With the capability of inducing elevated expression of ACE2 (angiotensin-converting enzyme 2), the cellular receptor for severe acute respiratory syndrome coronavirus 2, angiotensin II receptor blockers (ARBs) or ACE inhibitors treatment may have a controversial role in both facilitating virus infection and reducing pathogenic inflammation. We aimed to evaluate the effects of ARBs/ACE inhibitors on coronavirus disease 2019 (COVID-19) in a retrospective, single-center study. One hundred twenty-six patients with COVID-19 and preexisting hypertension at Hubei Provincial Hospital of Traditional Chinese Medicine in Wuhan from January 5 to February 22, 2020, were retrospectively allocated to ARBs/ACE inhibitors group (n=43) and non-ARBs/ACE inhibitors group (n=83) according to their antihypertensive medication. One hundred twenty-five age- and sex-matched patients with COVID-19 without hypertension were randomly selected as nonhypertension controls. In addition, the medication history of 1942 patients with hypertension that were admitted to Hubei Provincial Hospital of Traditional Chinese Medicine from November 1 to December 31, 2019, before the COVID-19 outbreak were also reviewed for external comparison. Epidemiological, demographic, clinical, and laboratory data were collected, analyzed, and compared between these groups. The frequency of ARBs/ACE inhibitors usage in patients with hypertension with or without COVID-19 were comparable. Among patients with COVID-19 and hypertension, those received either ARBs/ACE inhibitors or non-ARBs/ACE inhibitors had comparable blood pressure. However, ARBs/ACE inhibitors group had significantly lower concentrations of hs-CRP (high-sensitivity C-reactive protein; P=0.049) and PCT (procalcitonin, P=0.008). Furthermore, a lower proportion of critical patients (9.3% versus 22.9%; P=0.061) and a lower death rate (4.7% versus 13.3%; P=0.216) were observed in ARBs/ACE inhibitors group than non-ARBs/ACE inhibitors group, although these differences failed to reach statistical significance. Our findings thus support the use of ARBs/ACE inhibitors in patients with COVID-19 and preexisting hypertension.

新冠疫情突发,在校教学无法如期开展,在线教学成为医学免疫学理论课的首选.为保证教学任务的顺利完成,本教学团队采用了多平台混合式在线授课方式,即依托腾讯课堂,开展线上面对面直播教学,同时依托慕课课堂进行预习、复习和测验,依托QQ群通讯联络.该方式保证了课程的整体性、系统性和教学质量,达到了预期教学目标.

diabetes; dendritic lipopolysaccharide; damage-associated molecular pancreatic

Objective To investigate the effects of HLA-G1 molecule on inducing human anti- murine cvtotoxic T lymphocytes (CTL) and xenogeneic cytotoxic response.Methods G1-NIT-1,a NIT cell line expressing HLA-G1,was used as stimulator in xenogeneic mixed lymphocyte-tumor cell cuhures (XMLTC) to generate human anti-murine CTL.Cytolytic activity of the CTL targeting NIT-1 cell was determined by cytotoxicity assay with MTI.Results Human anti-murine CTL raised with NIT-1 showed a similar level of cytolytic activity against NIT-1 to that against G1-NIT-1 [(50.03±18.98) % vs (52.54±17.90) %,P>0.05],while the cytolytic activity of the CTL raised with G1-NIT-1 was sig- nificantly reduced compared with the CTL raised with NIT-1-pcDNA3 [(32.21±20.52)% vs (56.41± 14.80) %,P<0.05].Conclusions HLA-C1 could indirectly inhibit the cytolytic activity of the hu- man anti-murine CTL by suppressing its generation in xenogeneic rejection.

Since the discovery of EB virus, we have accumulated more and more information about it .So for, it has been found that many diseases are related to it such as nasopharyngeal carcinoma,lymphoma,infectious monorucleosis and so on and the pathogentic roles of EB virus have been researched intensively, especially,the molecular biology specificity of EB virus, immune response and the immune evasion induced by EB viruss antigen all paly importemt roles in disease development and progression. Pathogenesis and the type of infection differ in diseases that were caused by EB virus.

