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Positive Cells (positive + cell)
Kinds of Positive Cells Selected AbstractsChemokine receptor CXCR3 expression in inflammatory bowel diseaseINFLAMMATORY BOWEL DISEASES, Issue 4 2001Yu-Hong Yuan Abstract CD4+ T lymphocytes in the lamina propria (LP) of the gut play a central role in the immune response in inflammatory bowel disease (IBD). CXCR3 is a chemokine receptor expressed on activated T lymphocytes, and a key component for the recruitment of T helper (Th1) effector cells to the site of inflammation. To determine if CXCR3 is involved in localization of T cells to the gut in IBD patients, we investigated the expression of CXCR3 on CD4+ T lymphocytes in the LP and in the submucosa of resection specimens from 51 IBD patients and 15 control patients. Positive cells were microscopically scored using a semiquantitative analysis on a five-point scale. We found that CD4+ T cells, CXCR3+ cells, and CD4+CXCR3+ T cells in the LP were slightly increased in both IBD groups compared with control non-IBD specimens. In addition, CD4+ and CXCR3+ cells in the submucosa were significant increased in the CD group compared with the control group. CD4+ and CXCR3+ expression was not statistically different between CD and UC. Flow cytometry was used to analyze the percentage of CXCR3+ cells within the CD4+ T-cell population isolated from biopsy specimens and peripheral blood from IBD patients and control patients. There was no difference in the percentage of CD4+CXCR3+ cells between the different groups in the gut as well as in the circulation. These results suggest that CD4+CXCR3+ T cells migrate to the normal and inflamed intestinal mucosa, indicating a role in maintaining normal gut homeostasis. The selective expression of CXCR3+ cells in the submucosa of CD patients might also indicate that these cells play a role in inflammation. [source] TP53 mutations in clinically normal mucosa adjacent to oral carcinomasJOURNAL OF ORAL PATHOLOGY & MEDICINE, Issue 9 2010C. Thode J Oral Pathol Med (2010) 39: 662,666 Background:, The tumour-suppressor protein p53 often accumulates in histologically normal epithelium adjacent to oral squamous cell carcinomas (OSCC). We investigated whether this was associated with mutations in TP53, the gene for p53, and might implicate impending malignancy. Methods:, Specimens from 18 human squamous cell carcinomas were stained with monoclonal p53 antibodies. Positive cells were microdissected with laser-captured microscopy from the tumour and adjacent normal and dysplastic epithelium. DNA was extracted, and exons 5,9 of the TP53 gene were amplified by PCR. Amplified products were separated by denatured gradient gel electrophoresis. Fragments with a deviant DGEE pattern were sequenced. Results:,TP53 mutations were found in six of 18 tumours. Fourteen specimens contained histologically normal mucosa adjacent to the tumour; 13 of these showed small clusters of p53 positive cells. Seven specimens contained both histological normal and dysplastic epithelial tissues adjacent to the tumour. A TP53 mutation was found in only one specimen; this mutation appeared in the normal mucosa, the adjacent tumour, and the epithelial dysplasia. Conclusion:, We found that upregulation of p53 was a frequent event in histological normal mucosa adjacent to OSCC; however, it was rarely associated with a mutation in the TP53 gene. [source] Different distribution of immunocompetent cells in the dentogingival junction during root formation in rat molarsJOURNAL OF PERIODONTAL RESEARCH, Issue 1 2003Hiroshi Tamura The distribution of immunocompetent cells in the dentogingival junction of rat molars during root formation was investigated by immunocytochemistry using antibodies to class II major histocompatibility complex (MHC) molecules (OX6-antibody) and monocyte/macrophage lineage cells (ED1-antibody) as well as by histochemical reaction for periodic acid,Schiff (PAS). Two portions (the junctional epithelium in the mesial gingiva of the first molar, and the interdental gingiva between the first and second molars) were selected for observations. At the eruption stage of the first molar (16,18 days after birth), OX6-positive cells, dendritic or oval in shape, were abundantly distributed in the connective tissue between the oral epithelium and tooth germ. Positive cells with slender cell processes were also found beneath the ameloblast layer. At the commencement stage of the first molar occlusion (24,28 days after birth), numerous OX6-positive cells displaying a dendritic fashion existed preferentially in the mesial gingiva, but were fewer in the interdental gingiva. In contrast, the interdental gingiva showed a denser distribution of ED1-positive cells and PAS-reactive polymorphonuclear leukocytes (PMLs) than the mesial gingiva. At the completion stage of root formation (100,120 days after birth), the OX6-immunopositive cells invaded the deeper position of the mesial gingiva with the downgrowth of the epithelium; they had a considerably higher cell density compared with those in the interdental gingiva where PAS-reactive PMLs persisted. These findings indicated that the immunocompetent cells showed a region-specific distribution and cell density by their roles in immune response. [source] Down-regulation of the nonspecific and antigen-specific T cell cytokine response in ankylosing spondylitis during treatment with infliximabARTHRITIS & RHEUMATISM, Issue 3 2003Jianxiang Zou Objective Treatment of active ankylosing spondylitis (AS) with the monoclonal tumor necrosis factor , (TNF,) antibody infliximab is highly clinically effective. This study was undertaken to investigate the precise mechanism of action of anti-TNF, treatment in AS. Methods Cytokine expression of CD4+ and CD8+ T cells was investigated before and 6 and 12 weeks after the start of treatment in 10 patients treated with infliximab, and before and after 6 weeks of treatment and 6 weeks after placebo was switched to infliximab in 10 patients treated initially with placebo. Peripheral blood mononuclear cells (PBMCs) were stimulated for 6 hours either nonspecifically with phorbol myristate acetate (PMA)/ionomycin or antigen specifically with a pool of 46 overlapping 18-mer peptides derived from the G1 domain of aggrecan. Cells were stained for T cell surface markers CD4 and CD8 and for the intracellular