0.05).Conclusions With the increase of the ejaculatory frequency,human semen volumes and the total counts decrease,while sperm viability and motility increase.Successive two ejaculation does not affect the quality of human semen.

Objective To detect the frequency of peripheral blood CD4+ CD25high Tr and CD4+CD25lowT cells and expression of programmed death-1 ( PD-1 ) on their surface in the patients with systemic lupus erythematosus(SLE) and their clinical signifcance is analyzed.Methods The expression of PD-1 on the CD4+ CD25highTr and CD4+CD25lowT cells was examined in patients with 33 active SLE,18 inactive SLE and 38 healthy controls(HC) by flow cytometry.Clinical manifestations and laboratory findings of SLE were collected.Patients were divided into two groups according to their disease activity.SLE disease activity index(SLEDAI) score≥10 was defined as high disease activity and ＜10 as inactivity.The percentage of CD4+ CD25highTr and CD4+CD25lowT cells and proportions of expression of PD-1 on their surface were compared between not only inactive or active SLE patients and healthy controls (HC),but also between patients with lupus nephritis and without lupus nephritis.Correlation with clincal manifestations and laboratory findings was analyzed.Results (1)The proportions of CD4+CD25highTr and CD4+CD25low T cells were significantly decreased in active and inactive SLE patients as compared with HC.(2)The percentage of CD4+ CD25lowPD-1 +Tr and in active and inactive SLE patients were higher than HC.The proportions of CD4+CD25lowPD-1+ T cells were significantly increased in HC and inactive SLE patients as compared with inactive SLE patients.(3) The proportions of CD4+CD25highPD-1+Tr in SLE patients with nephritis were higher than those in patients without nephritis.But the percentage of CD4+ CD25 lowPD-1 + T cells was significantly decreased in SLE patients with nephritis as compared those without nephritis.(4)A positive correlation was observed for proportions of CD4+CD25highPD-1 + T cells with SLEDAI score.Proportions of CD4+ CD25high Tr,CD4+ CD25low PD-1 + T cells was inversely correlated with SLEDAI score.Conclusion The aberrations of proportions of CD4+CD25highpD-1+,CD4+CD25lowPD-1+,CD4+CD25high and CD4+CD25low T cells were observed in patients with SLE.The abnormality of PD-1 and PD-L1 pathway may play an important role in the function and number of Tr.

Objective:To observe the stimulating effect of C main Peptide purified from Mycobacterium tuberculosis peptide antigens(Mtb Ag) on human ??T cells, and the effect of C main peptide on the Mtb Ag activated ??T cells in vitro. Methods:C main peptide was used to stimulate fresh ??T cells of normal subjects in different doses for 10 d and the responded cells were phenotyped by flow cytometry. Furthermore,C main peptide was used to restimulate the activated ??T cells ,and then analyse the expression of early activation marker molecule CD69 in ??T cells by flow cytometry and the activity of proliferation of ??T cells by MTT assay. Results:C main peptide could stimulate the proliferation of fresh ??T cells predominantly, and it also could promote the expression of CD69 in activated ??T cells and enhance its activity of proliferation. Conclusion:C main peptide purified from Mtb Ag is a kind of specific stimulator of human ??T cell in vitro.

Objective: To investigate the mechanisms of anti-tumor cytotoxicity of human ??T cells activated by Mycobacterium tuberculosis antigen (Mtb-Ag) . Methods: The peripheral blood mononuclear cells (PBMC) of healthy donors were stimulated by Mtb-Ag in vitro to activate preferentially ??T cells that were expanded in IL-2 contained medium.The high purified ??T cells were isolated by the positive selection on MACS separator. The expression of mRNA of perforin, granzyme B and Fas Ligand in purified ??T cells were measured by RT-PCR technique. Fas Ligand on ??T cells and Fas molecule on tumor cells were detected by flow cytometry. The MTT assay was used to measureanti-tumor cytotoxicity of ??T cells. The inhibitory effects of concanamycin A (CMA) for perforin/granzyme pathway, and brefeldin A (BFA) for Fas/Fas ligand pathway on cytotoxicity of ??T cells were observed. Results: The mRNA of perforin and granzyme B and Fas ligand were highly expressed in purified ??T cells. The cytotoxicity of ??T cells against K562 and PG that expressed low level of Fas molecule (6.5% and 7.8%) was remarkably inhibited by pretreatment of the ??T cells with CMA (200 nmol/L) by 72% and 76%, respectively, while the BFA(30 ?mol/L) showed less inhibitory effects by 32% and 25%, respectively; However, it was showed that the BFA (30 ?mol/L) preferentially inhibited the cytotoxicity of ??T cells against tumor cell lines Raji and A375 that expressed high level of Fas molecule on cell surface (98.5% and 70.6%, respectively).Conclusion: Preferentially, granule exocytosis was a main mechanism by which the Mtb-Ag activated ??T cells showed cytotoxicity aganist tumor cells that expressed low level of Fas molecule, whereas both Fas/Fas ligand pathway and granule exocytosis were involved in the cytotoxicicty of Mtb-Ag activated ??T cells against the tumor cells that expressed high level of Fas molecule.