AIM:To form soluble HLA-G1-peptide complex by refolding in vitro, and to study its immune function. METHODS:The heavy chain and beta(2m) of sHLA-G1 were expressed as insoluble aggregates in E. coli, and then the two subunits were refolded to form HLA-G1-peptide complex by dilution method in the presence of specific peptide. The refolded product was purified through Sephadex G-75 gel filtration. The purified product was identified by Western blot with mAb W6/32. The function of soluble HLA-G1 was explored from following three aspects, namely, the influences on cytotoxicity of NK cells, on proliferation of T cells in mixed lymphocyte culture and apoptosis of activated T cells. RESULTS:The refolded complex was recognized by mAb W6/32. It effectively inhibited cytotoxicity of NK cells and proliferation of T cells, and induced apoptosis of activated T cells. CONCLUSION:The refolding of soluble HLA-G1-peptide complex has been successfully realized in vitro. The complex can inhibit the functions of NK cells and T cells.

目的:获取原核表达的可溶性HLA-G1重链分子(heavy chain of soluble HLA-G1,sHLA-G1-hc),为进一步构建可溶性HLA-G1单体分子奠定基础.方法:应用引物点突变技术,RT-PCR扩增去信号肽的可溶性HLA-G1重链分子的cDNA序列,构建表达载体pET22b/sHLA-G1-hc,导入大肠杆菌BL21(DE3)plys,IPTG诱导sHLA-G1-hc-6(his融合蛋白表达,提取包涵体蛋白,溶解后经Ni-NTA亲和层析纯化,透析后浓缩保存.SDS-PAGE、Western blot鉴定目的蛋白的表达和纯化.结果:克隆基因序列符合重链基因特征;表达产物以包涵体形式为主,表达量占菌体总蛋白的20%;纯化后证明表达产物具有较高纯度达到95%.结论:成功构建可溶性HLA-G1重链分子原核表达载体,为阐明可溶性HLA-G1的功能奠定基础.

AIM:To improve the refolding efficiency of soluble HLA-A2-peptide complex in vitro.METHODS:The heavy chain (HC) of MHC class I was extracted from bacteria under denaturing and non-reducing conditions. Anion-exchange and (NH4)2SO4 precipitation were applied to purify the HC. Then the purified HC, beta2m and an antigenic peptide (N-YMDGTMSQV-COOH of Try(369-377)) were refolded to form an HLA-A2-peptide complex by dilution method in the buffer of pH 6.6. The refolded products were detected by Western blot and ELISA with W6/32 and anti-human beta2m antibody.RESULTS:The refolded products consisted of HLA-A2-peptide complex, beta2m, and a little amount of HC polymer. The refolding efficiency was 2.5 fold higher than that of the conventional method.CONCLUSION:This study confirmed that the refolding efficiency of the method reported in this paper is higher as compared with the conventional method, which is of importance to the preparation of HLA-peptide tetramers and artificial antigen presenting cells.

Objective To study the inhibitory effects of HLA-G1 expressed in ECV304 on the proliferation of allogeneic T cells.Methods The recombinant plasmid pcDNA3-HLA-G1 which contained a full-length cDNA of HLA-G1 was constructed, and the ECV304 cell was transfected with the pcDNA3-HLA-G1 by using the lipofectin transfection. The expressed HLA-G1 on the cell surface was checked by specific monoclonal antibody (G11E5) with indirect immunofluorescence assay and FCM. The HLA-G1 expressed in ECV304 was used as stimulator in co-culture with allogeneic T cells, to perform allogeneic T cell proliferation assay and to generate ECV304-specific alloreactive CTL. The proliferation of T cells and the CTL’s cytotoxicity against ECV304 were tested by the MTT method.Results The expression of HLA-G1 on the surface of the ECV304 was verified with the immunofluorescent staining of the pcDNA3-HLA-G1 transfected cells. The proliferation intensity of allogeneic T cells was significantly decreased after the HLA-G1 expressed in ECV304, as the stimulation index of co-culture of allogeneic T cells with plasmid pcDNA3 transfected ECV304 was 1.59±0.41, meanwhile it was 1.33±0.46 to pcDNA3-HLA-G1 transfected ECV304, with the difference being significant (P 0.05). The cytotoxicity of allogeneic CTL was also inhibited by the HLA-G1 expressed on the targets, indicated by the specific lysis of specific CTL against pcDNA3-HLA-G1 transfected ECV304, which was (52.33±4.48) %, meanwhile the specific lysis against plasmid pcDNA3 transfected ECV304 was (64.81±3.07) %, with the difference being significant (P 0.05). Furthermore, this inhibition was blocked by addition of HLA-G specific mAb (G11E5).Conclusions The expression of HLA-G1 in the ECV-304 cells can inhibit the proliferation and cytotoxicity of allogeneic T cells. Our results support the hypothesis that HLA-G1 bears the tolerance-inducing effects in allogeneic T cell reaction. It is hopeful that HLA-G1 provides an alternative tool to treat autoimmune diseases and to induce and maintain tolerance to allografts.