cytokines interferon-, (IFN,), TNF,, interleukin-4 (IL-4), and IL-10. Positive cells were quantified by flow cytometry. For monocyte-derived cytokines, PBMCs were stimulated with lipopolysaccharide (LPS) for 18 hours and TNF, and IL-10 in the supernatant were measured by enzyme-linked immunosorbent assay. Results Compared with baseline, infliximab treatment induced a significant decrease at 12 weeks in the number of CD4+ and CD8+ T cells that were positive for IFN, and TNF, upon PMA/ionomycin stimulation (P = 0.005). A significant reduction had already begun to occur at 6 weeks. No change in the percent IFN, or TNF, positivity among CD4+ and CD8+ subpopulations was observed after 6 weeks in patients treated with placebo. However, when these patients began infliximab treatment after 6 weeks of receiving placebo, there was a similar significant decrease in IFN, and TNF, production by CD4+ and CD8+ T cells (P < 0.05). Furthermore, infliximab treatment induced a significant reduction in the number of IFN,+ and TNF,+ CD8+ T cells (P = 0.005 at week 6 and week 12) after antigen-specific in vitro stimulation with G1-derived peptides. Between-group analysis showed that the change in the expression of IFN, and TNF, in both CD4+ and CD8+ T cells was significantly different between the infliximab and placebo groups (P = 0.001 for all variables). There was no change in the number of IL-10+ or IL-4+ T cells during treatment. No significant change in the production of TNF, and IL-10 upon in vitro stimulation of PBMCs with LPS was detectable during infliximab treatment. Conclusion Infliximab down-regulates both IFN, and TNF, secreted by T cells but does not induce a change in cytokines produced by monocytes during 3 months of treatment. This is likely to be a relevant mechanism for the clinical efficacy of this therapy. [source] Inhibition of Alcohol-Associated Colonic Hyperregeneration by ,-Tocopherol in the RatALCOHOLISM, Issue 1 2003P. Vincon Background: Chronic alcohol consumption results in colorectal mucosal hyperregeneration, a condition associated with an increased risk for colorectal cancer. Possible mechanisms may involve the effects of acetaldehyde and/or free radicals generated during alcohol metabolism. Vitamin E is part of the antioxidative defense system, and its concentration is decreased or its metabolic utilization increased in various tissues after chronic alcohol consumption. We wondered whether ,-tocopherol supplementation may prevent ethanol-induced colorectal cell cycle behavior and whether these changes were related to alterations in protein synthesis. Methods: Five groups of male Wistar rats, each consisting of 14 animals, received liquid diets as follows: group 1, alcohol; group 2, alcohol +,-tocopherol; group 3, control (i.e., isocaloric glucose); group 4; control (i.e., isocaloric glucose) +,-tocopherol. Group 5 was fed a solid chow diet ad libitum. After 4 weeks of feeding, immunohistology was performed with anti-proliferating cell nuclear antigen (PCNA) or anti-BCL2 antibodies. Fractional (ks) and absolute (Vs) rates of protein synthesis and rates of protein synthesis relative to RNA (kRNA) and DNA (kDNA) were measured with a flooding dose of L-[4- 3H] phenylalanine with complementary analysis of protein and nucleic acid composition. Results: The PCNA index was increased significantly in the colon after ethanol administration compared with controls (ethanol, 10.3 ± 2.3 vs. control, 6.51 ± 1.6% PCNA positive cells, p < 0.05), although neither the protein, RNA, and DNA concentrations nor ks, kRNA, kDNA, and Vs were affected. This increase in PCNA index was significantly diminished by coadministration of ,-tocopherol (ethanol +, - tocopherol, 7.86 ± 1.71% PCNA positive cells, p < 0.05) without significant alterations in protein synthetic parameters. A similar result was obtained for the PCNA index in the rectal mucosa (ethanol, 14.6 ± 4.4 vs. control, 12.1 ± 4.2% PCNA positive cell), although this did not reach statistical significance. Neither ethanol nor , - tocopherol feeding had any significant effect on BCL-2 expression in the colorectal mucosa. As with the colon, protein synthetic parameters in the mucosa were not affected by alcohol feeding at 4 weeks. These effects on colonic cell turnover without corresponding changes in protein synthesis thus represent a specific localized phenomenon rather than a general increase in anabolic processes in the tissue and reaffirm the hyperregenerative properties of chronic alcohol consumption. Conclusions: Alcohol-associated hyperproliferation could be prevented, at least in part, by supplementation with ,-tocopherol. This may support the hypothesis that free radicals are involved in the pathogenesis of alcohol-associated colorectal hyperproliferation. [source] Perinatal development of the rat kidney: Apoptosis and epidermal growth factorCONGENITAL ANOMALIES, Issue 3 2003Toshiya Okada ABSTRACT, Localization of apoptotic cells in the kidney of perinatal rats was examined by the terminal deoxynucleotidyl transferase,mediated d,UTP,biotin nick end labeling (TUNEL) method and electron microscopy. Perinatal changes in the percentage of kidney cells with DNA fragmentation were determined by flow cytometric analysis. Through observation of two successive sections, the relationship between the localization of the epidermal growth factor receptor (EGFR) positive cells and TUNEL positive cells in the kidney was determined. From fetal day 18 to neonatal day 5, TUNEL positive cells were noted in immature glomeruli, collecting ducts and interstitium. Electron microscopically, chromatin condensed nuclei and apoptotic bodies were seen in the same tissue component as the TUNEL positive cells. The percentage of DNA fragmented cells significantly increased from fetal days 18 to 20 and significantly decreased from fetal days 20 to 22, while they still remained low in the neonatal period. The TUNEL positive cells in immature glomeruli and collecting ducts were not reactive to the EGFR antibody. The TUNEL positive cells were not observed in the proximal tubular cells, which were positive to EGFR antibody. These results indicate that apoptotic cells are present in the kidney throughout the perinatal period in the rat and that EGF plays an important role in perinatal development of the rat kidney. [source] Flow cytometric differential of leukocyte