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Lymphocytes Isolated (lymphocyte + isolated)
Selected AbstractsDNA damage and repair capacity in lymphocytes from obstructive sleep apnea patientsENVIRONMENTAL AND MOLECULAR MUTAGENESIS, Issue 9 2007Konstantina Kontogianni Abstract Obstructive sleep apnea (OSA) syndrome is a respiratory disease that is linked to heart attacks and high blood pressure. In the present study, we used the Comet assay to compare basal DNA damage and DNA damage induction by hydrogen peroxide, ethanol, and ,-irradiation in lymphocytes from 35 OSA patients and 35 controls. We also measured the apoptosis and necrosis produced by these agents and the ability of the lymphocytes to repair the induced DNA damage. It was found that lymphocytes isolated from OSA patients had higher basal levels of DNA damage and were more sensitive to the effects of the DNA-damaging agents than lymphocytes from controls. OSA patients also had a reduced capacity to repair the DNA damage induced by the three agents, but apoptosis and necrosis were similar in OSA patients and the controls. Environ. Mol. Mutagen., 2007. © 2007 Wiley-Liss, Inc. [source] Expression of individual immunoglobulin genes occurs in an unusual system consisting of multiple independent lociEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 9 2004Donna Abstract Humoral immunity is effected through the rearrangement of immunoglobulin (Ig) genes in individual somatic cells committed to the B,lymphocyte lineage. Haplotype or allelic exclusion restricts B,lymphocytes to the expression of a single Ig receptor that can sustain further somatic modification. In most species, a specific Ig chain is encoded at a single genetic locus. However, in cartilaginous fish, hundreds of independent Ig heavy- (IgH) and Ig light-chain (IgL) gene loci are present, many of which are joined in the germ line. Ig gene transcripts have been amplified from single peripheral blood lymphocytes isolated from the clearnose skate (Raja eglanteria) using reverse-transcription PCR, and a single productive IgH transcript was detected in the majority of cells analyzed. Similarly, only a single IgL transcript was detected in over half of the individual cells. Taken together, these findings suggest that a mechanism for haplotype exclusion arose early in the evolution of antibody diversity and is independent of a single genetic locus. [source] Characterization of the migration of lung and blood T cells in response CXCL12 in a three-dimensional matrixIMMUNOLOGY, Issue 4 2010Caroline E. Day Summary The ability of T cells to microlocalize within tissues, such as the lung, is crucial for immune surveillance and increased T-cell infiltration is a feature of many inflammatory lung conditions. T-cell migration has mainly been studied in two-dimensional assays. Using three-dimensional collagen gels to mimic the extracellular matrix of lung tissue, we have characterized the migration of T lymphocytes isolated from peripheral blood (PBT) and lung (LT) in response to interleukin-2 (IL-2) and CXCL12. Freshly isolated PBT and LT showed a low degree of migration (blood 4·0 ± 1·3% and lung 4·1 ± 1·7%). Twenty-four hours of culture increased the percentage of migrating PBT and LT (blood 17·5 ± 2·9% and lung 17·7 ± 3·8%). The IL-2 stimulation modestly increased migration of PBT after 6 days (32·3 ± 6·0%), but had no effect on the migration of LT (25·5 ± 3·2%). Twenty-four hours of stimulation with anti-CD3/CD28 caused a small but significant increase in the migration of PBT (to 36·4 ± 5·8%). In a directional three-dimensional assay, CXCL12 failed to induce migration of fresh PBT or LT. Twenty-four hours of culture, which increased CXCR4 expression of PBT 3·6-fold, significantly increased the migration of PBT in response to CXCL12. Migration of PBT to CXCL12 was blocked by pertussis toxin, but not by the phosphoinositide 3-kinase inhibitor wortmannin. Twenty-four-hour cultured LT did not respond to CXCL12. CD3/CD28-stimulation inhibited CXCL12-mediated migration of PBT. These results suggest that the migration pattern of PBT is distinct from that of LT. [source] Partial tolerance of subcutaneously transplanted xenogeneic tumour cell graft by Fas-mediated immunosuppressionIMMUNOLOGY, Issue 1 2001Takahiro Sawada Summary Certain anti-Fas antibodies, such as RMF2, induce apoptosis of Fas-expressing cells. We applied the Fas/anti-Fas system to induce killing of Fas-expressing immunocytes with resultant immunosuppression. W7TM-1 tumour cells, a rat T-cell