Expressions of LMP-1,LMP-2A,and LMP-2B in EBV-transformed lymphoblasts
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We aim to detect differential expressions of LMP genes(LMP-1,LMP-2A,LMP-2B) in normal human lymphocytes and EBV-transformed lymphoblasts in vitro.Firstly,expression levels of LMP genes(LMP-1,LMP-2A,LMP-2B) in the two kinds of cells were detected by using real-time PCR.Then LMP-1 protein expression was detected by Western bolt in the normal lymphocytes and EBV-transformed lymphoblasts.It was found that LMP-1 mRNA expression was 863 times up-regulated in the transformed lymphoblasts,as compared with that in the normal lymphocytes(P 0.01).Consequently,the expression of LMP-1 protein in EBV-transformed lymphoblasts was higher than that in the normal lymphocytes.LMP-2A mRNA expression was 1763 times up-regulated in the transformed lymphoblasts as compared with,that in the normal lymphocytes(P 0.05);LMP-2B mRNA expression was 90078 times up-regulated in the transformed lymphoblasts as compared with that in the normal lymphocytes(P 0.05).All the result show that the expressions of LMP-1,LMP-2A and LMP-2B genes would be upward in EBV-transformed lymphoblasts.Keywords:
Lymphoblast
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Tumor-associated lymphocytes (TALs) freshly isolated from patients with cancer usually manifest reduced proliferative and cytolytic functions. To determine whether alterations in signal transduction contribute to functional impairments seen in TALs, we purified populations of T and natural killer (NK) cells by negative selection from ascites of seven patients with ovarian carcinoma. The average purity was 84 +/- 5% for CD3(+) TALs and 77 +/- 10% for CD3(-)CD56(+)CD16(+) TALs. Expression of several signal transduction molecules, including the CD3-epsilon, CD3-zeta, and FcepsilonRI-gamma chains, p56(lck) protein tyrosine kinase, and phospholipase C-gamma1, was studied in these cells using Western blotting. A marked decrease in expression of zeta and FcepsilonRI-gamma associated with CD3 or FcgammaRIIIA was observed in T or NK cells obtained from TALs, as compared to T or NK cells purified from normal peripheral blood. Expression of CD3-epsilon, as assessed using flow cytometry, Western blotting, or ELISA was also reduced in purified TAL-T cells relative to that in normal peripheral blood T cells. Surface expression of CD3 on T cells and FcgammaRIIIA on NK cells obtained from TALs was significantly decreased in comparison to normal peripheral blood lymphocytes (PBLs): the mean fluorescence intensity of CD3 was 277 +/- 18 for TAL-T (n = 7) versus 349 +/- 13 for PBL-T (n = 9) and that of CD16 was 58 +/- 1 for TAL-NK (n = 7) versus 385 +/- 55 for PBL-NK (n = 23) cells. These observations suggest a defect in assembly of T cell receptor and FcgammaRIIIA multicomponent transmembrane receptors, which are zeta and gamma dependent. In addition to alterations in expression, the function of these receptors was also modified, since cross-linking of CD3 on TAL-T and CD16 on TAL-NK cells with the respective monoclonal antibodies resulted in a pattern of protein phosphorylation that was distinct from that observed in normal PBLs. Expression of tyrosine kinase p56(lck) and its kinase activity were also depressed, while expression of phospholipase C-gamma1 appeared to be normal in most preparations of the TALs tested. In vitro proliferation of TAL-T in response to anti-CD3 monoclonal antibody and TAL-NK cells to interleukin 2 were significantly depressed as was the ability to produce IFN-gamma. In contrast, TAL-T cells were able to produce interleukin 10 at levels similar to those secreted by normal PBLs. Thus, in TALs obtained from patients with advanced ovarian cancer, alterations in expression and activity of signaling molecules were associated with reduced cellular functions such as proliferation and production of certain cytokines.
CD16
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In the present study, the expressions of B cell activating factor belonging to the tumor necrosis factor family (BAFF) and its receptors (BAFF-R and TACI) on T lymphocytes from malignant pleural effusion (MPE) were examined by fluorescence-activated cell sorting (FACS) analysis, and compared with those on the T lymphocytes from non-malignant pleural effusion (NMPE) and healthy controls. It was found that CD3 positive T lymphocytes (including CD4, CD8, and part of CD25 and CD69 positive cells) of MPE in lung cancer highly and consistently expressed the BAFF molecule, while high expressions of BAFF could only be found in phytohemagglutinin (PHA) or interleukin 2 (IL-2) induced T lymphocytes from NMPE or healthy controls. These results were consistent with the results from BAFF mRNA detection by real-time PCR. In addition, T lymphocytes from MPE expressed significantly more BAFF-R than those from NMPE or healthy controls, while the expression of TACI was increased on CD4+ T cells but decreased on CD8+ T cells when compared with controls. The Annexin/PI assay suggested that recombinant human BAFF (rhBAFF) could promote the survival rate of T lymphocytes from MPE, while the decoy receptor TACI-Fc fusion protein could promote the apoptosis rate of T lymphocytes. Cytokines in the supernatant detected by ELISA assay showed that rhBAFF could significantly upregulate the secretion of IFN-gamma in vitro, and the IFN-gamma level in the TACI-Fc-treated group resembled that of the control groups. All of these results indicated that the abnormally high expression of BAFF on T lymphocytes from MPE may play a role of anti-tumor effect.