populations in normal bone marrow: Influence of peripheral blood contamination1,CYTOMETRY, Issue 1 2009R. A. Brooimans Abstract Background: Availability of immunophenotypic reference values for the various leukocyte populations distributed in bone marrow may be helpful to recognize abnormal bone marrow development and, therefore, useful as first screening of individuals with suspected hematological malignancies or other hematopoietic disorders. Methods: A single tube four-color staining panel (CD66abce/CD14/CD45/CD34) together with a predefined gating strategy was utilized to immunologically differentiate the distribution of the major leukocyte populations in bone marrow aspirates of healthy donors. The sample-blood erythrocyte ratio was applied to assess the amount of blood contamination of marrow and account for this in the marrow value estimates. Results: The frequency of the major leukocyte populations in bone marrow of 134 normal donors were for granulocytes: mean, 69.4%; SD, 10.3%; monocytes: mean, 4.7%; SD, 2.3%; lymphocytes: mean, 18.3%; SD, 8.7%. The frequency of the immature cell population that included precursor cells of each of the cell lineages among other cell types were mean 5.0%; SD 2.2%. The mean percentage of CD34 positive cells was 1.5%; SD 0.7%. Our results showed further that the frequency of cell populations, of which the presence is restricted to the bone marrow (e.g., CD34+ progenitor cells), is influenced by the degree of peripheral blood admixture. Between the total immature cells and purity of the bone marrow, there was a significant positive correlation demonstrated, whereas a negative correlation was found between the percentages of both lymphocytes as monocytes and the purity of the bone marrow. Conclusions: With a single tube-staining panel, we obtained reference values for flow cytometric assessment of all relevant leukocyte populations present in bone marrow that can be used as a frame of reference for better recognition of individuals with abnormal hematopoiesis. In addition, we have demonstrated the influence of the degree of peripheral blood admixture in the bone marrow aspirates on those reference values. © 2008 Clinical Cytometry Society [source] Use of a blocking antibody method for the flow cytometric measurement of ZAP-70 in B-CLLCYTOMETRY, Issue 4 2006Mark Shenkin Abstract Background: In this study we developed a method to measure the amount of ZAP-70 [zeta accessory protein] in B-CLL cells without relying on the ZAP-70 expression of patient B or T cells to normalize fluorescence intensity. Methods: B-CLL cells were fixed with formaldehyde before surface staining with gating antibodies CD19PC5 and CD5FITC. The cells were permeabilized with saponin, and the ZAP-70 antigen was blocked in one tube with unlabeled antibody to ZAP-70 [clone 1E7.2]. Zap-70-PE was then added to this tube. ZAP-70-PE was added to a second tube without unlabeled antibody to ZAP-70. The mean fluorescence intensity of the ZAP-70 in the tube without unlabeled antibody divided by the mean fluorescence intensity of the ZAP-70 in the tube with unlabeled antibody equals the RATIO of total fluorescence to non-specific ZAP-70 fluorescence in the B-CLL cells. In a second method of analysis, a region is created in the histogram showing ZAP-70 fluorescence intensity in the tube with unlabeled antibody to ZAP-70. This region is set to 0.9% positive cells. This same region is then used to measure the % positive [%POS] ZAP-70 cells in the tube without unlabeled antibody to ZAP-70. The brighter the ZAP-70 fluorescence above the non-specific background, the higher the %POS. Results: Due to the varying amount of non-specific staining between patient B-CLL cells and other cells, the blocking antibody method yielded a more quantitative and reproducible measure of ZAP-70 in B-CLL cells than other methods, which use the ratio of B-CLL fluorescence to normal B or T-cell fluorescence. Using this improved method, ZAP-70 was determined to be negative if the RATIO was less than 2:1 and positive if the RATIO was greater than 2:1. ZAP-70 was determined to be negative if the %POS was less than 5% and positive if the %POS was greater than 5%, a cut-off value lower than previous values published, due to exclusion of non-specific staining. Both cut-offs were based upon patient specimen distribution profiling. Conclusions: Use of a blocking antibody resulted in a robust, reproducible clinical B-CLL assay that is not influenced by the need to measure the amount of ZAP-70 in other cells. ZAP-70 results segre gate patients into indolent and aggressive groups suggested by published clinical outcomes. © 2006 International Society for Analytical Cytology [source] Comparison of bone marrow and peripheral blood ZAP-70 status examined by flow cytometric immunophenotyping in patients with chronic lymphocytic leukemiaCYTOMETRY, Issue 4 2006Rachel Sheridan Abstract Background: The mutational status of the immunoglobulin heavy chain variable gene in patients with chronic lymphocytic leukemia correlates with prognosis. Patients with mutated IgVH genes fare better than those with unmutated genes. Gene expression profiling studies identified the tyrosine kinase ZAP-70 to be expressed in unmutated CLL samples. Flow cytometric examination of ZAP-70 expression in tumor cells has been proposed to be a convenient surrogate marker for IgVH mutational status. However, a few studies have shown a small number of discordant results between ZAP-70 positivity, IgVH mutational status, and clinical outcome. There have been no reported studies comparing bone marrow samples with peripheral blood for ZAP-70 expression in CLL patients. Methods: We searched our flow cytometry files from October 2004 through April 2006 and identified CLL in 311 bone marrow and peripheral blood specimens from 256 patients. We defined ZAP-70 positivity as greater than 30% of the CD19+ B-cells above the isotype control value that coexpress ZAP-70. Statistical analyses were performed using the Fisher exact test and student t -test. Results: A significantly greater number of bone marrow specimens were positive for ZAP-70 when compared with the number of peripheral blood specimens. Of all the ZAP-70 negative specimens, CLL cells from bone marrow had a greater mean percentage of ZAP-70 positive cells when compared with the CLL cells from peripheral