Objective To evaluate the effects of HLA-G on the Th1/Th2 type cytokines produced by allogeneic peripheral blood monoeuclear cells (PBMCs). Methods The recombinant plasmid pcDNA3-HLA-G1 was transfected into the ECV304 cells using the method of lipofectin transfection, and the expression of membrane-bound HLA-G1 molecule in ECV304 cells was detected by indirect immunofluores-(cence (IIF)). The HLA-G-transfected ECV304 was inactivated, and then co-cultured with normal human PBMCs. The concentration of type cytokines in the supernatants was determined by ELISA. Results The production of IL-10 was significantly increased (P 0.05) but the production of IL-4, IL-2, and IFN-γ had no significant difference in the supernatants, compared with the controls. Conclusion HLA-G may be one of the factors that can regulate cytokine balance by shifting the Th1/Th2 balance toward Th2 polarization, which is one of important mechanisms of HLA-G in inducing and maintaining immune tolerance to allogeneic antigens.

AIM:To study the effect of HLA-G1 molecule expressed by an endothelial cell line (ECV304) on the cytotoxic activity of allogeneic NK cells.METHODS:ECV304 cells were transfected with recombinant plasmid pcDNA3-HLA-G1 by the liposome transfection, and the expressed HLA-G1 on the cell surface was detected by indirect immunofluorescent assay and flow cytometry. The cytotoxic activity of allogeneic NK cells against ECV304 cells was analyzed by the MTT method.RESULTS:HLA-G1 was expressed on the surface of the transfected ECV304 cells. The specific lysis of NK cells against plasmid pcDNA3 transfected ECV304 was (50.6+/-18.1)%, while the specific lysis against pcDNA3-HLA-G1 transfected ECV304 was (29.7+/-11.4)%, which was significantly lower than the former (P<0.001).CONCLUSION:HLA-G1 expressed by the ECV304 cells can inhibit cytotoxicity of allogeneic NK cells.

OBJECTIVETo study whether the porcine endothelial cells (PECs) lines transfected by HLA-G1 can alter the lysis mediated by human peripheral blood mononuclear cell (PBMC) and natural killer cell 92 (NK-92).METHODSBy use of liposomes pack, the pcDNA3. 0 eukaryotic expression vector carrying HLA-G1 was transfected into PECs. Using indirect immunofluorescence and RT-PCR assays, the HLA-G1 expression in PECs was detected. The alteration of the lysis mediated by PBMC and NK-92 was detected by 51Cr-release assays.RESULTSHLA-G1 expression could be detected in PECs after transfection of HLA-G1 at the levels of protein and RNA. It also could be found that the survival rate of transfected PECs was much higher than that of non-transfected PECs, when both of them faced the lysis mediated by human PBMC and NK-92. After transfecting the expression of HLA-G1 could be found in the transfected PECs and the lysis mediated by PBMC and NK-92 to PECs decreased obviously (P<0.05).CONCLUSIONThe PECs transfected by HLA-G1 can decrease the NK lysis, so that it may provide us a new thought to inhibit the xeno-cell-rejection.

目的克隆和表达HLA Ⅰ类分子轻链(β2m)基因,为人工制备HLA Ⅰ类分子提供基础.方法利用逆转录PCR技术从Jeg-3细胞株中克隆β2m基因,与质粒pET22b(+)重组,构建原核表达载体pET22b(+)-β2m,转化宿主菌E coli BL21(DE3),诱导β2m基因高效表达,并采用双抗体夹心ELISA法对表达产物进行抗原性和功能鉴定.结果酶切和测序证实β2m基因克隆和载体构建成功,目的蛋白在宿主菌中高效表达于包涵体中;通过包涵体的分离和纯化获得初步纯化的β2m,所制备的β2m能够与特异性抗体结合,并能与HLA Ⅰ类分子重链形成具有天然构象的HLA Ⅰ类分子.结论人β2m基因能够在原核宿主中高效表达,表达产物具有形成HLA Ⅰ类分子的功能.

