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Cell Fraction (cell + fraction)
Selected AbstractsSpecific Determination of Endothelial Cell Viability in the Whole Cell Fraction from Cryopreserved Canine Femoral Veins Using Flow CytometryARTIFICIAL ORGANS, Issue 10 2000Jong-Chul Park Abstract: An efficient method for specifically determining the viability of endothelial cells (EC) from cells dissociated from the human saphenous vein was investigated. Three different methods, trypan blue staining assay, [3H]-proline incorporation assay, and flow cytometry (FCM), combined with the fluorescein isothiocyanate conjugated with Griffonia simplicifolia agglutins (GS1-FITC)/propidium iodide (PI) double staining, were used. Both trypan blue staining and [3H] proline incorporation assays demonstrated less sensitivity to determine viability of EC differentially from the other cells. FITC-GS1 showed prominent binding to the vascular EC and could be counted by FCM including PI on dead cells. Following the cryopreservation process, the GS1-FITC/PI FCM analytical method was adopted to test simultaneously the viability of whole cells and EC from the same tissue, human saphenous veins, and mongrel dogs' femoral veins after harvesting, antibiotic solution treatment, and thawing. The viability of the whole cells from veins decreased with a significant difference (p < 0.05) from that of EC after thawing. [source] Human laminin-332 degradation by Candida proteinasesJOURNAL OF ORAL PATHOLOGY & MEDICINE, Issue 6 2008P. Pärnänen Background:, Human laminin-332 (Lm-332) degradation by 12 Candida strains and effects of synthetic proteinase inhibitors [Ilomastat (ILM), EDTA, chemically modified tetracycline-3(CMT-3), CMT-308, synthetic peptide CTT-2, and Pefabloc] were studied. Materials and methods:, Laminin-332 was incubated with sonicated cell fractions and 10 times concentrated cell-free fractions of reference and clinical strains of C. albicans, C. dubliniensis, C. guilliermondii, C. glabrata, C. krusei, and C. tropicalis. Proteolysis, pH effects, and inhibitors were analyzed by fluorography and zymography. Results:, Cell fractions of all species except C. guilliermondii and cell-free fractions of C. albicans, and C. dubliniensis showed 20,70 kDa gelatinases at pH 5.0 and 6.0. At pH 7.6, C. glabrata, C. krusei, and C. tropicalis cell fractions and C. tropicalis cell-free fractions showed 55,70 kDa gelatinases. CMT-3, CMT-308, and CTT-2 inhibited Candida gelatinases slightly better than Pefabloc, ILM, and EDTA. No Candida fractions degraded Lm-332 at pH 7.6, but at pH 5.0, 100 kDa bands were generated by cell fractions of C. dubliniensis and C. tropicalis; C. albicans and C. glabrata clinical strains; and C. guilliermondii reference strain. C. krusei reference strain yielded three 100,130 kDa bands. C. albicans, C. dubliniensis, and C. tropicalis reference and clinical strain's cell-free fractions generated 100 kDa band. Conclusions:, Laminin-332 degradation is pH-dependent and differences exist between studied Candida strains. Lm-332 degradation can exert functional disturbances on basement membrane integrity, possibly aiding Candida cell invasion into tissues. Certain synthetic matrix metalloproteinase inhibitors (CMTs, CTT) can inhibit Candida proteinases and may be therapeutically useful in future. [source] Analysis of proliferating cell fraction determined by monoclonal antibody to M1-subunit ribonucleotide reductase and Ki-67 in relation to p53 protein expression in fine-needle aspirates from non-Hodgkin's lymphomasCYTOPATHOLOGY, Issue 5 2000V. Sviatoha Analysis of proliferating cell fraction determined by monoclonal antibody to M1-subunit ribonucleotide reductase and Ki-67 in relation to p53 protein expression in fine-needle aspirates from non Hodgkin's lymphomas The purpose of this study was to analyse the proliferative fraction with the monoclonal antibody M1-R-R to M1-subunit ribonucleotide reductase and with MIB-1 to Ki-67 antigen in relation to p53 protein expression in fine needle aspirates from B-cell non-Hodgkin's lymphomas. One hundred and thirty-seven cases, previously diagnosed and sub-typed according to the Kiel classification and characterized by immunophenotyping, were included in the study. The M-1 subunit ribonucleotide reductase (M1 -R-R), Ki-67 and p53 antigens were detected using monoclonal antibodies on stored cytospin preparations. There was a good correlation (r = 0.72) between Ki-67 and M1 -R-R positive cell fraction in both high and low grade lymphomas. High-grade lymphomas had a median percentage of M1 -R-R/MIB-1 positive cells of 53.0/73.0 for lymphoblastic, 61.0/52.0 for immunoblastic and 33.5/41.0 for centroblastic lymphomas, respectively. In low grade lymphomas figures of median percentage of M1 -R-R/MIB-1 were 9.0/15.0 for centroblastic/centrocytic, 11.0/9.5 for chronic lymphocytic leukaemia, 16.0/27.0 for centrocytic and 12.0/9.0 for immunocytomas, respectively. The median percentages of M1 -R-R/MIB-1 for high and low grade lymphomas were 37.0/50.5 and 11.0/12.0, respectively. In the p53 positive cases the proliferation rate as measured by staining for M1 -R-R and MIB-1 was higher than in p53 negative cases, but the difference was not statistically significant. The results show that cytospin material obtained by fine needle aspiration and stored at ,70 °C for years can be used reliably for both peroxidase-avidin-biotin and three-step alkaline phosphatase immunocytochemical staining. In addition, proliferation fraction determined by M1 -R-R monoclonal antibody staining correlates well with that measured by an established marker for cell proliferation, the Ki-67 antibody. However, the proliferation fraction as measured by the two antibodies differs in the various subtypes of non-Hodgkin's lymphoma which indicates that they may contribute different prognostic information. [source] Hepatotoxicity assay using human hepatocytes trapped in microholes of a microfluidic deviceELECTROPHORESIS, Issue 18 2010Ju Hun Yeon Abstract Hepatocytes have been used for in vitro hepatotoxicity assays because of their ability to sustain intact liver-specific functions. Here, we demonstrate a hepatotoxicity assay system using primary human hepatocytes trapped in microholes of a microfluidic device, providing