Objective The aim of this study is to examine the expressions of Toll like receptor (TLR) 7 and TLR9 in the peripheral blood B lymphocytes of SLE patients and to analyze the correlation between TLR7/9 and clinical parameters. Methods lntracellular expression of TLR7/9 in the peripheral blood CD19+Blymphocytes was analyzed in 50 SLE patients and 30 healthy controls by flow cytometry. The difference of intracellular TLR7/9 expression levels in two groups was compared. Furthermore,the correlation between TLR7/9 expression and clinical parameters such as ESR, CRP, complement 3 (C3), complement 4 (CA), the level of serum IgG, anti-double stranded DNA antibody, anti-nuclear antibodies, SLEDAI score and urine protein excretion level, were analyzed. Results Compared with healthy subjects, the proportion of B cells expressing TLR7 and TLR9 was higher among SLE patients. Positive correlation was observed between TLR7 expression levels and clinical measurement of the SLEDAI and ESR. Negative correlation was observed between TLR7 expression levels and serum C3 levels. Positive correlation was observed between TLR9 expression levels and SLEDAI scores. Negative correlation was observed between TLR9 expression levels and serum C3 levels. Conclusion TLR7 and TLR9 expression is increased in the peripheral blood B cells of SLE patients, and correlates well with clinical parameters.

Objective To explore the roles of PKCθ(protein kinase Cθ)signaling pathway on the activation,proliferation and TH1/TH2 cytokines production of the γδT lymphocytes stimulated by Mycobacterium tuberculosis antigen(Mtb-Ag) in vitro.Methods Peripheral blood mononuclear cells(PBMC)were pretreated with or without Rottlerin at 5.0 μmol/L,and stimulated with Mtb-Ag and cultured in rhIL-2 containing medium.After different time of culture,activation molecules and cytokines production of γδT cells were measured by flow cytometry.The proliferation proportion and the percentage of each generation of γδT cells were determined by carboxylfluoreseein diacetate succinmidyl ester(CFSE)labeling technique and flow cytometry.Results After 3d of stimulation with Mtb-Ag,the expression rates of CD69 and CD25 of γδT cells were 46.2%and 45.6%,respectively.Pretreatment of 5.0 μmol/L Rottlerin markedly inhibited the both expressions of CD69 and CD25 in γδT cells(P＜0.01).After stimulation and expansion in vitro for 5,10,and 15 d,the percentages of the γδT cell were 9.6%,54.6%and 82.4%,respectively.There was a few γδT cells in propagation on the 5th day of culture,and almost all γδT cell divisions were above 6 generations on the 10th and 15th day of culture.Pretreatment of the Rotflerin significantly suppressed the γδT cell proliferation,but after 10 d culture,there were still a few parts of γδT cells in propagation.Meanwhile,after 7,14,and 21d of culture,upon stimulation with PMA+Ionomycin,the IFN-γ producing-γδT cells were about 80%at all times.But only after 21d culture,IL-4-producing γδT cells was 2.6%.,The percentage of IFN-γ producing γδT cells markedly reduced in Rottlerin group(P＜0.01).IL-4 secretion of the γδT cells was almost completely blocked.Conclusion PKCO signal pathway involves in activation,proliferation and differentiation of the γδT lymphocytes stimulated by Mtb-Ag.

Background. Our previous studies indicated that the increased arginine vasopressin(AVP) in ischemic brain regions of gerbils could exacerbate the ischemic brain edema. This experiments is further clarify the relation between AVP and cerebral ischemia at the molecular level. Methods. The contents of AVP, AVP mRNA, AVP immunoreactive(ir) neurons in supraoptic nucleus(SON)and paraventricular nucleus(PVN) after cerebral ischemia and reperfusion were respectively determined by radioim-munoassay(RIA), immunocytochemistry( Ⅱ C), situ hybridization and computed image pattem analysis. Results. The contents of AVP in SON, PVN were increased, and the AVP ir positive neurons in SON and PVN were also significantly increased as compared with the controls after ischemia and reperfusion. And there were very light staining of AVP ir positive neurons in the other brain areas such as suprachiasmatic nucleus (SC) and periven-tricular hypothalamic nucleus (PE), but these have no significant changes as compared with the controls. During dif-ferent periods of cerebral ischemia (30～ 120 min) and reperfusion (30 min), AVP mRNA expression in SON and PVN were more markedly increased than the controls. Condusions. The transcription of AVP gene elevated, then promoting synthesis and release of AVP in SON,PVN. Under the specific condition of cerebral ischemia and repeffusion, the activity and contents of central AVP in-creased abnormally is one of the important factors which causes ischemia brain damage.