blood. Finally, six patients were identified who were ZAP-70 positive in the bone marrow but ZAP-70 negative in the peripheral blood. Conclusions: These results may be due to either an increase in the false positive rate in bone marrow specimens or to an intrinsic feature of CLL cells in the compartment that is biologically distinct from peripheral tumor cells. As prognosis and treatment decisions may be based on ZAP-70 results from either specimen type, it is prudent to further examine this observation. © 2006 International Society for Analytical Cytology [source] JC-1, a sensitive probe for a simultaneous detection of P-glycoprotein activity and apoptosis in leukemic cellsCYTOMETRY, Issue 3 2006Driss Chaoui Abstract Background JC-1 probe has been successfully used for the analysis of either apoptosis or P-glycoprotein (P-gp) activity. Therefore, we wanted to see if JC-1 could also simultaneously assess both, P-gp activity and apoptosis, in acute myeloid leukemia (AML) cells. Methods P-gp activity was measured using JC-1 and compared to the results of the Rhodamine 123 (Rh 123) assay in P-gp negative and P-gp positive cell lines, and 12 AML samples. For apoptosis, spontaneous apoptosis, as well as, apoptosis induced by Cytosine Arabinosine and Homoharringtonine were analyzed. Both mitochondrial red fluorescence and cytoplasmic green fluorescence of JC-1 with and without a P-gp inhibitor (Cyclosporine A : CsA) were used for the identification of apoptotic cells, and this was compared to Annexin V/PI staining. Results (1) We found a good correlation between JC-1 and Rh 123 in viable cells. Even in a small population of viable cells, P-gp positive cells emitting low red fluorescence, gained on red fluorescence after P-gp inhibition with CsA permitting an evaluation of P-gp activity. (2) We found a good correlation between the Annexin V/PI staining and JC-1 (P < 0.0001) in the assessment of apoptotic cells. Most importantly, the apoptotic cells could be distinguished by the loss of red fluorescence and the increase of green fluorescence without any change after P-gp inhibition with CsA. Conclusions JC-1 can simultaneously evaluate two important parameters involved in drug resistance in AML cells, P-gp activity and apoptosis. © 2006 International Society for Analytical Cytology [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] Cleavage-like cell division and explosive increase in cell number of neonatal gonocytesDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 1 2004Yasuhiro Sakai Based on previous conventional quantitative observations of rat testes, it was proposed that large numbers of gonocytes degenerate after birth and this notion was widely accepted. However, many studies show that neonatal gonocytes display high levels of mitotic activity. In order to resolve the apparent contradiction of increased mitotic activity in gonocytes despite a decrease in their numbers at the neonate stage, quantitative analysis using a marker of suitably higher resolution is required. It has been shown that the vasa protein could be used as a marker of germ cells. In this study, quantitative changes in gonocytes were re-examined using a germ-cell-specific marker in order to delineate more clearly the process of development from gonocytes to spermatogonia after birth. The vasa -positive cells, which correspond to gonocytes and spermatogonia, increased exponentially after birth. This observation suggests that all gonocyte divide actively after birth and do not degenerate as previously believed. Surprisingly, the cell volume of gonocytes decreased during their division. The largest population size was 2000,4000 µ3 at day 2, 1000,2000 µ3 at day 4 and 500,1000 µ3 at day 6. This finding suggests that gonocytes divide in a similar way to cleavage, which can be considered a special mode of fertilized eggs. Judging from the growth of seminiferous tubules and the degree of volume reduction, 60% of the contribution rate is estimated to be due to ordinal cell growth, and 40% due to volume reduction as in cleavage of a fertilized egg. This unique cleavage-like division may contribute to the supply of large numbers of spermatogonia. [source] Impaired lactation in mice expressing dominant-negative FADD in mammary epitheliumDEVELOPMENTAL DYNAMICS, Issue 4 2009Mark Shackleton Abstract The Fas-associated death domain (FADD/Mort1) adaptor protein was originally identified as a key mediator of apoptosis, although pleiotropic functions for FADD have also been reported. FADD-mediated tumoricidal effects have been described in breast cancer cells; however, its physiological role in normal mammary gland epithelium is not well understood. To determine the role of FADD signaling during mammary gland development, we generated transgenic mice overexpressing dominant-negative FADD (DN-FADD) in mammary epithelium, using the steroid responsive mouse mammary tumor virus promoter. Transgenic mice exhibited a perturbation in lactation resulting in impaired milk production and pup growth retardation. Reduced expansion of alveoli was evident during early lactation with extensive shedding of luminal alveolar cells. Significantly more TUNEL (terminal deoxynucleotidyl transferase,mediated deoxyuridinetriphosphate nick end-labeling)-positive cells were present at this time point and a subsequent increase in bromodeoxyuridine-positive cells was observed. These findings suggest a role for FADD in maintaining the survival of mammary secretory alveolar cells after the establishment of lactation. Developmental Dynamics 238:1010,1016, 2009. © 2009 Wiley-Liss, Inc. [source] C-myc as a modulator of renal stem/progenitor cell populationDEVELOPMENTAL DYNAMICS, Issue 2 2009Martin Couillard Abstract The role of c - myc has been well-studied in gene regulation and oncogenesis but remains elusive in murine development from midgestation. We determined c - myc function during kidney development, organogenesis, and homeostasis by conditional loss of c - myc induced at two distinct phases of nephrogenesis, embryonic day (e) 11.5 and e17.5. Deletion of c - myc in early metanephric mesenchyme (e11.5) led to renal hypoplasia from e15.5 to e17.5 that was sustained until adulthood (range, 20,25%) and, hence, reproduced the human pathologic condition of renal hypoplasia. This phenotype resulted from depletion of c - myc,positive cells in cap mesenchyme, causing