a microscale in vivo liver-like environment. We performed microfluidic hepatotoxicity assays of several drugs, including acetaminophen, verapamil, diclofenac, and benzopyrene, all of which are known to specifically affect hepatic function. The drug sensitivities in hepatocytes and HepG2 cells were measured by calculating the live cell fraction at various drug concentrations. The results indicated that hepatocytes were more sensitive to these drugs than HepG2 cells. The lethal concentration 50 values for all drugs tested were similar to those from the in vitro toxicity data with human hepatocytes obtained from the literature. Furthermore, we developed a mathematical hepatotoxicity model based on the time-dependent cell death profiles measured by our device. This novel assay system enabled us to analyze in vivo -like hepatotoxicity in a microfluidic device by exploiting microstructures to mimic the microenvironment of the liver. [source] Functional characterization of highly adherent CD34+ keratinocytes isolated from human skinEXPERIMENTAL DERMATOLOGY, Issue 7 2010Araika Gutiérrez-Rivera Please cite this paper as: Functional characterization of highly adherent CD34+ keratinocytes isolated from human skin. Experimental Dermatology 2010; 19: 685,688. Abstract:, Compared to murine models, data on cells responsible for the homeostasis of human epidermis are scarce and often contradictory. Given the conflicting results and the availability of clinical grade protocols to purify CD34 cells from a given tissue, we pursued to phenotypically characterize human epidermal CD34+ population. After magnetic separation of whole skin CD34+ and CD34, cell fractions and selection for cells highly adherent to extracellular matrix, both CD34± fractions retained the ability to form a stratified epidermis in organotypic cultures and presented similar in vitro migratory phenotypes. However CD34, cells showed higher clonogenic potential and in vitro proliferative capacity. These results indicated that CD34, cell fraction contains stem/early progenitor cells, while CD34+ cells might be a transit-amplifying precursor for hair follicle (HF) sheath cells. The ability to isolate living cells using differential cell adhesion and surface markers provides an opportunity to study cells from different morphological regions of the HF. [source] Transplantation of an acutely isolated bone marrow fraction repairs demyelinated adult rat spinal cord axonsGLIA, Issue 1 2001Masanori Sasaki Abstract The potential of bone marrow cells to differentiate into myelin-forming cells and to repair the demyelinated rat spinal cord in vivo was studied using cell transplantation techniques. The dorsal funiculus of the spinal cord was demyelinated by x-irradiation treatment, followed by microinjection of ethidium bromide. Suspensions of a bone marrow cell fraction acutely isolated from femoral bones in LacZ transgenic mice were prepared by centrifugation on a density gradient (Ficoll-Paque) to remove erythrocytes, platelets, and debris. The isolated cell fraction contained hematopoietic and nonhematopoietic stem and precursor cells and lymphocytes. The cells were transplanted into the demyelinated dorsal column lesions of immunosuppressed rats. An intense blue ,-galactosidase reaction was observed in the transplantation zone. The genetically labeled bone marrow cells remyelinated the spinal cord with predominately a peripheral pattern of myelination reminiscent of Schwann cell myelination. Transplantation of CD34+ hematopoietic stem cells survived in the lesion, but did not form myelin. These results indicate that bone marrow cells can differentiate in vivo into myelin-forming cells and repair demyelinated CNS. GLIA 35:26,34, 2001. © 2001 Wiley-Liss, Inc. [source] Establishment, characterization and drug sensitivity testing in primary cultures of human thymoma and thymic carcinomaINTERNATIONAL JOURNAL OF CANCER, Issue 12 2008Volker Ehemann Abstract Thymomas and thymic carcinomas are peculiar epithelial tumors of the anterior mediastinum. They may show aggressive clinical behavior and are a paradigm for the interaction between the tumor and the immune system. So far, adequate functional studies enabling a better understanding of this malignancy have not been performed, since human thymoma/thymic carcinoma cell lines have not been available. Here, the authors describe the establishment, characterization and functional analyses of epithelial cell lines from a Type B1-thymoma and a poorly differentiated thymic carcinoma. By Fluorescence-activated cell sorting (FACS) analyses, both cell lines were aneuploid. The aneuploid cell fraction of the thymic carcinoma cell line was characterized by a high proliferation index of 55.9%, in contrast to a lower proliferation rate of the aneuploid cell fraction of the thymoma (19.7%). Array-based comparative genomic hybridization (aCGH) and conventional cytogenetic analysis of the thymoma revealed only minor imbalances whereas the thymic carcinoma was characterized by a complex karyotype in the hyperdiploid range that was readily defined with multicolor FISH (mFISH). Application of a selective COX-2 inhibitor reduced cell viability in both cell lines in a dose-dependent manner. In conclusion, these first cell lines of a thymoma and a CD5-positive thymic carcinoma are useful tools for further in vitro studies of cellular, molecular and genetic aspects of the disease and for functional tests to evaluate new therapeutic targets. © 2008 Wiley-Liss, Inc. [source] In vitro haem solubility of red cell fraction of porcine blood under various treatmentsINTERNATIONAL JOURNAL OF FOOD SCIENCE & TECHNOLOGY, Issue 4 2010Tong-Xun Liu Summary An in vitro peptic digestion at gastric pH 2.0 was used to assess the haem solubility of red cell fraction (RCF) of porcine blood derived samples. The in vitro haem solubility of the RCF decreased greatly in the denaturated form of haemoglobin. However, the native haemoglobin was susceptible to be hydrolysed by enzyme mixture of Flavourzyme and Alcalase. The in vitro digestion of the hydrolysates showed that the highest haem solubility was reached at degree of hydrolysis between 8.75% and 12.33%. The in vitro haem solubility was positively correlated with content of the highly soluble peptides