44 mAb inhibits their ability to mount a cutaneous delayed-type hypersensitivity (DTH) response within the first 24 h after hapten challenge. This inhibition reflects a block in both the edema and leukocyte infiltration of the cutaneous site of DTH, whereas the extravasation and accumulation of leukocytes in the draining lymph nodes progress normally. After 72 h, the leukocytes that extravasate into the site of antigen challenge express CD44. These results indicate that CD44 is not necessary for normal leukocyte circulation but is required for leukocyte extravasation into an inflammatory site involving nonlymphoid tissue.
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Romeijn, P., R. Lenthall, D. Stavrou, D. Melcher, H. Ladyman und MA Ritter. „Identification of glioma-associated antigen MUC 2-63 as CD44“. British Journal of Cancer 70, Nr. 5 (November 1994): 799–803. http://dx.doi.org/10.1038/bjc.1994.402.
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Utama, Bobby Indra. „ISOLATION OF AMNIOTIC FLUID MESENCHYMAL STEM CELLS (AF-MSCs) OBTAINED FROM CAESAREAN SECTIONS“. JOURNAL OBGIN EMAS 2, Nr. 1 (28.11.2019): 1–9. http://dx.doi.org/10.25077/aoj.2.1.1-9.2018.
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Amniotic fluid is a liquid that fills the amniotic cavity which has defense and nutritional functions in fetal development. Human aterm amniotic fluid can be an ideal alternative as a source of mesenchymal stem cells, originating from the neonate. Preclinical studies of second and third trimester amnion fluid cells confirmed the number of potential donors from this wasted material. In several studies, AF-MSCs express mesenchymal markers such as CD90, CD73 (SH3, SH), CD105 (SH2), CD29, CD166, CD49e, CD58 and CD44 (MHC class I). These cells also express HLA-ABC antigens, CD 34, CD 45 which are hematopoietic markers, and endothelial CD31 markers. There is no expression of CD10, CD11b, CD14, CD34, CD117, EMA and HLA-DR, DP, DQ antigens. Most of AF-MSCs have pluripotent properties which are characterized by the discovery of octamer binding protein 3/4 (Oct-3/4), transcription factors Nanog (Nanog), and stage-specific embryonic antigen 4 (SSEA-4) on RT-PCR examination. From this study, 8 million cells was isolated. These cells will be used for research on pelvic organ prolapse therapy by using AF-MSCs. AF-MSCs isolation totally takes 6 weeks. From 1 flask, 2 million of stem cells was obtained. Keywords: amniotic fluid, AF-MSCs
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Maltzman, J. S., J. A. Carman und J. G. Monroe. „Role of EGR1 in regulation of stimulus-dependent CD44 transcription in B lymphocytes.“ Molecular and Cellular Biology 16, Nr. 5 (Mai 1996): 2283–94. http://dx.doi.org/10.1128/mcb.16.5.2283.
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The immediate-early gene egr-1 encodes a transcription factor (EGR1) that links B-cell antigen receptor (BCR) signals to downstream activation events through the regulation of previously unidentified target genes. Here we identify the gene encoding the lymphocyte homing and migration protein CD44 as a target of EGR1 regulation in B cells. BCR-induced increases in CD44 mRNA expression and transcription levels are shown to occur in EGR1-expressing but not in nonexpressing subclones of the B-cell line WEHI-231. Kinetics of egr-1 transcription and the appearance of nuclear EGR1 protein precede CD44 induction and occur within 30 min after stimulation in the EGR1-expressing subclone. A single EGR1 binding motif is demonstrated at bp -301 of the human CD44 promoter. Cotransfection of a CD44 promoter-chloramphenicol acetyltransferase reporter construct with an egr-1 expression vector resulted in a 6.5- to 8.5-fold induction of transcriptional activity relative to an empty expression vector. The EGR1 binding motif was shown to be necessary for stimulus-induced expression of a CD44 promoter-chloramphenicol acetyltransferase reporter construct in nontransformed B lymphocytes and was required for transactivation by an EGR1 expression vector in a B-cell line. These studies identify EGR1 as an intermediary linking BCR-derived signals to the induction of CD44. The relevance of these molecular events to BCR signal transduction and antigen-stimulated B-cell-mediated immune responses is discussed.