a ,35% marked decrease of Six2- and Cited1-stem/progenitor population and of proliferation that likely impaired self-renewal. By contrast, c - myc loss from e17.5 onward had no impact on late renal differentiation/maturation and/or homeostasis, providing evidence that c - myc is dispensable during these phases. This study identified c - myc as a modulator of renal organogenesis through regulation of stem/progenitor cell population. Developmental Dynamics 238:405,414, 2009. © 2009 Wiley-Liss, Inc. [source] Development of lymphatic vessels in mouse embryonic and early postnatal heartsDEVELOPMENTAL DYNAMICS, Issue 10 2008Juszy, Micha Abstract We aimed to study the spatiotemporal pattern of lymphatic system formation in the embryonic and early postnatal mouse hearts. The first sign of the development of lymphatics are Lyve-1,positive cells located on the subepicardial area. Strands of Lyve-1,positive cells occur first along the atrioventricular sulcus of the diaphragmatic surface and then along the great arteries. Lumenized tubules appear, arranged in rows or in a lattice. They are more conspicuous in dorsal atrioventricular junction, along the major venous and coronary artery branches and at the base of the aorta and the pulmonary trunk extending toward the heart apex. At later stages, some segments of the lymphatic vessels are partially surrounded by smooth muscle cells. Possible mechanisms of lymphangiogenesis are: addition of Lyve-1,positive cells to the existing tubules, elongation of the lymphatic lattice, sprouting and coalescence of tubules. We discuss the existence of various subpopulations of endothelial cells among the Lyve-1,positive cells. Developmental Dynamics 237:2973,2986, 2008. © 2008 Wiley-Liss, Inc. [source] Two DM domain genes, DMY and DMRT1, involved in testicular differentiation and development in the medaka, Oryzias latipesDEVELOPMENTAL DYNAMICS, Issue 3 2004Tohru Kobayashi Abstract The recent discovery of the DMY gene (DM domain gene on Y chromosome and one of the DMRT1 family genes) as a key determinant of male development in the medaka (Oryzias latipes) has led to its designation as the prime candidate gene for sex-determination in this species. This study focused on the sites and pattern of expression of DMY and DMRT1 genes during gonadal differentiation of medaka to further determine their roles in testis development. DMY mRNA and protein are expressed specifically in the somatic cells surrounding primordial germ cells (PGCs) in the early gonadal primordium, before morphological sex differences are seen. However, somatic cells surrounding PGCs never express DMY during the early migratory period. Expression of DMY persists in Sertoli cell lineage cells, from PGC-supporting cells to Sertoli cells, indicating that only DMY -positive cells enclose PGCs during mitotic arrest after hatching. DMRT1 is expressed in spermatogonium-supporting cells after testicular differentiation (20,30 days after hatching), and its expression is much higher than that of DMY in mature testes. In XX sex-reversed testes, DMRT1 is expressed in the Sertoli cell lineage, similar to the expression of DMY in XY testes. These results suggest strongly that DMY regulates PGC proliferation and differentiation sex-specifically during early gonadal differentiation of XY individuals and that DMRT1 regulates spermatogonial differentiation. Developmental Dynamics 231:518,526, 2004. © 2004 Wiley-Liss, Inc. [source] Increased infiltration of Chlamydophila pneumoniae in the vessel wall of human veins after perfusionEUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 7 2008K. Kupreishvili ABSTRACT Background Several studies have suggested an association between Chlamydophila pneumoniae (Cp) infection and atherosclerosis. A recent study detected Cp DNA in the saphenous vein of 12% of all patients before bypass grafting and in 38% of failed grafts. We used a system in which human veins were perfused with autologous blood under arterial pressure. Materials and methods, Veins were surplus segments of saphenous veins of coronary artery bypass grafting (CABG) patients. Vein grafts were perfused with the blood of the same patient after CABG procedures. Veins were analysed for Cp -specific membrane protein using immunohistochemical and PCR analysis. Veins were analysed before and after perfusion (up to 4 h). The number of Cp positive cells was then quantified in the vein layers. Results Cp protein was detected within macrophages only. In non-perfused veins, Cp was present in the adventitia in 91% of all patients, in the circular (64%) and longitudinal (23%) layer of the media. No positivity was found in the intima. Perfusion subsequently resulted in a significant increase of Cp positive cells within the circular layer of the media that, however, differed strongly between different patients. Cp DNA was not detected by PCR in those specimens. Conclusion Cp protein was present in 91% of veins, but the number of positive cells differed remarkably between patients. Perfusion of veins resulted in increased infiltration of Cp into the circular layer. These results may point to a putative discriminating role of Cp with respect to graft failure between different patients. [source] Expression of angiogenic factors in chronic myeloid leukaemia: role of the bcr/abl oncogene, biochemical mechanisms, and potential clinical implicationsEUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 2004C. Sillaber Abstract Chronic myeloid leukaemia (CML) is a stem cell disease characterized by an increased production and accumulation of clonal BCR/ABL,positive cells in haematopoietic tissues. The chronic phase of CML is inevitably followed by an accelerated phase of the disease, with consecutive blast crisis. However, depending on genetic stability, epigenetic events, and several other factors, the clinical course and survival appear to vary among patients. Recent data suggest that angiogenic cytokines such as vascular endothelial growth factor (VEGF), are up-regulated in CML, and play a role in the pathogenesis of the disease. These factors appear to be produced and released in leukaemic cells in patients with CML. In line with this notion, increased serum-levels of angiogenic growth factors are measurable in CML patients. In this study we provide an overview of angiogenic growth factors expressed in CML cells, discuss