with molecular weights ranged from 7.5 kDa to 1 kDa, but negatively with peptides fractions >7.5 kDa and <1 kDa, mostly due to the precipitation of the highly molecular weight fraction (>7.5 kDa) and part of small peptides (<1 kDa) with higher haem/peptide ratio, which was confirmed by gel filtration chromatograms and by the analysis of the precipitate at pH 2.0. [source] In vitro characterization of a human neural progenitor cell coexpressing SSEA4 and CD133JOURNAL OF NEUROSCIENCE RESEARCH, Issue 2 2007Perrine Barraud Abstract The stage-specific embryonic antigen 4 (SSEA4) is commonly used as a cell surface marker to identify the pluripotent human embryonic stem (ES) cells. Immunohistochemistry on human embryonic central nervous system revealed that SSEA4 is detectable in the early neuroepithelium, and its expression decreases as development proceeds. Flow cytometry analysis of forebrain-derived cells demonstrated that the SSEA4-expressing cells are enriched in the neural stem/progenitor cell fraction (CD133+), but are rarely codetected with the neural stem cell (NSC) marker CD15. Using a sphere-forming assay, we showed that both subfractions CD133+/SSEA4+ and CD133+/CD15+ isolated from the embryonic forebrain are enriched in neurosphere-initiating cells. In addition CD133, SSEA4, and CD15 expression is sustained in the expanded neurosphere cells and also mark subfractions of neurosphere-initiating cells. Therefore, we propose that SSEA4 associated with CD133 can be used for both the positive selection and the enrichment of neural stem/progenitor cells from human embryonic forebrain. © 2006 Wiley-Liss, Inc. [source] Quantitative PSA RT-PCR for preoperative staging of prostate cancerTHE PROSTATE, Issue 4 2003Ralf Kurek Abstract BACKGROUND The clinical value of detecting prostate specific antigen (PSA) mRNA in the peripheral blood mononuclear cell fraction of patients (pts) by standard RT-PCR assays with localized prostate cancer remains controversial. We used a quantitative RT-PCR assay to measure the PSA mRNA copy number in addition to the qualitative PSA RT-PCR and correlated the results with clinical parameters. METHODS Total RNA was extracted from the peripheral blood mononuclear cell fraction of 115 prostate cancer pts prior to radical retropubic prostatectomy (RP) who received 3 months of neoadjuvant androgen deprivation. For quantitative RT-PCR, a PSA-like internal standard (IS) was added to each sample prior to reverse transcription and the PCR products for PSA and IS were selectively detected with fluorescent europium chelates after hybridization. Corresponding qualitative PSA,RT-PCR was performed for all samples. RESULTS The median PSA copy number was 126 (range: 0,37988). There were no significant correlations established between qualitative or quantitative RT-PCR results and given clinical parameters. Corresponding quantitative and qualitative RT-PCR results were significantly associated (P,=,0.01). CONCLUSIONS We were unable to show any additional value of quantitative as well as qualitative PSA RT-PCR for preoperative staging of prostate cancer so far. Nevertheless, the long-term follow up of the patients has to be awaited. Prostate 56: 263,269, 2003. © 2003 Wiley-Liss, Inc. [source] Decrease in intrahepatic CD56+ lymphocytes in gastric and colorectal cancer patients with liver metastasesAPMIS, Issue 12 2009MAYA GULUBOVA The aim of the study was to examine the main intrahepatic lymphocyte subpopulations, namely CD3+ lymphocytes, natural killer (NK)-like T lymphocytes (NKT) expressing the CD3+ CD56+ phenotype, CD56+ NK cells, CD4+, and CD8+ T cells in livers of patients with gastric and colorectal cancer with and without hepatic metastases. The proportion of each lymphocyte subset was determined in 34 patients with gastric or colorectal cancer (18 with and 16 without liver metastasis) by two-color flow cytometry after extraction of hepatic mononuclear cell fraction. The distribution of lymphocyte subpopulations in selected areas of liver metastases and adjacent liver tissue was evaluated using immunohistochemistry for CD4, CD8, and CD56. Flow cytometry analysis revealed a significant decrease in the proportion of CD3+ CD56+ cells in metastatic livers, but not in nonmetastatic livers (11.9 ± 10.3 vs 24.2 ± 13.6%, p = 0.02). The percentage of intrahepatic CD3,CD56+ cells was also decreased in patients with metastases compared to those without (10.1 ± 11.6 vs 16.6 ± 8.9%, p = 0.039). Immunohistochemically, three types of lymphocytes (CD4+, CD8+, and CD56+) were present in the metastatic tissue, although the number of CD56+ cells was almost twice lower. We found a low prevalence of tumor-infiltrating CD4+, CD8+, and CD56+ cells in livers with multiple metastases, whereas in cases with solitary metastasis a higher degree of lymphocyte infiltration was observed. The number of CD3,CD56+ and CD3+ CD56+ cells was decreased in metastatic livers compared to those unaffected by metastases. Therefore the prevalence of tumor-infiltrating lymphocytes seems to be related to the progression of metastatic liver disease. Depletion of hepatic innate lymphocytes may reveal susceptibility to metastatic liver disease and could be a reason for the escape of metastatic cells from the mechanisms of liver immune control. [source] Efficacy of Bone Marrow Mononuclear Cells to Promote Bone Regeneration Compared With Isolated CD34+ Cells From the Same Volume of AspirateARTIFICIAL ORGANS, Issue 7 2010Shinji Yasuhara Abstract Autologous bone marrow mononuclear cell (BMMNC) transplantation is currently an emerging clinical treatment in the orthopedic as well as cardiovascular fields. It is believed that the therapeutic effect of the BMMNCs is due to neovascularization enhanced by the CD34+ cells contained therein, which include endothelial progenitor cells. However, isolation of the CD34+ cell fraction for clinical application has many disadvantages such as cost and invasiveness related to cell mobilization with cytokine. To investigate whether a purification step is in fact necessary for bone regeneration, we separated BMMNCs, CD34+, and CD34 - cells from the same initial volume of rabbit bone marrow aspirates. We then transplanted them back into a femoral bone defect of the same rabbit together with atelocollagen gel