line, were inoculated subcutaneously in BALB/c mice and tumour growth was monitored in untreated mice and in mice treated with RMF2. Prior to treatment with RMF2, we examined the expression of Fas in isolated splenocytes and in tumour-infiltrating lymphocytes by flow cytometry and immunohistochemistry, respectively. There was a remarkable increase in Fas-positive lymphocytes, including natural killer (NK) cells, among splenocytes at day 5 after tumour cell inoculation. The number of Fas-positive infiltrating lymphocytes also increased markedly, from day 5 to day 10. We then examined whether RMF2 could induce apoptosis of Fas-positive activated lymphocytes isolated from the spleen at day 5 in vitro. Terminal deoxy (d) -UTP nick end labelling (TUNEL) and Annexin V staining methods showed apoptosis of isolated cells when incubated with RMF2, and typical apoptotic features were confirmed by 4,,6-diamidino-2-phenylindole dihydrochloride (DAPI) staining. Furthermore, suppression of cellular and humoral immunity was noted in RMF2-treated mice by mixed lymphocyte reaction and assay of serum levels of immunoglobulin G, respectively. Finally, treatment of animals with RMF2 daily from day 5 to day 9 could maintain the tumour size, while the tumour mass began to diminish in untreated mice immediately after reaching a maximum size. We confirmed the enhancing effects of long-term treatment with RMF2, through the induction of immunosuppression, on the growth of unvascularized xenogeneic tumour cell grafts. [source] Gene Expression Profiling in Paget's Disease of Bone: Upregulation of Interferon Signaling Pathways in Pagetic Monocytes and Lymphocytes,,§JOURNAL OF BONE AND MINERAL RESEARCH, Issue 2 2008Zsolt B Nagy Abstract We examined the gene expression profile of genes involved in bone metabolism in 23 patients with PD compared with 23 healthy controls. We found a significant overexpression of the genes of the IFN pathway along with a downregulation of tnf-,. Our result suggest that IFN-mediated signaling may play important roles in aberrant osteoclastogenesis of PD. Introduction: Paget's disease of bone (PD) is characterized by focal regions of highly exaggerated bone remodeling and aberrant osteoclastogenesis. Under physiological conditions, circulating monocytes may serve as early progenitors of osteoclasts and along with peripheral blood lymphocytes produce a wide variety of factors important in bone metabolism. Nevertheless, little is known about the roles of circulating monocytes and lymphocytes in relation to the pathological bone turnover in PD. Materials and Methods: In this study, we aimed at investigating the gene expression pattern of PD using quantitative real-time PCR in monocytes and lymphocytes isolated from peripheral blood mononuclear cells (PBMCs). Fifteen genes known to be involved in osteoclastogenesis were studied in cells from 23 patients with PD and in cells from 23 healthy controls. Eight human genes including ifn-, (3.48-fold, p < 0.001), ifn-, (2.68-fold, p < 0.001), ifn-, (1.98-fold, p = 0.002), p38 ,2 mapk (2.47-fold, p = 0.002), ifn-,r1 (2.03-fold, p = 0.01), ifn-,r2 (1.81-fold, p = 0.02), stat1 (1.57-fold, p = 0.037), and tnf-, (,2.34, p < 0.001) were found to be significantly altered in pagetic monocytes compared with monocytes of healthy controls. Results: In pagetic lymphocytes, significant changes in the expression of ifn-, (2.17-fold, p < 0.001), ifn-, (2.13-fold, p = 0.005), ifn-, (1.89-fold, p < 0.001), ifn-,r1 (1.02-fold, p = 0.04), ifn-,r2 (1.01-fold, p = 0.031), stat2 (1.79-fold, p < 0.001), and tnf-, (,1.49, p < 0.001) were found compared with lymphocytes of healthy controls. Furthermore, IFN-, protein was significantly elevated in the sera of PD patients (18.7 ± 6.69 pg/ml) compared with healthy controls (3.87 ± 6.48 pg/ml, p = 0.042). Conclusions: In conclusion, our data suggest that novel pathways mainly related to the IFN-mediated signaling may play important roles in the aberrant osteoclastogenesis of PD. [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] Macaques co-immunized with SIVgag/pol-HIVenv and IL-12 plasmid have increased cellular responsesJOURNAL OF MEDICAL PRIMATOLOGY, Issue 4-5 2007T.M. Robinson Abstract Background, The cell mediated immune profiles following immunization with a recombinant DNA vaccine was assessed in the simian-human immunodeficiency virus (SHIV) and Macaque model. Earlier work demonstrated increased numbers of antigen specific CD8 and CD4 effector cells able to secrete IFN- ,. Method, The vaccine strategy