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Isolated Lymphocytes (isolated + lymphocyte)
Selected AbstractsDNA damage and repair measurements from cryopreserved lymphocytes without cell culture,A reproducible assay for intervention studiesENVIRONMENTAL AND MOLECULAR MUTAGENESIS, Issue 7 2006Jyh-Lurn Chang Abstract Single-cell gel electrophoresis (the Comet assay) can be used to measure DNA damage and DNA repair capacity (DRC). However, to test DRC of cryopreserved lymphocytes, published methods include steps for cell culturing and phytohemagglutinin stimulation, which may limit use of this assay in intervention studies. We developed a modified Comet assay protocol that allows us to measure DRC from cryopreserved lymphocytes without these in vitro manipulations. Assay reproducibility was evaluated by performing the assay six times on different dates using six aliquots from one blood draw of one individual. The interindividual variation was assessed by performing the assay using one aliquot from six individuals. When ,-irradiation was used as the mutagen, intra-assay coefficients of variation (CVs.) for baseline DNA damage, damage after ,-irradiation exposure, and DRC,measured as tail moment,were 8, 31, and 10%, respectively. Interindividual CVs. were higher. When H2O2 was used as the mutagen, intra-assay CVs. for damage measurements were lower for a protocol modification that included damage and repair at 37°C (CVs. ranging from 8 to 35%) than for the more standard 4°C protocol. Analyzing moment arm,the average distance of DNA migration within the tail,yielded similar results. DNA repair was successfully detected in each experiment. Comparing freshly isolated lymphocytes to cryopreserved lymphocytes from the same individuals' blood draw indicated that DRC was highly correlated when determined using moment arm values. This modified protocol extends the use of the Comet assay to measuring DRC in intervention studies (e.g., dietary interventions) in that it assesses cellular response after cryopreservation without cell culture or other extensive manipulation. Environ. Mol. Mutagen., 2006. © 2006 Wiley-Liss, Inc. [source] Intrinsic genetic instability of normal human lymphocytes and its implication for loss of heterozygosityGENES, CHROMOSOMES AND CANCER, Issue 4 2001Arnolda G. de Nooij-van Dalen A combination of flow cytometry and microsatellite analysis was used to investigate loss of expression of HLA-A and/or HLA-B alleles and concurrent LOH at polymorphic chromosome 6 loci both in freshly isolated lymphocytes (in vivo mutations) and in lymphocytes cultured ex vivo. The fraction of in vivo mutants that showed LOH at 6p appeared to vary from 0%,49% for various donors. During culturing ex vivo, HLA-A, cells arose at a high rate and showed simultaneous loss of expression at the linked HLA-B locus. Up to 90% of the ex vivo arisen HLA-A2, cell population showed LOH of multiple 6p markers, and 50% had lost heterozygosity at 6q. This ex vivo spectrum resembles that found in HLA-A2 mutants obtained from lymphoblastoid cells. The HLA-A2 mutants present in vivo may reflect only a small fraction of the mutants that can be detected ex vivo. In normal lymphocytes, in vivo only mitotic recombination appears to be sustained, indicating the importance of this mechanism for tumor initiation in normal cells. Although mutations resulting in LOH at both chromosome 6 arms were shown to result in nonviable cells in normal lymphocytes, they have been shown to result in viable mutants in lymphoblastoid cells. We hypothesize that these types of mutations also occur in vivo but only survive in cells that already harbor a mutated genetic background. In light of the high rate at which these types of mutations occur, they may contribute to cancer progression. © 2001 Wiley-Liss, Inc. [source] Natural Killer Cell Receptor+ T-Lymphocytes in Normal and Helicobacter pylori -Infected Human Gastric MucosaHELICOBACTER, Issue 6 2008Joan O'Keeffe Abstract Background:,Helicobacter pylori infection is associated with development of chronic inflammation and infiltration of immune cells into the gastric mucosa. As unconventional T-lymphocytes expressing natural killer cell receptors are considered to play central roles in the immune response against infection, a study investigating their frequencies in normal and H. pylori -infected gastric mucosa was undertaken. Materials and Methods:, Flow cytometry was used to quantify T-cells expressing the natural killer cell markers CD161, CD56, and CD94 in freshly isolated lymphocytes from the epithelial and lamina propria layers of gastric mucosa. Thirteen H. pylori -positive and 24 H. pylori -negative individuals were studied. Results:, CD94+ T-cells were the most abundant (up to 40%) natural killer receptor-positive T-cell population in epithelial and lamina propria layers of H. pylori -negative gastric mucosa. CD161+ T-cells accounted for about one-third of all T-cells in both compartments, but the lowest proportion were of CD56+ T-cells. Compared with H. pylori -negative mucosa, in H. pylori -infected mucosa the numbers of CD161+ T-cells were significantly greater (p = .04) in the epithelium, whereas the numbers of CD56+ T-cells were lower (p = .01) in the lamina propria. A minor population (< 2%) of T-cells in both mucosal layers of H. pylori -negative subjects were natural killer T-cells, and whose proportions were not significantly different (p > .05) to those in H. pylori -infected individuals. Conclusions:, The predominance, heterogeneity, and distribution of natural killer cell receptor-positive T-cells at different locations within the gastric mucosa reflects a potential functional role during H. pylori infection and warrants further investigation. [source] Proteomic