and basic fibroblast growth factor (bFGF) and evaluated neovascularization and bone regeneration up to 8 weeks after transplantation. The greatest potential for neovascularization and bone regeneration medicated by cells from the same volume of bone marrow aspirate was found in the BMMNC group. Although purified CD34+ cells might be an ideal cell source, BMMNCs could be a practical and feasible cell source for bone regeneration in present clinical settings with limited cost, availability of materials, and technical issues for transplantation. [source] Supplementation-dependent differences in the rates of embryonic stem cell self-renewal, differentiation, and apoptosisBIOTECHNOLOGY & BIOENGINEERING, Issue 5 2003Sowmya Viswanathan Abstract Although it is known that leukemia inhibitory factor (LIF) supports the derivation and expansion of murine embryonic stem (ES) cells, it is unclear whether this is due to inhibitory effects of LIF on ES cell differentiation or stimulatory effects on ES cell survival and proliferation. Using an ES cell line transgenic for green fluorescent protein (GFP) expression under control of the Oct4 promoter, we were able to simultaneously track the responses of live Oct4-GFP-positive (ES) and -negative (differentiated) fractions to LIF, serum, and other growth factors. Our findings show that, in addition to inhibiting differentiation of undifferentiated cells, the administration of LIF resulted in a distinct dose-dependent survival and proliferation advantage, thus enabling the long-term propagation of undifferentiated cells. Competitive responses from the differentiated cell fraction could only be elicited upon addition of serum, fibroblast growth factor-4 (FGF-4), or insulin-like growth factor-1 (IGF-1). The growth factors did not induce additional differentiation of ES cells, but rather they significantly improved the proliferation of already differentiated cells. Our analyses show that, by adjusting culture conditions, including the type and amount of growth factors or cytokines present, the frequency of media exchange, and the presence or absence of serum, we could selectively and specifically alter the survival, proliferation, and differentiation dynamics of the two subpopulations, and thus effectively control population outputs. Our findings therefore have important applications in engineering stem cell culture systems to predictably generate desired stem cells or their derivatives for various regenerative therapies. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng84: 505,517, 2003. [source] Changes in the Sensitivity of Intratumor Cells during Fractionated Tirapazamine AdministrationCANCER SCIENCE, Issue 7 2000Shin-ichiro Masunaga Mice bearing solid tumors received 10 intraperitoneal administrations of 5-bromo-2,-deoxyuridine (BrdU) to label the proliferating (P) tumor cells. Then, as a priming treatment, tirapazamine (TPZ) was intraperitoneally administered. Further, 0 through 48 h later, the tumor-bearing mice received TPZ again at various doses. The tumor cells were isolated and incubated with a cytokinesis blocker. The micronucleus (MN) frequencies in cells with and without BrdU labeling, which were regarded as P and quiescent (Q) cells at the priming treatment, respectively, were determined using immunofluorescence staining for BrdU. The MN frequency in the total (P+Q) tumor cells was determined from the tumors that were not pretreated with BrdU. In addition, P cell ratios in the tumors at the second treatment were determined using immunofluorescence staining for P cell nuclear antigen. In each cell fraction, the longer the interval between the two treatments, the higher was the sensitivity to TPZ, except 1 h after the priming treatment. More than 24 h later, total and P cells, especially P cells, showed significantly higher sensitivity to TPZ than in the case of a single TPZ treatment. The longer the period between the two TPZ treatments, the lower was the P cell ratio at the second treatment. These findings were thought to indicate that the use of TPZ in the treatment of solid tumors causes a shift from the P to the Q state in vivo. [source] Frequencies and role of regulatory T cells in patients with (pre)malignant cervical neoplasiaCLINICAL & EXPERIMENTAL IMMUNOLOGY, Issue 2 2007J. Visser Summary Oncogenic human papillomavirus (HPV)-infection is crucial for developing cervical cancer and its precursor lesions [cervical intraepithelial neoplasia (CIN)]. Regulatory T cells (Tregs) might be involved in the failure of the immune system to control the development of HPV-induced cancer. We investigated frequencies, phenotype and activity of Tregs in patients with cervical neoplasia. CIN and cervical cancer patients showed increased CD4+/CD25high T cell frequencies in peripheral blood and CD4+ T cell fraction. These CD4+/CD25high T cells represent Tregs as demonstrated by their low proliferation rate, low interferon (IFN)-,/interleukin (IL)-10 ratio, high expression of CD45RO, GITR, CTLA-4, forkhead box P3 (FoxP3) and low CD45RA expression. Moreover, in HPV16+ cervical cancer patients, in-vitro depletion of CD25+ T cells resulted in increased IFN-, T cell responses against HPV16 E6- and E7 peptides. Thus, increased frequencies of Tregs in cervical cancer patients may indeed suppress HPV-specific immunity. Longitudinal analysis of CD4+/CD25high T cell frequencies in patients showed a modest decline 1 year after curative surgery or chemoradiation. This study demonstrates increased frequencies and suppressive activity of Tregs in cervical cancer. These results imply that Tregs may suppress the immune control of cervical neoplasia and furthermore that suppression of immunity by Tregs will be another hurdle to overcome in therapeutic immunization strategies against cervical neoplasia. [source] Subcellular compartmentalization of aromatase is sexually dimorphic in the adult zebra finch brainDEVELOPMENTAL NEUROBIOLOGY, Issue 1 2007Kevin N. Rohmann Abstract The vertebrate brain is a source of estrogen (E) via the expression of aromatase (E-synthase). In the zebra finch (Taeniopygia guttata), despite documented dimorphisms in E-action, no differences are detectable in circulating E, or the neural levels of aromatase transcription, activity, or somal protein expression. Studies