Analysis of influencing factors on hepatic encephalopathy in patients with non-cirrhotic portal hypertension / 中华肝脏病杂志

Objective@#To investigate the incidence of hepatic encephalopathy (HE) in patients with non-cirrhotic portal hypertension (NCPH) and to explore its risk factors.@*Methods@#The incidence rate of HE in 150 cases with NCPH was evaluated in two hospitals, and 188 cases of compensated cirrhosis patients were taken as control. Logistic regression was used to screen for independent risk factors for HE in patients with NCPH.@*Results@#The incidence of overt hepatic encephalopathy (OHE) in patients with NCPH was not statistically significantly different from that in patients with cirrhosis (4.7% vs. 6.9%, P = 0.682). The incidence of mild hepatic encephalopathy (MHE) was significantly lower than that of cirrhosis patients (32.7% vs. 46.3%, P < 0.05). The presence of upper gastrointestinal bleeding, infection and portosystemic venous shunt were the main independent factors for HE in NCPH patients (OR > 1, P < 0.05).@*Conclusion@#HE is one of the important complications of NCP, and may be influenced by factors such as upper gastrointestinal bleeding, infection and portosystemic venous shunt.

Immunoregulation effect of TIGIT-Fc on macrophages / 中国免疫学杂志

Objective:To prepare the production of TIGIT-Fc fusion protein using H22 cells stably integrated the gene by lentivirus vector , and to explore the immunoregulatory effect on macrophages by TIGIT-Fc.Methods: TIGIT-Fc fusion gene were constructed by molecular cloning.The fusion gene was then subcloned to plasmids contained the secretion signaling peptide .The secrected TIGIT-Fc fusion gene was inserted into the lentivirus backbone vector.The purified lentivirus vector was the used to infect the murine H22 cell line.TIGIT-Fc protein was purified by protein A column from the ascites of H 22-injected C57BL/6 mice.Macrophages stimulated by lipopolysaccharide ( LPS ) was challenged to TIGIT-Fc treatment or control.Cytokine levels was then detected by ELISA.Results: TIGIT-Fc protein was purified from the ascites of H 22-injected mice.PVR was upregulated in LPS-treated macrophages.IL-10 level was upregulated in TIGIT-Fc treated macrophages.Conclusion: TIGIT-Fc promotes the mature macrophages to secrete anti-inflammatory cytokine IL-10.

Apoptosis of Tim-3~- T cells induced by secretion of galectin-9 in mice / 中华微生物学和免疫学杂志

Objective To prepare recombinant adenovirus pAd-gal-9 containing murine galectin-9 and explore galectin-9's pro-apoptotic effect on T lymphocytes. Methods The recombinant adenovirus plas-mid pAd/CMV/V5-DEST-gal-9 was prepared by conventional molecular cloning and LR reaction. The pAd/ CMV/V5-DEST-gal-9 linearlized by Pac I was transfected into 293A cells with Lipofectin 2000. Eight days after transfection, the 293A cells were subjected to freeze/thraw circle for three times and the supernatant was collected after centrifugation. Higer titer pAd-gal-9 was produced by large-scale infection of 293A cells with the supernatant containing pAd-gal-9. The supernatant was condensed to get purified pAd-gal-9 by CsCl density gradient centrifugation. After titer determination with gradient dilution of harvested pAd-gal-9 infec-tion in 293A-seeded 96-wells, pAd-gal-9 was used to infect the CHO cell line. Immunohistological assay, Western blot and flow cytometry were employed to ascertain the subcellular location expression of galectin-9. We added solid-phase transgenic CHO cells or freshly-cultured supernatant to medium containing activated T cells to detect the pro-apoptotic effect of galectin-9. Results The pAd-gal-9 was prepared successful. Im-munohistochemical staining of CHO infected with pAd-gal-9 confirmed that galectin-9 was expressed in the cytosol. Intercellular staining indicated that mean fluorescence intensity of galectin-9 was significantly higher in pAd-gal-9-infected CHO group than control group. Supernatant from pAd-gal-9-infected CHO promoted the apoptosis of T cells. The percent of apoptotic T cells was higher than the Tim-3 positive T cells. Conclu-sion CHO infected with pAd-gal-9 can secret galectin-9 to promote the apoptosis of activated T cells via Tim-3-independent mechanisms.