the possible pathogenetic role of these cytokines, the biochemical basis of their production in leukaemic cells, and their potential clinical implications. [source] Human parathyroid cell proliferation in response to calcium, NPS R-467, calcitriol and phosphateEUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 7 2001M-C. Roussanne It remains uncertain how calcium, phosphate and calcitriol regulate parathyroid cell growth. The present study was aimed at examining possible direct effects of these modulators and of the calcimimetic NPS R-467 on parathyroid cell growth in vitro. Cell proliferation was determined by [3H]thymidine incorporation and cell cycle antigen Ki 67 expression in a parathyroid cell culture model derived from uraemic patients. The effect of NPS R-467 on parathyroid hormone (PTH) secretion and intracellular [Ca2+]i response was also examined. Increasing the [Ca2+] in the medium from 0·5 to 1·7 mM increased DNA synthesis (P < 0·005) and the number of Ki 67-positive cells (P < 0·005). However, NPS R-467 (0·01,1 µM) inhibited 3[H]thymidine incorporation by 35% in the presence of 0·5 mM [Ca2+]e. Exposure of cells to Ca2+ or NPS R-467 led to a rapid increase of intracellular Ca2+, although the pattern of increase differed. Addition of calcitriol (10,10,10,7 M) to the culture medium suppressed [3H]thymidine incorporation dose-dependently. Finally, high levels of phosphate (3·5 mM) in the medium led to a significant (P < 0·05) increase in [3H]thymidine incorporation. The observed stimulatory effect of Ca2+ in the medium in vitro appears to be at variance with the inhibitory effect of calcimimetic NPS R-467 in vitro. In an attempt to solve these apparent discrepancies, and based on the notion of a reduced calcium-sensing receptor (CaR) expression in parathyroid tissues of uraemic patients, we hypothesize that Ca2+ may regulate parathyroid cell proliferation via two different pathways, with predominant growth inhibition in cases of high CaR expression or activation, but prevailing stimulation of proliferation in cases of low CaR expression. [source] Kinetics of stem cell engraftment and clearance of leukaemia cells after allogeneic stem cell transplantation with reduced intensity conditioning in chronic myeloid leukaemiaEUROPEAN JOURNAL OF HAEMATOLOGY, Issue 1 2002Karl-Anton Kreuzer Abstract: It is hypothesised that an effective graft-vs.-leukaemia reaction contributes substantially to the therapeutic effect of reduced intensity conditioning stem cell transplantation in chronic myeloid leukaemia. However, kinetic data on eradication of leukaemia cells and stem cell engraftement which could support this assumption are lacking . Thus, we investigated bcr/abl fusion transcripts and haematopoietic chimerism in 14 patients undergoing such a transplantation protocol. Ten of them obtained a complete molecular remission, and two patients achieved haematologic remissions but remained bcr/abl positive. Weekly determinations of bcr/abl transcript numbers by qualitative and quantitative polymerase chain reaction and donor chimerism revealed that 10 responders cleared bcr/abl positive cells from the peripheral blood within a median of 9 wk (range 3,22 wk). The close relation (P = 0.0075) between the firstoccurrence of graft-vs.-host disease and the complete clearance ofbcr/abl positive blood cells argues in favour of an effective graft-vs.-leukaemia reaction. [source] Frequency and prognostic relevance of cyclin D1 dysregulation in multiple myelomaEUROPEAN JOURNAL OF HAEMATOLOGY, Issue 5-6 2001Thomas Rasmussen Abstract:Objective: Cyclin D1 dysregulation has been found with varying frequencies in multiple myeloma (MM) and has been suggested to be associated with a poor prognosis. The aim of this study was to investigate the frequency of cyclin D1 dysregulation in patients being treated for MM and to test whether cyclin D1 dysregulation is a prognostic factor for MM patients. Methods: To achieve the above aims we designed a highly sensitive and reproducible real-time reverse-transcription polymerase chain reaction (RT-PCR) assay for quantitation of cyclin D1 mRNA. Using this assay, 110 diagnostic bone marrow (BM) samples from patients with MM were screened for cyclin D1 dysfuntion. Results: The real-time assay was able to detect the presence of 0.01% cyclin D1 positive cells allowing a safe detection in MM BM samples. In 42% (46/110) of MM BM samples a ,,3-fold increase in cyclin D1 mRNA was observed compared to the cyclin D1 level in normal BM. In the remaining group of MM patients the cyclin D1 mRNA levels were comparable to normal donors. Follow-up of 76 MM patients showed no significant (P = 0.35) difference in survival between cyclin D1 positive and negative MM patients. In addition, cyclin D1 dysregulation did not correlate with known prognostic factors. Conclusion: The developed real-time RT-PCR assay for detection of cyclin D1 mRNA levels offers a fast and safe screening for cyclin D1 dysfunction. When a large cohort of MM patients was screened, the cyclin D1 gene was found to be frequently dysregulated, but there was no significant correlation to survival or known prognostic parameters. [source] Extracellular lysosome-associated membrane protein-1 (LAMP-1) mediates autoimmune disease progression in the NOD model of type 1 diabetesEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 5 2005Marcelo De Carvalho Bittencourt Abstract Treatment (from 5 to 25,weeks of age) with a novel blocking monoclonal antibody, mAb I-10, directed against the plasma membrane (pm) form of LAMP-1, protected against development of autoimmune diabetes in the NOD mouse. A shorter course of treatment, i.e. from 5 to 12,weeks of age, significantly reduced the occurrence of insulitis as well as disease onset. Interfering with pm-LAMP-1 required continuous treatment as tolerance was not observed when treatment was stopped, and no higher proportion of cells with a T regulatory phenotype (e.g. CD4+CD25+) were induced. The mechanism appears to involve modulating a proinflammatory cytokine, as the proportion of pancreatic-infiltrating IFN-,-positive cells was significantly reduced in the mAb I-10-treated group. These results demonstrate an unexpected role for pm-LAMP-1 in autoimmune disease progression, and suggest that further investigation should be performed to understand how this molecule modulates