of aromatase expression at the light- and electron-microscope levels reveal greater numbers of fibers and presynaptic boutons in adult males relative to females. We assayed aromatase activity and content in synaptosomes and microsomes from the anterior [containing lMAN and Area X (males)] and posterior telencephalon (containing HVC and RA) of adult birds. In contrast to non-song birds and mammals, both cell fractions contain abundant aromatase measurable in terms of activity (enzyme assays) and content (Western blots) with minimal enrichment in microsomes. From brain homogenates of identical concentration, aromatase activity was higher in the synaptosomal relative to the microsomal fraction, in males relative to females, and in the posterior compared to anterior telencephalon. These effects were driven by high levels of synaptosomal aromatase in the male posterior telencephalon. These data suggest that males possess more aromatase per presynaptic bouton, or a greater number of aromatase-containing presynaptic boutons than females in the posterior telencephalon. Further, the present report reveals synaptic aromatization as a considerable source of E in the zebra finch brain, and supports the idea that telencephalic synapses in and around the adult male song production nuclei may be exposed to higher levels of E compared to the female brain. © 2006 Wiley Periodicals, Inc. J Neurobiol 67: 1,9, 2007 [source] Functional characterization of highly adherent CD34+ keratinocytes isolated from human skinEXPERIMENTAL DERMATOLOGY, Issue 7 2010Araika Gutiérrez-Rivera Please cite this paper as: Functional characterization of highly adherent CD34+ keratinocytes isolated from human skin. Experimental Dermatology 2010; 19: 685,688. Abstract:, Compared to murine models, data on cells responsible for the homeostasis of human epidermis are scarce and often contradictory. Given the conflicting results and the availability of clinical grade protocols to purify CD34 cells from a given tissue, we pursued to phenotypically characterize human epidermal CD34+ population. After magnetic separation of whole skin CD34+ and CD34, cell fractions and selection for cells highly adherent to extracellular matrix, both CD34± fractions retained the ability to form a stratified epidermis in organotypic cultures and presented similar in vitro migratory phenotypes. However CD34, cells showed higher clonogenic potential and in vitro proliferative capacity. These results indicated that CD34, cell fraction contains stem/early progenitor cells, while CD34+ cells might be a transit-amplifying precursor for hair follicle (HF) sheath cells. The ability to isolate living cells using differential cell adhesion and surface markers provides an opportunity to study cells from different morphological regions of the HF. [source] Down-regulation of reduced folate carrier may result in folate malabsorption across intestinal brush border membrane during experimental alcoholismFEBS JOURNAL, Issue 24 2007Abid Hamid Folate plays a critical role in maintaining normal metabolic, energy, differentiation and growth status of all mammalian cells. The intestinal folate uptake is tightly and diversely regulated, and disturbances in folate homeostasis are observed in alcoholism, attributable, in part, to intestinal malabsorption of folate. The aim of this study was to delineate the regulatory mechanisms of folate transport in intestinal absorptive epithelia in order to obtain insights into folate malabsorption in a rat model of alcoholism. The rats were fed 1 g·kg,1 body weight of ethanol daily for 3 months. A reduced uptake of [3H]folic acid in intestinal brush border membrane was observed over the course of ethanol administration for 3 months. Folate transport exhibited saturable kinetics and the decreased intestinal brush border membrane folate transport in chronic alcoholism was associated with an increased Km value and a low Vmax value. Importantly, the lower intestinal [3H]folic acid uptake in ethanol-fed rats was observed in all cell fractions corresponding to villus tip, mid-villus and crypt base. RT-PCR analysis for reduced folate carrier, the major folate transporter, revealed that reduced folate carrier mRNA levels were decreased in jejunal tissue derived from ethanol-fed rats. Parallel changes were observed in reduced folate carrier protein levels in brush border membrane along the entire crypt,villus axis. In addition, immunohistochemical staining for reduced folate carrier protein showed that, in alcoholic conditions, deranged reduced folate carrier localization was observed along the entire crypt,villus axis, with a more prominent effect in differentiating crypt base stem cells. These changes in functional activity of the membrane transport system were not caused by a general loss of intestinal architecture, and hence can be attributed to the specific effect of ethanol ingestion on the folate transport system. The low folate uptake activity observed in ethanol-fed rats was found to be associated with decreased serum and red blood cell folate levels, which might explain the observed jejunal genomic hypomethylation. These findings offer possible mechanistic insights into folate malabsorption during alcoholism. [source] Secretion of proteases in serglycin transfected Madin,Darby canine kidney cellsFEBS JOURNAL, Issue 3 2006Lillian Zernichow Madin,Darby canine kidney (MDCK) cells, which do not normally express the proteoglycan (PG) serglycin, were stably transfected with cDNA for human serglycin fused to a polyhistidine tag (His-tag). Clones with different levels of serglycin mRNA expression were generated. One clone with lower and one with higher serglycin mRNA expression were selected for this study. 