analysis of proteins differentially expressed in uterine lymphocytes obtained from wild-type and NOD mice,JOURNAL OF CELLULAR BIOCHEMISTRY, Issue 2 2009Cui Li Abstract Non-obese diabetic (NOD) mice exhibit impaired fertility and decreased litter size when compared to wild type (WT) mice. However, it is unclear why allogeneic pregnant NOD mice are prone to spontaneous embryo loss. Herein, two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) were used to detect differentially expressed proteins in the uterine lymphocytes isolated from these mice and WT BALB/c controls. We found 24 differentially expressed proteins. The differential expression of 10 of these proteins was further confirmed by Western blot analysis. Out of the 24 identified proteins, 20 were expressed in uterine lymphocytes of WT mice at a level at least 2 times higher than in NOD mice, whereas 4 were down-regulated. Western blot analysis confirmed that 8 proteins were up-regulated and 2 proteins were down-regulated in WT mice compared with NOD mice, consistent with the results of 2-DE and MS. Additionally, most of the highly expressed proteins in WT uterine lymphocytes were expressed at a significantly lower level in the corresponding splenic group (17/20). These results suggest that up-regulated expression of these proteins may be specific to uterine lymphocytes. Reported functions of the highly expressed proteins affect key functions during pregnancy, including cell movement, cell cycle control, and metabolisms. Finally, we analyzed the constitutional ratio of CD3+ and CD49b+ cells in the isolated lymphocytes by flow cytometry. Our results suggest that the differentially expressed proteins may participate in the modulation of embryo implantation and early-stage development of embryos, and subsequently influence pregnancy outcome. J. Cell. Biochem. 108: 447,457, 2009. © 2009 Wiley-Liss, Inc. [source] Liquid chromatography,tandem mass spectrometry method for simultaneous determination of cyclosporine A and its three metabolites AM1, AM9 and AM4N in whole blood and isolated lymphocytes in renal transplant patientsJOURNAL OF SEPARATION SCIENCE, JSS, Issue 15 2010Hana Brozmanová Abstract A LC-MS/MS method was developed and validated for the determination of cyclosporine A (CsA) and its three phase 1 metabolites AM1, AM9, and AM4N in whole blood and lymphocytes isolated on the Histopaque gradient. 200,,L of whole blood was precipitated with 10,mol/L zinc sulfate in acetonitrile/methanol (40:60, v/v) and lymphocytes isolated from 1.5,mL blood were extracted with acetonitrile/methanol (40:60, v/v). The analytes and internal standard cyclosporine D were separated on RP column BEH C18, 2.1×50,mm, 1.7,,m using gradient LC-MS/MS analysis in positive electrospray mode. Time of analysis was 5,min. Linearity in blood was 5,2000,,g/L for CsA, AM1, and AM9; 2,500,,g/L for AM4N; and 2,500,,g/L for all substances in lymphocytes. Coefficient of variations was 1.8,9.8% and recovery was 92.0,110.0%. The method was used in early and chronic renal transplant patients for therapeutic drug monitoring of CsA to compare either its share in lymphocytes as target organ or binding to one lymphocyte. The same parameters were calculated for all metabolites tested. [source] Identification of the testis-specific protein 10 (TSGA10) as serologically defined tumour-associated antigen in primary cutaneous T-cell lymphomaBRITISH JOURNAL OF DERMATOLOGY, Issue 3 2005S.M. Theinert Summary Background, The number of identified tumour-associated antigens for cutaneous lymphoma is still very restricted, which limits the elucidation of the tumour immunology of these malignancies and the development of specific immunotherapies and immunodiagnostics. Objectives, To identify new serologically defined antigens associated with cutaneous lymphoma. Methods, A phage expression library of the human testis transcriptom was established and immunoscreened with sera from 100 patients with cutaneous lymphoma and nine with parapsoriasis, and 81 age-matched control donors. Positive expression clones were sequenced to identify the respective antigen. Results, The testis-specific protein 10 (TSGA10) was identified as an antigen recognized by sera of two patients with Mycosis fungoides but not by sera from healthy donors. By reverse transcription,polymerase chain reaction analysis, TSGA10 was found expressed in all cutaneous lymphoma samples tested, various tumour cell lines, testis, peripheral blood mononuclear cells, skin, isolated lymphocytes, keratinocytes and fibroblasts. TSGA10 overexpression had previously been reported for other cancers. Conclusions, TSGA10 is a new tumour-associated antigen of cutaneous lymphoma. [source] High-potentiality preliminary selection criteria and transformation time-dependent factors analysis for establishing Epstein,Barr virus transformed human lymphoblastoid cell linesCELL PROLIFERATION, Issue 6 2006I.-C. Chang Techniques for optimal infection of the lymphocytes are vital for the establishment of a human biobank. The present study found that more than half (58,86%) of such established LCLs had transport times of less than 48 h, cell densities exceeding 106 cells/ml and cell viabilities greater than 90%. After EBV infection, 3306 freshly isolated lymphocytes required 30.0 ± 0.1 days to become LCLs. Conversely, 1210 cryopreserved lymphocytes required 36.2 ± 0.4 days. Cell density and viability of the culture affected transformation time in freshly isolated lymphocytes. On the other hand, blood transport time, cryopreservation time and initial cell viability were major factors in cryopreserved specimens. These results contribute to general information concerning the establishment of a human biobank for EBV infected cells. [source] | |||||||||||||