AIM To refold and biotinylate HLA-A2-peptide complex in-vitro. METHODS The BirA substrate peptide (BSP) containing H chain of HLA-A2 and beta(2m) were expressed highly as insoluble aggregates in E.coli, and then the two subunits were refolded to form an HLA-A2-peptide complex by dilution method in the presence of an antigenic peptide (NH(2)-CLGGLLTMV-COOH of EB virus latent membrane protein 2A LMP2A). Then the BirA enzyme was used to biotinylate the refolded complex. The refolded and biotinylated products were detected by ELISA and Western blot with mAb W6/32 and rabbit anti-human beta(2m) antibody and streptavidin. RESULTS The refolded complex was composed of H chain aggregate, HLA-A2-peptide complex and beta(2m). Both HLA-A2-peptide complex and the H chain aggregate could be biotinylated. CONCLUSION The refolding and biotinylation of HLA-A2-peptide complex were successfully performed and the products were confirmed by our practical immunological method. This study laid the foundation for the preparation of HLA-peptide tetramer and artificial antigen presenting cells.

In this study,the prokaryotically expressed sHLA-A*0201-BSP was used as heavy chain and β 2m as light chain to form the sHLA-A*0201 antigenic peptide complex,refolded in the presence of an synthetic EBV antigenic peptide (LMP2A 426-434 NH 2CLGGLLTMV-COOH). The conformation of the refolded complex was identified with monoclonal antibody W6/32 that recognized the natural conformations of HLA class I molecules from human cells by means of dot-ELISA and Western blotting analysis. Using the method mentioned above,a soluble HLA-A*0201-antigenic peptide complex was successfully obtained and it may offer the basis for the preparation of soluble HLA-A peptide tetramer and artificial antigen presenting cells.

我们在成功建立大鼠腹部、颈部及小鼠颈部心脏移植模型的基础上,建立了小鼠心脏移植急性排斥反应体外模型,现将结果报道如下.

OBJECTIVE:To investigate the immune tolerance inducing effects of soluble human leucocyte antigen G1 (sHLA-G1) on natural killer (NK) cells and T cells.METHODS:A recombinant plasmid expressing sHLA-G1 was constructed and transfected into human lymphoblastoid cells LCL721.221. sHLA-G1 in the supernatant was purified by immuno-affinity chromatography and then added into the culture of NK cells obtained from the peripheral blood mononuclear cells of 3 unrelated individuals. Target cells, K562 cells, were added too. The killing rate of NK was calculated. Peripheral blood lymphocytes (PBLs) were obtained and stimulated by Ebstein-Barr-virus-transformed B lymphoblastoid cell line (EBV-LCL). The proliferation of the T cells in the mixed lymphocyte culture was examined by enzyme linked immunosorbent assay. The antigen-specific T cells in the peripheral blood was activated. sHLA-G1 was added into the culture. Then the T cells were suspended in the solution of fluorescence isothiocyanate (FITC)-annexin-V. Flow cytometry was used to detect the fluorescent intensity of FITC so as to examine the apoptosis of T cells.RESULTS:sHLAG-1 inhibited the cytotoxicity of NK cells dose-dependently. sHLAG-1 inhibited the proliferation of activated T cells, and induced the apoptosis of T cells dose-dependently, with a dose-saturation character and without antigen-specificity.CONCLUSION:sHLAG-1 is a kind of immune tolerance inducing molecule.

目的:分析湖北汉族人群甘露糖结合凝集素(Mannose-Binding Lectin或Mannose-Binding Protein,MBL或MBP)基因外显子1多态性,探讨其与系统性红斑狼疮(Systemic Lupus Erythematosus,SLE)的易感性关系.方法:采用异源双链杂交技术对111例健康正常人和41例SLE患者的MBL基因外显子1多态性进行分析.结果:①共检测出两种等位基因:野生型A和变异型B(在54位密码子由GGC→GAC),未检出变异型C、D等位基因.②正常对照中A等位基因及B等位基因的基因频率分别为0.910和0.090,符合Hardy-Wernberg定律;与此前在日本人中报道的A及B等位基因频率0.767和0.233相比,变异型B的等位基因频率明显低于后者(P＜0.05).③SLE病人组中B等位基因频率为0.146,高于正常对照组,但差异无显著性意义.结论:在湖北汉族人群中MBL基因外显子1具有遗传多态性,而且与已知其他人群的分布频率有一定差异.MBL基因外显子1的遗传多态性并不与系统性红斑狼疮相关联.

甘露糖结合凝集素(mannose-binding lectin, MBL)是一种由肝脏合成和分泌的急性期蛋白,可激活补体,溶解病原菌[1],并具有调理吞噬作用.是参与非特异性免疫防御的重要分子.