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Huang, Tzuu-Yuan, Jun-ichi Kuratsu, Hideo Takeshima, Toru Nishi und Yukitaka Ushio. „Expression of CD44 adhesion molecules in intracranial germinomas“. Neurosurgical Focus 5, Nr. 1 (Juli 1998): E5. http://dx.doi.org/10.3171/foc.1998.5.1.6.
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Adhesion molecules play a role in tumor growth, invasiveness, and the metastatic process. The expression of CD44 adhesion molecules in 11 intracranial germinoma specimens was investigated using anti-CD44 monoclonal antibody and immunohistochemical methods. In six of 11 specimens studied, CD44 antibodies were bound to the membrane of tumor cells; in five of six specimens, CD44 antigen was also present in the cytoplasm of tumor cells. The only three patients who showed CD44-positive expression in tumor cells, lymphocytes, and extracellular matrix (ECM) exhibited either cerebrospinal fluid dissemination or multiple tumors at different locations. In all 11 specimens, no expression of CD44 in normal glial cells or capillary endothelium was detected. According to the authors' findings, the expression of CD44 in intracranial germinomas is similar to that of gonadal seminomas. Analysis of the results further suggests the possibility that the expression of CD44 in intracranial germinoma tumor cells, lymphocytes, and ECM may contribute to tumor cell migration, adhesion to cerebrospinal fluid dissemination, and/or multiple tumor locations.
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McKinney-Freeman, Shannon L., Olaia Naveiras, Frank Yates, Sabine Loewer, Marsha Philitas, Matthew Curran, Peter J. Park und George Q. Daley. „Surface antigen phenotypes of hematopoietic stem cells from embryos and murine embryonic stem cells“. Blood 114, Nr. 2 (09.07.2009): 268–78. http://dx.doi.org/10.1182/blood-2008-12-193888.
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Abstract Surface antigens on hematopoietic stem cells (HSCs) enable prospective isolation and characterization. Here, we compare the cell-surface phenotype of hematopoietic repopulating cells from murine yolk sac, aorta-gonad-mesonephros, placenta, fetal liver, and bone marrow with that of HSCs derived from the in vitro differentiation of murine embryonic stem cells (ESC-HSCs). Whereas c-Kit marks all HSC populations, CD41, CD45, CD34, and CD150 were developmentally regulated: the earliest embryonic HSCs express CD41 and CD34 and lack CD45 and CD150, whereas more mature HSCs lack CD41 and CD34 and express CD45 and CD150. ESC-HSCs express CD41 and CD150, lack CD34, and are heterogeneous for CD45. Finally, although CD48 was absent from all in vivo HSCs examined, ESC-HSCs were heterogeneous for the expression of this molecule. This unique phenotype signifies a developmentally immature population of cells with features of both primitive and mature HSC. The prospective fractionation of ESC-HSCs will facilitate studies of HSC maturation essential for normal functional engraftment in irradiated adults.
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Kube, D., M. Vockerodt, O. Weber, K. Hell, J. Wolf, B. Haier, F. A. Grässer et al. „Expression of Epstein-Barr Virus Nuclear Antigen 1 Is Associated with Enhanced Expression of CD25 in the Hodgkin Cell Line L428“. Journal of Virology 73, Nr. 2 (01.02.1999): 1630–36. http://dx.doi.org/10.1128/jvi.73.2.1630-1636.1999.
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ABSTRACT Epstein-Barr virus is associated with several human malignancies including Burkitt’s lymphoma, nasopharyngeal carcinoma, and Hodgkin’s disease (HD). To examine the effect of Epstein-Barr virus nuclear antigen 1 (EBNA-1) in the pathogenesis of HD, we transfected the gene into the HD cell line L428. EBNA-1 expression was associated with significantly enhanced CD25 expression (interleukin 2 [IL-2]-receptor α chain) in transient and stably transfected L428 cells but did not affect the expression of IL-2 receptor β and γ chains. There was no up-regulation of the B-cell activation molecules CD23, CD30, CD39, CD40, CD44, CD71, and CD54 (intercellular adhesion molecule 1) or enhanced production of IL-6, IL-10, lymphotoxin alpha, and the soluble form of CD25. Stable EBNA-1-expressing L428 cells were nontumorigenic in SCID mice but showed enhanced lymphoma development in nonobese diabetic-SCID mice compared to mock-transfected cells.