included co-immunization of a DNA based vaccine alone or in combination with a macaque IL-12 expressing plasmid (pmacIL12). Antigen activated lymphocytes were studied for activation of a set of immunological molecules. Results, The current study demonstrates lymphocytes isolated and activated from the group that was immunized with DNA and pmacIL12 had a higher level of IFN- , producing cells. We also observed a different immunological profile when comparing the cells isolated from macaques immunized with DNA as compared to those animals that also received pmacIL12. Conclusion, The observed immune profiles are reflective of the co-delivery of pmacIL12 and demonstrates that IL-12 can increase the magnitude and polyfunctionality of the cellular immune response. [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] Silencing S1P1 Receptors Regulates Collagen-V Reactive Lymphocyte-Mediated Immunobiology in the Transplanted LungAMERICAN JOURNAL OF TRANSPLANTATION, Issue 3 2008M. Chiyo Type V collagen (col[V])-reactive lymphocytes contribute to lung transplant rejection, but the mechanisms for emigration into the graft are unknown. Sphingosine-1-phosphate-1 receptors (S1P1R) are believed to be required for lymphocyte emigration in other studies, but their role in col(V)-reactive lymphocyte rejection responses is not known. Utilizing small interfering RNA (siRNA) to reduce S1P1R expression on col(V)-reactive lymphocytes, we examined the role of S1P1R in the rejection response. Quantitative polymerase chain reaction (PCR) revealed strong expression of S1P1R messenger RNA (mRNA)on col(V)-reactive lymphocytes isolated from immunized rats. S1P1R -specific siRNA (S1P1R siRNA) reduced expression of S1P1R mRNA and protein, whereas scramble siRNA (SC siRNA) had no effect. Adoptive transfer of lymphocytes treated with S1P1R siRNA to rat Wistar Kyoto (WKY) lung isograft recipients resulted in retention of cells within the liver with fewer cells in mediastinal lymph nodes when compared to cells exposed to SC siRNA. S1P1R -deficient cells proliferated in response to alloantigens, but not in response to col(V), and produced less interferon (IFN)-, in response to col(V) compared to controls. Downregulating S1P1R did not affect production of interleukin (IL)-10and tumor necrosis factor (TNF)-,, or expression of adhesion molecules critical for migration, but prevented rejection pathology and lowered local levels of IFN-, post adoptive transfer. These data demonstrate novel roles of S1P1R, which include regulating emigration and modulating lymphocyte activation. [source] Immunostimulatory cellular responses of cured Leishmania -infected patients and hamsters against the integral membrane proteins and non-membranous soluble proteins of a recent clinical isolate of Leishmania donovaniCLINICAL & EXPERIMENTAL IMMUNOLOGY, Issue 1 2005R. Garg Summary Development of an effective immunoprophylactic agent for visceral leishmaniasis (VL) has become imperative due to the increasing number of cases of drug resistance and relapse. Live and killed whole parasites as well as fractionated and recombinant preparations have been evaluated for vaccine potential. However, a successful vaccine against the disease has been elusive. Because protective immunity in human and experimental leishmaniasis is predominantly of the Th1 type, immunogens with Th1 stimulatory potential would make good vaccine candidates. In the present study, the integral membrane proteins (IMPs) and non-membranous soluble proteins (NSPs), purified from promastigotes of a recent field isolate, Leishmania donovani stain 2001, were evaluated for their ability to induce cellular responses in cured patients (n = 9), endemic controls (n = 5) of visceral leishmaniasis (VL) and treated hamsters (n = 10). IMPs and NSPs induced significant proliferative responses (SI 6·3 ± 4·1 and 5·6 ± 2·3, respectively; P < 0·01) and IFN-, production (356·3 ± 213·4 and 294·29 ± 107·6 pg/ml, respectively) in lymphocytes isolated from cured VL patients. Significant lymphoproliferative responses against IMPs and NSPs were also noticed in cured Leishmania animals (SI 7·2 ± 4·7 & 6·4 ± 4·1, respectively; P < 0·01). In addition, significant NO production in response both IMPs and NSPs was also noticed in macrophages of hamsters and different cell lines (J774A-1 and THP1). These results suggest that protective, immunostimulatory molecules are present in the IMP and NSP fractions, which may be exploited for development of a subunit vaccine for VL. [source] | |||||||||||||