IFN-,-driven responses. [source] The role of Notch and IL-7 signaling in early thymocyte proliferation and differentiationEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 4 2005Gina Balciunaite Abstract We have analyzed the roles of Notch and IL-7 signaling in the proliferation and differentiation of mouse progenitor thymocyte subpopulations cultured on Notch delta-like-1 ligand-expressing OP9 stromal cells. Using bulk and limiting dilution cultures, we show that DN1 and DN2 cells require both Notch and IL-7 signaling for efficient proliferation and differentiation into cytoplasmic TCR, and surface TCR,/, and TCR,/, expressing T cells. Selection for cytoplasmic TCR,-positive cells is dependent on preT, expression. Both ,/, and ,/, TCR expressing T cells arising in culture can be efficiently stimulated by anti-CD3 cross-linking, suggesting that they might be functional. The differentiation of adult, but not fetal, DN1 and DN2 thymocytes into CD4 and/or CD8 expressing cells is inhibited by IL-7. Finally, efficient proliferation and differentiation of DN3 cells requires Notch signaling and preTCR expression, but is independent of IL-7. [source] Involvement of mitochondrial signaling pathways in the mechanism of Fas-mediated apoptosis after spinal cord injuryEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 1 2009Wen Ru Yu Abstract Activation of the Fas receptor has been recently linked to apoptotic cell death after spinal cord injury (SCI). Although it is generally considered that Fas activation mediates apoptosis predominantly through the extrinsic pathway, we hypothesized that intrinsic mitochondrial signaling could be involved in the underlying mechanism of Fas-induced apoptosis after SCI. In the present study, we utilized the FejotaTM clip compression model of SCI at T5,6 in C57BL/6 Fas-deficient (lpr) and wild-type mice. Complementary studies were conducted using an in vitro model of trauma or a Fas-activating antibody to induce apoptosis in primary neuronal,glial mixed spinal cord cultures. After in vivo SCI, lpr mice, in comparison with wild-type mice, exhibited reduced numbers of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells at the lesion, reduced expression of truncation of Bid (tBid), apoptosis-inducing factor, activated caspase-9 and activated caspase-3, and increased expression of the antiapoptotic proteins Bcl-2 and Bcl-xL. After in vitro neurotrauma or the induction of Fas signaling by the Jo2 activating antibody, lpr spinal cord cultures showed an increased proportion of cells retaining mitochondrial membrane integrity and a reduction of tBid expression, caspase-9 and caspase-3 activation, and TUNEL-positive cells as compared to wild-type spinal cord cultures. The neutralization of Fas ligand (FasL) protected against traumatically induced or Fas-mediated caspase-3 activation and the loss of mitochondrial membrane potential and tBid expression in wild-type spinal cord cultures. However, in lpr spinal cord cultures, FasL neutralization had no protective effects. In summary, these data provide direct evidence for the induction of intrinsic mitochondrial signaling pathways following Fas activation after SCI. [source] Olfactory bulb hypoplasia in Prokr2 null mice stems from defective neuronal progenitor migration and differentiationEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 12 2007Haydn M. Prosser Abstract New neurons are added on a daily basis to the olfactory bulb (OB) of a mammal, and this phenomenon exists throughout its lifetime. These new cells are born in the subventricular zone and migrate to the OB via the rostral migratory stream (RMS). To examine the role of the prokineticin receptor 2 (Prokr2) in neurogenesis, we created a Prokr2 null mouse, and report a decrease in the volume of its OB and also a decrease in the number of bromodeoxyuridine (BrdU)-positive cells. There is disrupted architecture of the OB, with the glomerular layer containing terminal dUTP nick-end labeling (TUNEL) -positive nuclei and also a decrease in tyrosine hydroxylase-positive neurons in this layer. In addition, there are increased numbers of doublecortin-positive neuroblasts in the RMS and increased PSA-NCAM (polysialylated form of the neural cell adhesion molecule) -positive neuronal progenitors around the olfactory ventricle, indicating their detachment from homotypic chains is compromised. Finally, in support of this, Prokr2-deficient cells expanded in vitro as neurospheres are incapable of migrating towards a source of recombinant human prokineticin 2 (PROK2). Together, these findings suggest an important role for Prokr2 in OB neurogenesis. [source] A modified MPTP treatment regime produces reproducible partial nigrostriatal lesions in common marmosetsEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 4 2005Mahmoud M. Iravani Abstract Standard MPTP treatment regimens in primates result in >,85% destruction of nigral dopaminergic neurons and the onset of marked motor deficits that respond to known symptomatic treatments for Parkinson's disease (PD). The extent of nigral degeneration reflects the late stages of PD rather than events occurring at its onset. We report on a modified MPTP treatment regimen that causes nigral dopaminergic degeneration in common marmosets equivalent to that occurring at the time of initiation of motor symptoms in man. Subcutaneous administration of MPTP 1 mg/kg for 3 consecutive days caused a reproducible 60% loss of nigral tyrosine hydroxylase (TH)-positive cells, which occurred mainly in the calbindin-D28k -poor nigrosomes with a similar loss of TH-immunoreactivity (TH-ir) in the caudate nucleus and the putamen. The animals showed obvious motor abnormalities with reduced bursts of activity and the onset of motor disability. However, the loss of striatal terminals did not reflect early PD because a greater loss of TH-ir occurred in the caudate nucleus than in the putamen and a marked reduction in TH-ir occurred in striatal patches compared to the matrix. Examination of striatal fibres following a partial MPTP lesion showed a conspicuous increase in the number and the diameter of large branching fibres in the putaminal and to some extent caudatal matrix, pointing to a possible compensatory sprouting of dopaminergic terminals. In addition, these partially lesioned animals did not respond to acute treatment with L-DOPA. This primate