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Koopman, G., K. H. Heider, E. Horst, G. R. Adolf, F. van den Berg, H. Ponta, P. Herrlich und S. T. Pals. „Activated human lymphocytes and aggressive non-Hodgkin's lymphomas express a homologue of the rat metastasis-associated variant of CD44.“ Journal of Experimental Medicine 177, Nr. 4 (01.04.1993): 897–904. http://dx.doi.org/10.1084/jem.177.4.897.
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A recently described splice variant of CD44 expressed in metastasizing cell lines of rat tumors, has been shown to confer metastatic potential to nonmetastasizing rat pancreatic carcinoma and sarcoma cell lines. Using antibodies raised against a bacterial fusion protein encoded by variant CD44 sequences, we have explored the expression of variant CD44 glycoproteins on human lymphoid cells and tissues and on non-Hodgkin's lymphomas. Normal lymphohematopoietic cells express barely detectable low levels of variant CD44 glycoproteins, whereas T lymphocytes, upon activation by mitogen or antigen, transiently upregulate expression of specific CD44 variant glycoproteins. The reaction pattern of various antibodies indicates that these CD44 variants contain the domain encoded by exon v6, which is part of the variant that in the rat confers metastatic capability. It is interesting that overexpression of v6 was also found in several aggressive, but not low-grade, non-Hodgkin's lymphomas.
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Stanomir, Alina, Carmen Mihaela Mihu, Simona Rednic, Cristina Pamfil, Alexandra Roman, Andrada Soancă, Iulia Cristina Micu et al. „Oral Mesenchymal Stromal Cells in Systemic Sclerosis: Characterization and Response to a Hyaluronic-Acid-Based Biomaterial“. Applied Sciences 11, Nr. 17 (31.08.2021): 8101. http://dx.doi.org/10.3390/app11178101.
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Introduction. As oral mesenchymal stromal cells (MSCs) have not, to date, been isolated from systemic sclerosis (SSc) patients, the aim of this in vitro experiment was to characterize gingival MSCs (SScgMSCs) and granulation tissue MSCs (SScgtMSCs) from SSc and to evaluate their functionality in comparison to healthy MSCs (hMSCs), in normal or hyaluronic acid (HA) culture media. Materials and Methods. Isolated cells were described by immunophenotyping of surface antigen make-up and by trilineage mesenchymal differentiation capacity. Colony-Forming Unit-Fibroblast (CFU-F) test and migration potential evaluated MSC functionality. Results. All types of MSCs displayed positivity for the following surface markers: CD29, CD73, CD90, CD105, CD44, and CD79a. These cells did not express CD34, CD45, HL-DR, and CD14. Isolated MSCs differentiated into osteoblasts, adipocytes, and chondroblasts. The frequency of CFU-F for SScgtMSCs was significantly lower than that of hMSCs (p = 0.05) and SScgMSCs (p = 0.004) in normal medium, and also markedly lower than that of SScgMSCs (p = 0.09) in HA medium. Following HA exposure, both SScgMSCs and SScgtMSCs migrated significantly less (p = 0.033 and p = 0.005, respectively) than hMSCs. Conclusions. A reduced functionality of MSCs derived from SSc as compared to hMSCs was observed. HA in culture medium appeared to significantly stimulate the migration potential of hMSCs.
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Sun, Wanling, Yongji Wu, Hui Li, Xuan Wang, Nong Zou und Junling Zhuang. „Establishment and Characterization of a New Human Myeloma Cell Line WuS1.“ Blood 106, Nr. 11 (16.11.2005): 5114. http://dx.doi.org/10.1182/blood.v106.11.5114.5114.