Objective To investigate the clinical significance of abnormally expressed PD-1 on CD 4+CD 2 8+/-T cells in peripheral blood of patients with systemic lupus erythematosus ( SLE ) . Methods Peripheral blood samples were collected form 50 patients with primary SLE and 40 healthy subjects and used to isolated mononuclear cells. Expression of CD4+CD28-, CD4+CD28+, CD4+CD28+PD-1+and CD4+CD28-PD-1+T cells in peripheral blood samples of the two groups were detected by flow cytometry. Clinical data of SLE patients were collected. Based on SLE disease activity index (SLEDAI), SLE patients were classified into two groups: stable group (SLEDAI<10) and active group (SLEDAI≥10). Based on the condition of renal damage, they were also divided into two groups: lupus nephritis group and non-lupus ne-phritis group. Differences in T cell expression were compared among these groups. Statistical analysis was performed to analyze the relationships of different T cell subsets with laboratory and clinical parameters rela-ting to SLE and SLEDAI. Results The percentages of peripheral CD4+CD28-, CD4+CD28+PD-1+and CD4+CD28-PD-1+T cells of active group were higher than those of stable and healthy control groups ( P<0. 05). Moreover, patients with lupus nephritis had higher percentages of these T cell subsets than those without (P<0. 01). SLE patients who were positive for anti-dsDNA or anti-SmRNP antibody, or had de-creased complement C3, thrombocytopenia or decreased lymphocytes had higher percentages peripheral CD4+CD28-T cells than those in the corresponding negative group. SLE patients who were positive for anti-dsDNA or anti-SmRNP antibody, or had decreased complement C3, complement C4 or lymphocytes showed en-hanced expression of peripheral CD4+CD28+PD-1+T cells as compared with those in the corresponding nega-tive group. SLE patients positive for anti-dsDNA antibody, or with decreased complement C3 or lymphocytes or suffering from alopecia had higher percentages of peripheral CD4+CD28-PD-1+T cell than those in the cor-responding negative group. Differences between different groups were statistically significant (P<0. 05). Conclusion Abnormal expression of CD4+CD28-T cells and PD-1 on CD4+CD28-and CD4+CD28+T cells in peripheral blood of patients with SLE has certain correlation with laboratory parameters and clinical indicators.

Objective:To explore the association of DNA methylation with immune response to hepatitis B (HepB) vaccine in Han nationality children from Guangxi province.Methods:A total of 263 children aged 8-9 months who had completed HepB immunization program were recruited from three hospitals in Guangxi province by using unmatched case-control method. Children with the HepB surface antibody concentration(Anti -HBs)<100 mIU/ml was set as the case group and ≥100 mIU/ml as the control group. Multiplex PCR and heavy sulfite sequencing were used to treat the samples. Illumina platform was used for high-throughput DNA methylation sequencing of IFNG gene target regions and CpG sites. Unconditional logistic regression was used to analyze the association between cytosine-phospho-guanosine DNA methylation at 18 loci of IFNG gene and HepB immune response level. Results:There were 104 children in the case group and 159 in the control group. The median ( Q1, Q3) level of anti -HBs in two groups were 62.34 (30.06, 98.88) mIU/ml and 1 089.10 (710.35, 1 233.45) mIU/ml. The methylation levels of IFNG_1 gene 44 and 93 locus in the case group were higher than those in the control group ( P<0.05). The unconditional logistic regression model showed that the DNA methylation level of IFNG_1 gene at 44 ( OR=1.18, 95% CI: 1.03-1.35) and 93 ( OR=1.21, 95% CI: 1.07-1.38) locus was associated with the HepB response level. Conclusion:The changes of DNA methylation at locus 44 and 93 of IFNG_1 gene may be relevant factors affecting the response level of HepB in Han nationality children from Guangxi province.

Objective:To explore the association of DNA methylation with immune response to hepatitis B (HepB) vaccine in Han nationality children from Guangxi province.Methods:A total of 263 children aged 8-9 months who had completed HepB immunization program were recruited from three hospitals in Guangxi province by using unmatched case-control method. Children with the HepB surface antibody concentration(Anti -HBs)<100 mIU/ml was set as the case group and ≥100 mIU/ml as the control group. Multiplex PCR and heavy sulfite sequencing were used to treat the samples. Illumina platform was used for high-throughput DNA methylation sequencing of IFNG gene target regions and CpG sites. Unconditional logistic regression was used to analyze the association between cytosine-phospho-guanosine DNA methylation at 18 loci of IFNG gene and HepB immune response level. Results:There were 104 children in the case group and 159 in the control group. The median ( Q1, Q3) level of anti -HBs in two groups were 62.34 (30.06, 98.88) mIU/ml and 1 089.10 (710.35, 1 233.45) mIU/ml. The methylation levels of IFNG_1 gene 44 and 93 locus in the case group were higher than those in the control group ( P<0.05). The unconditional logistic regression model showed that the DNA methylation level of IFNG_1 gene at 44 ( OR=1.18, 95% CI: 1.03-1.35) and 93 ( OR=1.21, 95% CI: 1.07-1.38) locus was associated with the HepB response level. Conclusion:The changes of DNA methylation at locus 44 and 93 of IFNG_1 gene may be relevant factors affecting the response level of HepB in Han nationality children from Guangxi province.