partial lesions model may be useful for examining potential neuroprotective or neurorestorative agents for PD. [source] Neurogenesis in explants from the walls of the lateral ventricle of adult bovine brain: role of endogenous IGF-1 as a survival factorEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 2 2003M. Pérez-Martín Abstract Previous studies have shown the existence of proliferating cells in explants from bovine (Bos Taurus) lateral ventricle walls that were maintained for several days in vitro in the absence of serum and growth factors. In this study we have characterized the nature of new cells and have assessed whether the insulin-like growth factor-1 (IGF-1) receptor regulates their survival and/or proliferation. The explants were composed of the ependymal layer and attached subependymal cells. Ependymal cells in culture were labelled with glial markers (S-100, vimentin, GFAP, BLBP, 3A7 and 3CB2) and did not incorporate bromodeoxiuridine when this molecule was added to the culture media. Most subependymal cells were immunoreactive for ,III-tubulin, a neuronal marker, and did incorporate bromodeoxiuridine. Subependymal neurons displayed immunoreactivity for IGF-1 and its receptor and expressed IGF-1 mRNA, indicating that IGF-1 is produced in the explants and may act on new neurons. Addition to the culture media of an IGF-1 receptor antagonist, the peptide JB1, did not affect the incorporation of bromodeoxiuridine to proliferating subependymal cells. However, JB1 significantly increased the number of TUNEL positive cells in the subependymal zone, suggesting that IGF-1 receptor is involved in the survival of subependymal neurons. In conclusion, these findings indicate that neurogenesis is maintained in explants from the lateral cerebral ventricle of adult bovine brains and that IGF-1 is locally produced in the explants and may regulate the survival of the proliferating neurons. [source] Expression of the human Cathepsin L inhibitor hurpin in mice: skin alterations and increased carcinogenesisEXPERIMENTAL DERMATOLOGY, Issue 9 2007Markus Walz Abstract:, The serine protease inhibitor (serpin) hurpin (serpin B13) is a cross class-specific inhibitor of the cysteine protease Cathepsin (Cat) L. Cat L is involved in lysosomal protein degradation, hair follicle morphogenesis, epidermal differentiation and epitope generation of antigens. Hurpin is a 44 kDa protein which is expressed predominantly in epidermal cells. In psoriatic skin samples, hurpin was strongly overexpressed when compared with normal skin. Keratinocytes overexpressing hurpin showed increased resistance towards UVB-induced apoptosis. To further analyse the functional importance of this inhibitor, we have generated transgenic mice with deregulated Cat L activity by expressing human hurpin in addition to the endogenous mouse inhibitor. The three independent transgenic lines generated were characterized by identical effects excluding insertional phenotypes. Macroscopically, mice expressing human hurpin are characterized by abnormal abdominal fur. The number of apoptotic cells and caspase-3 positive cells was reduced after UV-irradiation in transgenic animals compared with wild-type mice. Interestingly, after chemical carcinogenesis, transgenic mice showed an increased susceptibility to develop skin cancer. Array analysis of gene expression revealed distinct differences between wild-type and hurpin-transgenic mice. Among others, differentially expressed genes are related to antigen presentation and angiogenesis. These results suggest an important role of Cat L regulation by hurpin which might be of clinical relevance in human skin diseases. [source] Generation of a transgenic mouse line expressing GFP-Cre protein from a Hoxb4 neural enhancerGENESIS: THE JOURNAL OF GENETICS AND DEVELOPMENT, Issue 2 2008Elena Rivkin Abstract Here, we describe a transgenic mouse line, in which expression of green fluorescent protein fused to Cre recombinase (GFP-Cre) is directed by the early neuronal enhancer (ENE) of Hoxb4. In E9.0,13.5 transgenic embryos, Cre activity coincided with endogenous Hoxb4 throughout the neural tube up to the r6/r7 boundary in the hindbrain, the dorsal root ganglia, and the Xth cranial ganglia. Unexpectedly, Cre activity was also consistently detected in the trigeminal (Vth) cranial nerve, which is devoid of endogenous Hoxb4 expression. Strong GFP dependent fluorescence appeared slightly later in E9.5,E11.5 embryos, and reflected the later expression pattern expected for Hoxb4-ENE directed expression in the neural tube up to the r7/r8 not r6/r7 boundary. Thus, with the exception of the trigeminal nerve, this reporter faithfully reproduces endogenous embryonic neural Hoxb4 expression, and provides an excellent reagent for in vivo gene manipulations in neuronal Hoxb4 positive cells as well as the developing trigeminal nerve. This transgenic mouse line should prove especially useful for determining the fate map of neuronal populations arising in rhombomeres 7 and 8 on its own and in combination with the small set of other existing rhombomere-specific Cre recombinase expressing lines. genesis 46:119,124, 2008. © 2008 Wiley-Liss, Inc. [source] An enhancer sequence directs LacZ expression to developing pharyngeal endoderm in transgenic miceGENESIS: THE JOURNAL OF GENETICS AND DEVELOPMENT, Issue 2 2001Hema Parmar Abstract Summary: The murine Hoxc-6 homeobox gene comprises three exons with two distinct promoters (PRI and PRII) located 9 kb apart. To characterise the PRII promoter, a region 3 kb upstream of the transcription start site was sequenced, and an Antananapedia-like consensus binding sequence was found (Coletta et al., 1991). A LacZ reporter gene construct, containing three copies of this sequence, directs highly specific expression in cells forming pharyngeal endoderm in transgenic mice. Expression was first detected in a few individual anterior endoderm cells at E7.5, which increase in number up to E9.5, where expression was clearly visible in the pharyngeal endoderm. Expression of the endodermal genes HNF3,, Pax-9, Shh, and Nkx2.5 showed colocalization with the LacZ -positive cells in the foregut and pharyngeal endoderm. This novel enhancer provides a means of tracking the morphogenetic movement of endodermal cells fated to form the foregut. genesis 31:57,63, 2001. © 2001 Wiley-Liss, Inc. [source] |