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Abstract A new human myeloma cell line WuS1 was established from the bone marrow of a 45-year-old Chinese male patient with IgGλ type multiple myeloma (stage IIIB). The growth of WuS1 cells is constitutively independent of exogenous growth factors of feeder cells. The WuS1 cell line proliferated consistently as free-floating single cells in suspension, sometimes in small clusters or slightly adherent on the bottom of the plastic culture flask, without forming clumps. The cell line has been maintained without any external growth factors for over a year, and cells frozen in liquid nitrogen can be revived successfully. The doubling time of the cells was about 11 hours and the colony-forming rate was 55.56±6.33%. WuS1 displayed immature plasma cell features with an obvious heterogeneity in size and a high nuclear-cytoplasmic ratio in Wright-Giemsa staining. Figure Figure They were positive for ALP, CE, ACP(not inhibited by tartrate) and PAS stainings and negative for POX and NBE. By transmission electron microscopy, the cytoplasm of WuS1 contained abundant mitochondria, and parallel endoplasmic reticulum or Golgi apparatus in some cells. The monoclonal immunoglobulin G and λ light chain were positive in cell lysate and not in cell culture supernatants by immnuoelectrophoresis. The cell surface antigens were positive for CD3, CD59, CD106 and CD138, and negative for CD4, CD5, CD8, CD10, CD13, CD14, CD19, CD20, CD22, CD29, CD31, CD33, CD34, CD38, CD44, CD49d, CD45, CD54, CD56 and HLA-DR by flow cytometry. Chromosomal analysis revealed a hypodiploidy and complex karyotype. WuS1 cells were negative for Epstein-Barr virus by PCR using EBV nuclear antigen-1 specific primers. Twelve SCID mice were injected with WuS1 cells intravenously or subcutaneously, and obvious tumor infiltration in bone marrow, liver, spleen, lung, kidney and injection site (subcutaneously group) were observed by pathologic examination. Figure Figure The novel WuS1 cell line will be useful in the study of the biology, etiology and treatment of multiple myeloma.
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Morimoto, K., E. Robin, MC Le Bousse-Kerdiles, Y. Li, D. Clay, C. Jasmin und F. Smadja- Joffe. „CD44 mediates hyaluronan binding by human myeloid KG1A and KG1 cells“. Blood 83, Nr. 3 (01.02.1994): 657–62. http://dx.doi.org/10.1182/blood.v83.3.657.657.
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Abstract Hyaluronan-binding function of the CD44 molecule has not been so far detected in myeloid cells. To study pure populations of primitive myeloid cells, we investigated the hyaluronan-binding function of the CD44 molecule from three myeloid cell lines: KG1a, KG1, and HL60. Both KG1a and KG1 cells express the CD34 antigen characteristic of the hematopoietic stem cells and HL60 cells do not; accordingly, KG1a and KG1 cells are generally considered as the most primitive and HL60 cells as the most mature of these cell lines. Measurement of cell adhesion to hyaluronan-coated surfaces (using 51Cr-labeled cells) and of aggregate formation in hyaluronan-containing solutions, showed that 45% of KG1 cells and 22% to 24% of KG1a spontaneously bind to hyaluronan, whereas HL60 cells do not either spontaneously or after treatment with a phorbol ester. Hyaluronan binding by KG1a and KG1 cells is mediated by CD44, because it is specifically abolished by monoclonal antibodies (MoAbs) to this molecule. The binding might require phosphorylation by protein kinase C and perhaps also by protein kinase A, because it is prevented by staurosporine, which inhibits these enzymes. 12-O- tetradecanoylphorbol-13-acetate (TPA) which activates protein kinase C, rises to 80% the proportion of KG1 and KG1a cells that bind hyaluronan; this activation is dependent on protein synthesis, for it is abrogated by cyclophosphamide, a protein synthesis inhibitor. Binding of TPA- treated cells to hyaluronan is only partly inhibited by MoAb to CD44: this suggests that TPA may induce synthesis of a hyaluronan-binding protein distinct from CD44. Considering the abundance of hyaluronan in human bone marrow, these results suggest that CD44 may be involved in mediating precursor-stroma interaction.
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Morimoto, K., E. Robin, MC Le Bousse-Kerdiles, Y. Li, D. Clay, C. Jasmin und F. Smadja- Joffe. „CD44 mediates hyaluronan binding by human myeloid KG1A and KG1 cells“. Blood 83, Nr. 3 (01.02.1994): 657–62. http://dx.doi.org/10.1182/blood.v83.3.657.bloodjournal833657.