35S-labelled serglycin in cell fractions and conditioned media was isolated using HisTrap affinity chromatography. Serglycin could also be detected in conditioned media using western blotting. To investigate the possible importance of serglycin linked to protease secretion, enzyme activities using chromogenic substrates and zymography were measured in cell fractions and serum-free conditioned media of the different clones. Cells were cultured in both the absence and presence of phorbol 12-myristate 13-acetate (PMA). In general, enzyme secretion was strongly enhanced by treatment with PMA. Our analyses revealed that the clone with the highest serglycin mRNA expression, level of HisTrap isolated 35S-labelled serglycin, and amount of serglycin core protein as detected by western blotting, also showed the highest secretion of proteases. Transfection of serglycin into MDCK cells clearly leads to changes in secretion levels of secreted endogenous proteases, and could provide further insight into the biosynthesis and secretion of serglycin and potential partner molecules. [source] Linear relationship between Wnt activity levels and apoptosis in colorectal carcinoma cells exposed to butyrateINTERNATIONAL JOURNAL OF CANCER, Issue 4 2004Darina L. Lazarova Abstract We have reported that butyrate, a fatty acid produced by dietary fiber that induces cell cycle arrest, differentiation and/or apoptosis in colorectal carcinoma (CRC) cells in vitro, modulates Wnt activity in 2 CRC cell lines (Bordonaro et al., Int. J. Cancer, 2002; 97:42,51). Our study determines how changes in the levels of Wnt activity induced by butyrate relate to the effects of butyrate on apoptosis, cell cycle arrest and differentiation of CRC cells. In 10 human CRC cell lines a direct relationship was shown between apoptosis and butyrate-induced increase in Wnt activity, as well as between suppressed clonal growth and increased Wnt activity. No correlation existed between butyrate-induced increase in Wnt activity and differentiation. The direct relationship between apoptosis and Wnt activity was supported by analyses of DLD-1 and HCT-116 cells expressing a dominant negative form of Tcf4, and therefore, with repressed Wnt activity, as well as by measuring the ratio of apoptotic to live cells in flow cytometry-sorted cell fractions with high and low Wnt activity. Novel flow cytometric methodology was utilized to show that butyrate differentially increases the number of cells with Wnt activity in different CRC cell lines. Thus, CRC cell lines in which butyrate upregulated Wnt activity to relatively high levels were most susceptible to the apoptotic effects of butyrate, whereas cell lines in which butyrate modestly modulated Wnt activity were less affected. © 2004 Wiley-Liss, Inc. [source] Nuclear and nuclear envelope localization of dystrophin Dp71 and dystrophin-associated proteins (DAPs) in the C2C12 muscle cells: DAPs nuclear localization is modulated during myogenesisJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 3 2008R. González-Ramírez Abstract Dystrophin and dystrophin-associated proteins (DAPs) form a complex around the sarcolemma, which gives stability to the sarcolemma and leads signal transduction. Recently, the nuclear presence of dystrophin Dp71 and DAPs has been revealed in different non-muscle cell types, opening the possibility that these proteins could also be present in the nucleus of muscle cells. In this study, we analyzed by Immunofluorescence assays and Immunoblotting analysis of cell fractions the subcellular localization of Dp71 and DAPs in the C2C12 muscle cell line. We demonstrated the presence of Dp71, ,-sarcoglycan, ,-dystrobrevin, ,-dystroglycan and ,-syntrophin not only in plasma membrane but also in the nucleus of muscle cells. In addition, we found by Immunoprecipitation assays that these proteins form a nuclear complex. Interestingly, myogenesis modulates the presence and/or relative abundance of DAPs in the plasma membrane and nucleus as well as the composition of the nuclear complex. Finally, we demonstrated the presence of Dp71, ,-sarcoglycan, ,-dystroglycan, ,-dystrobrevin and ,-syntrophin in the C2C12 nuclear envelope fraction. Interestingly, ,-sarcoglycan and ,-dystroglycan proteins showed enrichment in the nuclear envelope, compared with the nuclear fraction, suggesting that they could function as inner nuclear membrane proteins underlying the secondary association of Dp71 and the remaining DAPs to the nuclear envelope. Nuclear envelope localization of Dp71 and DAPs might be involved in the nuclear envelope-associated functions, such as nuclear structure and modulation of nuclear processes. J. Cell. Biochem. 105: 735,745, 2008. © 2008 Wiley-Liss, Inc. [source] In vitro multipotentiality and characterization of human unfractured traumatic hemarthrosis-derived progenitor cells: A potential cell source for tissue repairJOURNAL OF CELLULAR PHYSIOLOGY, Issue 3 2007Sang Yang Lee Mesenchymal progenitor cells (MPCs) are a very attractive tool in the context of repair and regeneration of musculoskeletal tissue damaged by trauma. The most common source of MPCs to date has been the bone marrow, but aspirating bone marrow from the patient is an invasive procedure. In an attempt to search for alternative sources of MPCs that could be obtained with minimal invasion, we looked into traumatic hemarthrosis of the knee. In this study, we determined whether a population of multipotent MPCs could be isolated from acute traumatic knee hemarthrosis in the absence of intra-articular fractures. Mononuclear cells were isolated from the aspirated hemarthrosis by density gradient separation, and cultured. We were able to obtain plastic adherent fibroblast-like cells from the mononuclear cell fractions. Flow cytometry analysis revealed that the adherent fibroblast-like cells were consistently positive for CD29, CD44, CD105, and CD166, and were negative for CD14, CD34, and CD45. These were similar to control bone marrow stromal cells. These cells could differentiate in vitro into osteogenic, adipogenic, and chondrogenic cells in the presence of lineage-specific induction factors. In conclusion, acute unfractured traumatic hemarthrosis of the knee contains MPCs with multipotentiality. Because knee hemarthrosis is easy to harvest with minimal pain and without unnecessary invasion, we regard hemarthrosis-derived cells as an additional progenitor cell source for future tissue engineering and cell-based therapy in knee injuries. J. Cell. Physiol. 