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Objective To investigate the influence of HCMV infection on expressions of MCM2 and MCM8 in human embryonic lung fibroblastic cells(HEL),and explore the possible pathogenesis of HCMV infection.Methods Synchronized HEL cells were infected by HCMV,simultaneously set up the mock-infected group as control group.Infected HEL cells were harvested at 12,24,48,72 and 96 hours post infection(h p.i.).The expression levels of MCM2 and MCM8 mRNA in HEL cell were detected with semi-quantitative RT-PCR method.Results The statistical analysis showed the expression levels of MCM2 and MCM8 mRNA in HCMV-infected group were statistically lower than the ones in the mock-infected group at 12,24 h p.i.(P0.05),but at 48,72 and 96 h p.i.,the expression levels of MCM2 and MCM8 mRNA in HCMV-infected group were statistically higher than the ones in the mock-infected group(P0.05);The graphs of the expression levels of MCM2 and MCM8 mRNA suggested that the expression levels reached a peak at 24 h p.i.in the mock-infected group,but in HCMV-infected group reached a peak at 48 h p.i.,which markedly lagged behind the ones in the mock-infected group.Conclusion HCMV infection could induce the abnormal expressions of MCM2 and MCM8 mRNA,and delays their expressions.It is presumably one of the multiple pathways by which the virus blocks the host cell cycle progression.
Pathogenesis
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CXCL16
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Levels of intracellular bcl-2 oncoprotein have been found to be increased in leukemic cells of CD5+ B-chronic lymphocytic leukemia (CLL) patients. However, it is not clear whether bcl-2 overexpression is a peculiar feature of CD5+ B-CLL. Based on this background we carried out a quantitative flow cytometric evaluation of intracellular bcl-2 levels on leukemic cells of CD5+ and CD5- B-CLL.We assessed in flow cytometry levels of bcl-2 protein using a quantitative indirect immunofluorescence assay (QIFI kit) on samples from 46 previously untreated CD5+ B-CLL patients. Results were compared with those obtained on either normal peripheral blood B-lymphocytes or leukemic cells from 7 CD5- B-CLL patients intentionally selected for statistical comparison.A relatively homogeneous amount of bcl-2 protein which did not reflect either clinical-biological features at the time of diagnosis nor in vivo response to therapy was found. Results expressed as antibody binding capacity (ABC) accounted for a mean value of 12.2 +/- 1.5 x 10(3) molecules/cell (range, 6.4-13 x 10(3) molecules/cell). Levels of bcl-2 detected on CD5+ B-CLL leukemic cells were significantly lower than those of B peripheral blood lymphocytes from healthy donors (p = 0.0001). The same applied when comparing CD5+ and CD5- B-CLL patients (bcl-2 ABC, 8.07 +/- 0.26 x 10(3) molecules/cell vs. 12.2 +/- 1.5 x 10(9) molecules/cell; p = 0.0001).According to the role of bcl-2 in preventing apoptosis, our results indicate that differences in the pattern of expression of such an oncoprotein, might, at least in part, explain the more aggressive clinical course of CD5- B-CLL forms.
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Immunoglobulin A
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Toll-like receptors (TLR) signaling plays an important role in the B cell biology.It induces the maturation, proliferation and antibody production in response to pathogen recognition.Interleukin-1 Receptor-associated Kinase-4 (IRAK-4) is TLR downstream molecule which is of strategic importance in starting a cascade of NF-kappaB (Nuclear factor-kappaB) and MAPK (Mitogen-activated protein kinase) intracellular signaling pathways.The aim of the study was to identify the differences in IRAK-4 expression in normal and chronic lymphocytic leukemia (CLL) lymphocytes.We have also studied the IRAK-4 expression alteration upon TLR2/4 ligand stimulation in leukemic and normal B cells.We carried out Real-Time PCR and Western blot analysis of IRAK4 mRNA (RQ) and protein expression.The study were performed on the isolated CD19 + normal B lymphocytes and CLL cells (CD5 + CD19 + ) before and 30 min after (Lipopolysaccharide) LPS and (Staphylococcus aureus strain Cowan I) SAC stimulation.IRAK-4 mRNA expression was significantly higher in normal CD19 + lymphocytes as compared to leukemic CD5 + CD19 + subpopulation (RQ median 0.90 vs 0.54; p=0.012).In normal lymphocytes, LPS and SAC treatment led to significant IRAK-4 down-regulation both at mRNA (RQ median 0.90 vs 0.53 vs 0.40; p=0.03, p=0.01) and protein level, whereas generally there were no significant changes in IRAK-4 expression neither on mRNA nor protein level.No trend or significant differences in IRAK-4 expression in leukemic lymphocytes after LPS and SAC stimulation may imply the presence of alleged defect of immune tolerance in CLL.
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Null cell
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The present study reports the surprising observation that IL-2-R+ cells can be detected in fresh, unstimulated, murine spleen T cells from unimmunized mice by flow cytometry using the monoclonal anti-receptor antibody 7D4. Also, unexpectedly, these cells were found exclusively in the L3T4+Lyt-2- population by two-color fluorescence, in contrast to receptor+ cells after stimulation, in which both L3T4+Lyt-2- and Lyt-2+L3T4- cells were found. The fraction of splenic T cells bearing IL-2-R reproducibly varies twofold under non-H-2-linked genetic control, with high expression in DBA/2 and BALB/c (approximately 6-7%) and low expression in B10.D2 and C57BL/6 (3%). This correlates quantitatively with a greater responsiveness of the DBA/2 and BALB/c splenic T cells to high doses of IL-2, compared with B10.D2 T cells; twice as many B10.D2 T cells as DBA/2 T cells were required to get the same response. Studies with 23 B X D RI strains revealed that the level of IL-2-R+ cells in unstimulated spleen cells was regulated by multiple genes, very likely including at least one gene on chromosome 7, near the HBB locus. The mapping makes novel use of nonparametric (Smirnov) statistics, which we suggest may be of general usefulness in similar analyses of RI strains.
Interleukin 3
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