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Hyaluronan-binding function of the CD44 molecule has not been so far detected in myeloid cells. To study pure populations of primitive myeloid cells, we investigated the hyaluronan-binding function of the CD44 molecule from three myeloid cell lines: KG1a, KG1, and HL60. Both KG1a and KG1 cells express the CD34 antigen characteristic of the hematopoietic stem cells and HL60 cells do not; accordingly, KG1a and KG1 cells are generally considered as the most primitive and HL60 cells as the most mature of these cell lines. Measurement of cell adhesion to hyaluronan-coated surfaces (using 51Cr-labeled cells) and of aggregate formation in hyaluronan-containing solutions, showed that 45% of KG1 cells and 22% to 24% of KG1a spontaneously bind to hyaluronan, whereas HL60 cells do not either spontaneously or after treatment with a phorbol ester. Hyaluronan binding by KG1a and KG1 cells is mediated by CD44, because it is specifically abolished by monoclonal antibodies (MoAbs) to this molecule. The binding might require phosphorylation by protein kinase C and perhaps also by protein kinase A, because it is prevented by staurosporine, which inhibits these enzymes. 12-O- tetradecanoylphorbol-13-acetate (TPA) which activates protein kinase C, rises to 80% the proportion of KG1 and KG1a cells that bind hyaluronan; this activation is dependent on protein synthesis, for it is abrogated by cyclophosphamide, a protein synthesis inhibitor. Binding of TPA- treated cells to hyaluronan is only partly inhibited by MoAb to CD44: this suggests that TPA may induce synthesis of a hyaluronan-binding protein distinct from CD44. Considering the abundance of hyaluronan in human bone marrow, these results suggest that CD44 may be involved in mediating precursor-stroma interaction.
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Dallas, Matthew R., Guosheng Liu, Wei‐Chiang Chen, Susan N. Thomas, Denis Wirtz, David L. Huso und Konstantinos Konstantopoulos. „Divergent roles of CD44 and carcinoembryonic antigen in colon cancer metastasis“. FASEB Journal 26, Nr. 6 (13.03.2012): 2648–56. http://dx.doi.org/10.1096/fj.12-203786.
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Migliaccio, Anna Rita F., Valentina Tirelli, Francesca Masiello, Massimo Sanchez, Carolyn Whitsett und Giovanni Migliaccio. „High Levels of CD44 Expression Identify Hematopoietic Cells Capable of Generating Great Numbers of Erythroid Cells Under HEMA Conditions“. Blood 116, Nr. 21 (19.11.2010): 3349. http://dx.doi.org/10.1182/blood.v116.21.3349.3349.
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Abstract Abstract 3349 Both mononuclear (MNC) and CD34pos cells from adult blood (AB) generate great numbers of erythroid cells (EBs) in cultures when stimulated with dexamethasone, estradiol, SCF, IL-3 and EPO (Human Erythroid Massive Amplification, HEMA). The total number of erythroid cells generated by CD34pos cells is, however, on average 1-log lower than that generated by MNC. Loss of erythroid progenitor cells during the CD34 selection procedure and/or existence of circulating CD34neg erythroid progenitors have been considered as possible explanations for this observation. To clarify the phenotype of the circulating hematopoietic cells that generate EBs under HEMA conditions, we compared progenitor cell activity (CFC, i.e. generation of BFU-E and CFU-GM derived colonies in semisolid media,) and erythroid precursor activity (EPC) (i.e. fold increase, FI, of EBs at day 15 in HEMA) of cell populations prospectively isolated from AB MNC by cell sorting according to expression of CD34, CD44 (the α integrin subunit which represents an early erythroid marker in mouse, Chen et al, PNAS 2009, 106:17413-17418), CD36 (the thrombospondin receptor whose expression is activated when hematopoietic progenitors are exposed to EPO) and CD42a (the α subunit of von Willebrand factor, a megakaryocyte marker as negative control). The results are summarized in Table I. Table I. Populations present in AB MNC with the potential to generate erythroid cells under HEMA conditions CD44 high high