210: 561,566, 2007. © 2006 Wiley-Liss, Inc. [source] Kinetic studies during peripheral blood stem cell collection show CD34+ cell recruitment intra-apheresisJOURNAL OF CLINICAL APHERESIS, Issue 3 2001Lene Meldgaard Knudsen Abstract A sufficient number of CD34+ cells in the peripheral blood stem cell product is important to achieve a rapid and sustained engraftment. The purpose of the present work was to study CD34+ cell kinetics during leukapheresis. Blood samples before and after leukapheresis were analysed for CD34+ cells in 205 procedures. The number of CD34+ cells after plus the number of CD34+ cells harvested was 1.5-fold greater than the number available at the beginning of the procedure, indicating recruitment of CD34+ cells during leukapheresis. In a subgroup of 66 procedures, granulocytes and platelets were measured. In contrast to CD34+ cells, these cell fractions were not recruited to the blood stream during leukapheresis. An additional nine patients were studied with serial blood measurements during leukapheresis, showing an initial decline that was followed by an increase in CD34+ cells during leukapheresis. In conclusion, CD34+ cells are recruited to the blood during the leukapheresis procedure in contrast to granulocytes and platelets. J. Clin. Apheresis. 16:114,119, 2001. © 2001 Wiley-Liss, Inc. [source] Human papillomavirus DNA detected in peripheral blood samples from healthy Australian male blood donorsJOURNAL OF MEDICAL VIROLOGY, Issue 10 2009Alice Che-Ha Chen Abstract Recent studies have shown that human papillomavirus (HPV) DNA can be found in circulating blood, including peripheral blood mononuclear cells (PBMCs), sera, plasma, and arterial cord blood. In light of these findings, DNA extracted from PBMCs from healthy blood donors were examined in order to determine how common HPV DNA is in blood of healthy individuals. Blood samples were collected from 180 healthy male blood donors (18,76 years old) through the Australian Red Cross Blood Services. Genomic DNA was extracted and specimens were tested for HPV DNA by PCR using a broad range primer pair. Positive samples were HPV-type determined by cloning and sequencing. HPV DNA was found in 8.3% (15/180) of the blood donors. A wide variety of different HPV types were isolated from the PBMCs; belonging to the cutaneous beta and gamma papillomavirus genera and mucosal alpha papillomaviruses. High-risk HPV types that are linked to cancer development were detected in 1.7% (3/180) of the PBMCs. Blood was also collected from a healthy HPV-positive 44-year-old male on four different occasions in order to determine which blood cell fractions harbor HPV. PBMCs treated with trypsin were negative for HPV, while non-trypsinized PBMCs were HPV-positive. This suggests that the HPV in blood is attached to the outside of blood cells via a protein-containing moiety. HPV was also isolated in the B cells, dendritic cells, NK cells, and neutrophils. To conclude, HPV present in PBMCs could represent a reservoir of virus and a potential new route of transmission. J. Med. Virol. 81:1792,1796, 2009. © 2009 Wiley-Liss, Inc. [source] Human laminin-332 degradation by Candida proteinasesJOURNAL OF ORAL PATHOLOGY & MEDICINE, Issue 6 2008P. Pärnänen Background:, Human laminin-332 (Lm-332) degradation by 12 Candida strains and effects of synthetic proteinase inhibitors [Ilomastat (ILM), EDTA, chemically modified tetracycline-3(CMT-3), CMT-308, synthetic peptide CTT-2, and Pefabloc] were studied. Materials and methods:, Laminin-332 was incubated with sonicated cell fractions and 10 times concentrated cell-free fractions of reference and clinical strains of C. albicans, C. dubliniensis, C. guilliermondii, C. glabrata, C. krusei, and C. tropicalis. Proteolysis, pH effects, and inhibitors were analyzed by fluorography and zymography. Results:, Cell fractions of all species except C. guilliermondii and cell-free fractions of C. albicans, and C. dubliniensis showed 20,70 kDa gelatinases at pH 5.0 and 6.0. At pH 7.6, C. glabrata, C. krusei, and C. tropicalis cell fractions and C. tropicalis cell-free fractions showed 55,70 kDa gelatinases. CMT-3, CMT-308, and CTT-2 inhibited Candida gelatinases slightly better than Pefabloc, ILM, and EDTA. No Candida fractions degraded Lm-332 at pH 7.6, but at pH 5.0, 100 kDa bands were generated by cell fractions of C. dubliniensis and C. tropicalis; C. albicans and C. glabrata clinical strains; and C. guilliermondii reference strain. C. krusei reference strain yielded three 100,130 kDa bands. C. albicans, C. dubliniensis, and C. tropicalis reference and clinical strain's cell-free fractions generated 100 kDa band. Conclusions:, Laminin-332 degradation is pH-dependent and differences exist between studied Candida strains. Lm-332 degradation can exert functional disturbances on basement membrane integrity, possibly aiding Candida cell invasion into tissues. Certain synthetic matrix metalloproteinase inhibitors (CMTs, CTT) can inhibit Candida proteinases and may be therapeutically useful in future. [source] SCF and c-kit expression profiles in male individuals with normal and impaired spermatogenesisANDROLOGIA, Issue 2 2010M. Bialas Summary The transcription levels of stem cell factor (SCF) and c-kit were examined using real-time RT PCR in interstitial and intratubular cell fractions, as well as in tissue homogenates from normal, azoospermic and neoplasmic patients. Peripheral blood mononuclear cells (PBMC) were used as a systemic control. The observed level of c-kit expression in all investigated groups was generally higher than the expression of SCF. The highest (statistically significant) level of c-kit was noted in testicular tumours (the greater part of which were represented by seminomas) in contrast to SCF mRNA, which may indicate an association between c-kit overexpression and seminoma development. In Sertoli cell only syndrome, almost equal levels of SCF and c-kit transcripts were noted. These results may indicate Leydig cells as the alternative source of c-kit gene transcription. SCF transcript values were low and comparable among the analysed subgroups except that in maturation arrest at spermatocyte stage, the SCF gene expression was statistically higher than in testicular tumours. It appears from the study that c-kit has been a dynamic gene, changing its activity in a variety of testicular pathologies while being expressed in all testicular compartments but clearly overexpressed in testicular tumours of seminomatous origin. [source] A2B5 Cells from Human Glioblastoma have Cancer Stem Cell PropertiesBRAIN PATHOLOGY, Issue 1 2010Aurélie Tchoghandjian