high neg CD34 pos neg neg neg CD36 neg neg low high CD235a neg neg neg neg CD42a neg neg neg pos Frequency 0.1% 78% 0.84% 0.56% CFC 160/500 plated cells (BFU-E:CFU-GM = 1:1) 17/10,000 cells (all BFU-E) – – EPC FI = 12,000 Day 15 FI = 15 Day 15 FI = 1,250 – Other Megakaryocyte precursors (?) At least three cell populations were recognized to exert EPC activity: CD34pos/CD44high cells, which grew in HEMA culture (FI = 12,000) and contained CFC (160 CFC/500 plated cells); CD34negCD44pos cells (78%) which generated EBs in HEMA (FI = 15) and contained CFC (16.5±0.5 BFU-E/10,000 plated cells) and CD34negCD44posCD36low cells which generated EBs in HEMA (FI = 1250) but did not contain CFC. An additional population of CD36high cells which did not express CD14 and were therefore not monocytes, did not express CD44 but expressed CD42a. These cells did not generate EBs in HEMA or CFC in semisolid cultures and were likely megakaryocyte precursors. Therefore, the three populations capable to generate erythroid cells in HEMA expressed CD44 at high levels. These results suggest a pattern of antigenic activation during erythroid commitment in which CD44 is already expressed by CD34pos cells. Its expression is retained at early stages of erythroid commitment until low levels of CD36 expression appear and is lost when cells upregulate CD36 expression to acquire MK markers (CD42a). To test this model, we analysed the changes in antigen expression profiles of cells generated by MNC during 13 days of HEMA culture. The results are summarized in Table II. Table II. Dynamic changes in antigen expression during the culture of MNC in HEMA (in percent of total cells) CD44 high high high high medium low CD34 pos neg neg neg neg CD36 neg low low high high low CD42a neg neg neg neg neg neg CD235a neg neg neg neg pos pos Day 0 0.1 – 0.84 – – – Day 1 0.1 ± 0.4 – – – Day 2 0.2 0.1 0.6 – – – Day 3 0.7 0.3 0.9 – – – Day 6 – – 1.1 – 0.2 – Day 8 – – – 19 39 – Day 10 – – – 19 63 – Day 13 – – – 13 77 3.6 Results are expressed as percent of the total population present in culture. CD44posCD34negCD36neg cells were not analysed in these experiments. Absorbance fluorescence intensity for CD44: High= >8.000; medium=1000–2000; low=500–800 CD34pos cells increased from 0.1 to 0.7% at day 3 of culture and became undetectable by day 6. CD34negCD36low cells increased in frequency until day 6 when they represented 1.1% of the total cell population. A CD34posCD36low population, not detectable in MNC, appeared in culture at day 1 and represented 0.3% of total cells at day 3. These cells may represent the progeny of CD34pos cells that have acquired CD36 expression. All these cells retained high levels of CD44 expression. Another population not detectable among MNC was represented by CD34negCD44highCD36high CD42aneg cells. These cells appeared at day 6 and became 19% of the total cells by day 8. Prospective isolation coupled with functional studies identified that these cells have EPC activity as demonstrated by their ability to generate additional EBs in HEMA. Cells expressing the erythroid marker CD235a (glycophorin A) began to be detectable by day 6 (0.2%) and reached 77% of the total population by day 13. These cells expressed medium levels of CD44 and when prospectively isolated were unable to generate additional EBs in HEMA. By day 13, few cells (3.6%) with the phenotype of mature EBs (CD36lowCD235ahigh) were detectable. These cells expressed low levels of CD44. Overall, these data indicate that high levels of CD44 expression mark in vivo and ex vivo generated cells with EPC activity. Disclosures: No relevant conflicts of interest to declare.
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Leone, Julieta, Juan E. Perez, Maria E. Dominguez, Julian Iturbe, José P. Leone, Maria C. Mac Donnell, Gabriel Grosman, Carlos T. Vallejo, Bernardo A. Leone und Ariel O. Zwenger. „Role of Difucosylated Lewis Y Antigen in Outcome of Locally Advanced Cervical Squamous Cell Carcinoma Treated with Cisplatin Regimen“. International Journal of Biological Markers 31, Nr. 3 (Juli 2016): 300–308. http://dx.doi.org/10.5301/jbm.5000206.
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Background Several mechanisms are involved in the development of resistance to therapy in locally advanced cervical squamous cell carcinoma (LACSCC). Studies have shown that CD44 and Lewis Y antigen (LeY) form a complex that is associated with chemoresistance, tumor invasion and metastasis. We assessed the role of CD44 and LeY in the outcome of LACSCC patients treated with different chemotherapy regimens. Methods 126 LACSCC patients at FIGO stages IIB-IVA were selected from the GOCS database: 74 patients included in 3 different prospective phase II trials in the neoadjuvant setting (vinorelbine, docetaxel, ifosfamide-vinorelbine-cisplatin) and 52 patients treated with standard radiochemotherapy based on cisplatin (RCBC). Clinical data at baseline, disease-free survival (DFS) and overall survival (OS) were recorded. Univariate and multivariate Cox models were employed. Results Median age was 45.6 years (range: 24.9-80.5). Sixty-three and 47 tumors were CD44+ and LeY+, respectively. Tumors with expansive growth showed higher grade (p = 0.0024), mitotic index (p = 0.0505), tumor necrosis (p = 0.0191), LeY+ (p = 0.0034) and CD44+/LeY+ coexpression (p = 0.0334). CD44+ cells were present in 91.3% of patients with local recurrence (p = 0.0317). Advanced stage was associated with LeY+ tumors. Patients treated with RCBC had worse DFS and OS when their tumors expressed LeY (p = 0.0083 and p = 0.0137, respectively). Pre-treatment hemoglobin level, FIGO stage and tumor response remained the most significant prognostic factors in Cox regression. Conclusions In our cohort of LACSCC patients, the coexpression of CD44 and LeY was not associated with worse outcome. However, in the subgroup of patients receiving RCBC, LeY expression was correlated with shorter DFS and OS.
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Jalkanen, S., und M. Jalkanen. „Lymphocyte CD44 binds the COOH-terminal heparin-binding domain of fibronectin.“ Journal of Cell Biology 116, Nr. 3 (01.02.1992): 817–25. http://dx.doi.org/10.1083/jcb.116.3.817.
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The lymphocyte-high endothelial venule (HEV) cell interaction is an essential element of the immune system, as it controls lymphocyte recirculation between blood and lymphoid organs in the body. This interaction involves an 85-95-kD class of lymphocyte surface glycoprotein(s), CD44. A subset of lymphocyte CD44 molecules is modified by covalent linkage to chondroitin sulfate (Jalkanen, S., M. Jalkanen, R. Bargatze, M. Tammi, and E. C. Butcher. 1988. J. Immunol. 141:1615-1623). In this work, we show that removal of chondroitin sulfate by chondroitinase treatment of lymphocytes or incubation of HEV with chondroitin sulfate does not significantly inhibit lymphocyte binding to HEV, suggesting that chondroitin sulfate is not involved in endothelial cell recognition of lymphocytes. Affinity-purified CD44 antigen was, on the other hand, observed to bind native Type I collagen fibrils, laminin, and fibronectin, but not gelatin. Binding to fibronectin was studied more closely, and it was found to be mediated through the chondroitin sulfate-containing form of the molecule. The binding site on fibronectin was the COOH-terminal heparin binding domain, because (a) the COOH-terminal heparin-binding fragment of fibronectin-bound isolated CD44 antigen; (b) chondroitin sulfate inhibited this binding; and (c) finally, the ectodomain of another cell surface proteoglycan, syndecan, which is known to bind the COOH-terminal heparin binding domain of fibronectin (Saunders, S., and M. Bernfield. 1988. J. Cell Biol. 106: 423-430), inhibited binding of CD44 both to intact fibronectin and to its heparin binding domain. Moreover, inhibition studies showed that binding of a lymphoblastoid cell line, KCA, to heparin binding peptides from COOH-terminal heparin binding fragment of fibronectin was mediated via CD44. These findings suggest that recirculating lymphocytes use the CD44 class of molecules not only for binding to HEV at the site of lymphocyte entry to lymphoid organs as reported earlier but also within the lymphatic tissue where CD44, especially the subset modified by chondroitin sulfate, is used for interaction with extracellular matrix molecules such as fibronectin.