Abstract Glioblastomas, like other cancers, harbor small cell populations with the capability of sustaining tumor formation. These cells are referred to as cancer stem cells. We isolated cells expressing the surface marker A2B5 from three human glioblastomas (GBM) and showed that after grafting into nude mice, they generated dense and highly infiltrative tumors. Then, we extensively studied A2B5+ cells isolated from 11 human GBM. These cells display neurosphere-like, self-renewal, asymmetrical cell division properties and have multipotency capability. Stereotactic xenografts of dissociated A2B5+ -derived secondary spheres revealed that as few as 1000 cells produced a tumor. Moreover, flow cytometry characterization of A2B5+ -derived spheres revealed three distinct populations of cells: A2B5+/CD133+, A2B5+/CD133 - and A2B5 - /CD133 - , with striking proportion differences among GBM. Both A2B5+/CD133+ and A2B5+/CD133 - cell fractions displayed a high proliferative index, the potential to generate spheres and produced tumors in nude mice. Finally, we generated two green fluorescent protein-cell lines that display,after serum induction,distinct proliferative and migratory properties, and differ in their CD133 level of expression. Taken together, our results suggest that transformed A2B5+ cells are crucial for the initiation and maintenance of GBM, although CD133 expression is more involved in determining the tumor's behavior. [source] Cyclooxygenase-2 expression and angiogenesis in squamous cell carcinoma of the skin and its precursors: a paired immunohistochemical study of 35 casesBRITISH JOURNAL OF DERMATOLOGY, Issue 4 2004T. Nijsten Summary Background, Cyclooxygenase (COX)-2 expression and tumour-induced angiogenesis appear to be increased in squamous cell carcinoma (SCC) of the skin. In other cancers, COX-2 is a pro-angiogenic factor. The association between angiogenesis and COX-2 has not been studied in skin cancer. Objectives, To assess the onset of increased COX-2 expression and angiogenesis in the multistage carcinogenesis of SCC as well as the correlation between those two parameters. Patients/methods, We performed a retrospective paired immunohistochemical analysis of normal skin, actinic keratosis (AK), Bowen's disease (BD) and SCC among 35 individuals. Specimens were considered COX-2 immunopositive when 5% or more of the tumour cells showed clear evidence of immunostaining. To quantify active angiogenesis, we used a Ki-67,CD34 double-labelling immunohistochemical stain and calculated the fraction of proliferating endothelial cells. The Chalkley method was used to determine the microvessel density. To detect hypoxia, a carboanhydrase IX immunostain was used. Results, Compared with normal epidermis (0%), AK (31%), BD (22%) and SCC (40%) were significantly more likely to be COX-2 immunopositive (P < 0·01). The fraction of proliferating endothelial cells and the Chalkley scores paralleled multistage carcinogenesis (P < 0·05 between different stages). COX-2 immunopositivity was fairly correlated with hypoxia and higher proliferating endothelial cell fractions but not with Chalkley counts. Conclusions, Induction of COX-2 expression and angiogenesis are both early events in the development of SCC. In addition to ultraviolet light, hypoxia and COX-2 may be involved in skin tumour angiogenesis. [source] Alteration of Ca2+ -ATPase activity in the homogenate, plasma membrane and microsomes of the salivary glands of streptozotocin-induced diabetic ratsCELL BIOCHEMISTRY AND FUNCTION, Issue 3 2009José Nicolau Abstract Diabetes has been implicated in the dryness of the mouth, loss of taste sensation, sialosis, and other disorders of the oral cavity, by impairment of the salivary glands. The aim of the present study was to examine the plasma membrane, microsomal, and homogenate Ca2+ -ATPase activity in the rat submandibular and parotid salivary glands of streptozotocin-induced diabetes. We have also examined the influence of the acidosis state on this parameter. Diabetes was induced by an intraperitoneal injection of streptozotocin and acidosis was induced by daily injection of NH4Cl. At 15 and 30 days after diabetes induction, the animals were euthanized and the submandibular and parotid salivary glands were removed and analyzed. Ca2+ -ATPase (total, independent, and dependent) was determined in the homogenate, microsomal, and plasma membranes of the salivary glands of diabetic and control rats. Calcium concentration was also determined in the glands and showed to be higher in the diabetic animals. Ca2+ -ATPase activity was found to be reduced in all cell fractions studied in the diabetic animals compared with control. Similar results were obtained for the submandibular salivary glands of acidotic animals; however in the parotid salivary glands it was found an increase in the enzyme activity. Copyright © 2009 John Wiley & Sons, Ltd. [source] Differential effect of cholera toxin on CD45RA+ and CD45RO+ T cells: specific inhibition of cytokine production but not proliferation of human naive T cellsCLINICAL & EXPERIMENTAL IMMUNOLOGY, Issue 2 2000K. Eriksson We have studied how cholera toxin (CT) and its non-toxic cell-binding B-subunit (CTB) affect the activation of pure human T cells in an anti-CD3-driven system. CT, as opposed to CTB, strongly suppressed the proliferative responses as well as cytokine production in CD4+ and CD8+ T cells. CT however, had a differential effect on naive and activated/memory T cell subsets. Costimulation through exogenous IL-2 or through CD28 cross-linking rescued the proliferation of CT-treated naive CD45RA+ T cells, but not of activated/memory CD45RO+ cells. IL-2 production and IL-2 receptor expression were markedly reduced by CT in all T cell fractions, i.e. also in CD45RA+ cells which had maintained proliferative responses. However, the proliferative responses of CT-treated CD45RA+ T cells were IL-2-dependent, as shown by blocking experiments using anti-IL-2 antibodies. These results indicate (i) that CTB has no cytostatic effect on human T cells, (ii) that CT affects proliferation and cytokine production by two different signal pathways, and (iii) that CT might interact with a signal pathway generated through or influenced by CD45. [source] | ||||||||||||||||||||||||||||