Changes of Tim-3 expression In T lymphocytes from different sites in mice heart-transplant recipients / 中华器官移植杂志

Objective To explore the expression level of Tim-3,the marker of activated T_H 1 cells.in T lymphocytes in different sites from recipients with acute rejection.Methods The model of cervical heterotopic heart transplantation was established in mice Two groups were get up:the isograft group(C57BL/6→C57BL/6) and the allograft group (Balb/c→C57BL/6).Lymphocytes were isolated from peripheral blood,spleens,draining lymph nodes and grafts 3 or 6 days after transplantation.The expression of TIM-3 in CD4~+ and CD8~+ T subsets was detected by flow cytometry.Results There was no significant difference in Tim-3~+/CD4~+ and Tim-3~+/CD8~+ ratio in peripheral blood or spleens between two groups.As compared with the isograft group,the proportion of Tirn-3~+/CD4~+ cells was slightly elevated in draining lymph node(P<0.05),but the percentage of Tim-3~+/CD4~+ cells had no significant change between 3 days and 6 days in the allograft group(P>0.05).The expression of Tim-3 in CD4~+ and CD8~+ of graft infiltrating T cells was obviously increased in allograft group(P<0.01),and it was significantly (P<0.01) up-regulated on the 6th day as compared with that on the 3rd day.Conclusion The dynamic changes of Tim-3 expression in T lymphocytes in draining lymph node and graft were correlated with the progresston oi acute rejection in mice.

Characterization of acute renal allograft rejection by human serum proteomic analysis / 华中科技大学学报（医学）（英德文版）

To identify acute renal allograft rejection biomarkers in human serum, two-dimensional differential in-gel electrophoresis (2-D DIGE) and reversed phase high-performance liquid chromatography (RP-HPLC) followed by electrospray ionization mass spectrometry (ESI-MS) were used. Serum samples from renal allograft patients and normal volunteers were divided into three groups: acute rejection (AR), stable renal function (SRF) and normal volunteer (N). Serum samples were firstly processed using Multiple Affinity Removal Column to selectively remove the highest abundance proteins. Differentially expressed proteins were analyzed using 2-D DIGE. These differential protein spots were excised, digested by trypsin, and identified by RP-HPLC-ESI/MS. Twenty-two differentially expressed proteins were identified in serum from AR group. These proteins included complement C9 precursor, apolipoprotein A-IV precursor, vitamin D-binding protein precursor, beta-2-glycoprotein 1 precursor, etc. Vitamin D-binding protein, one of these proteins, was confirmed by ELISA in the independent set of serum samples. In conclusion, the differentially expressed proteins as serum biomarker candidates may provide the basis of acute rejection noninvasive diagnosis. Confirmed vitamin D-binding protein may be one of serum biomarkers of acute rejection. Furthermore, it may provide great insights into understanding the mechanisms and potential treatment strategy of acute rejection.

Galectin-9(Tim-3L)significantly attenuates allogeneic immune response in mice / 中华微生物学和免疫学杂志

Objective To explore the subcellular localization of Galectin-9 and its effect on allogeneic immune response.Methods The plasmid pEGFP-N1 was inserted with Galectin-9 fragment which was amplified from pBKCMV-Galectin-9 by PCR.The recombinant plagmid wag then transfected into CHO cells using JetPEI in vitro.The cells were cultured in G418 selecting mediam to obtain the stably-transfected cells.The transcription and expression of Galectin-9 gene were verified by immunohistochemical staining and RT-PCR.The solid-phase transgenic CHO cells or freshly-cultured supernatant wag added into the mixed lymphocyte response system to detect the inhibitory effect of Galectin-9.Galectin-9 protein wag administered intraperitoneally for 7d consecutively.Results The expression of Galectin-9 wag localized in the cytosol of CHO.The allogeneic mix lymphocyte proliferation was dose-dependently inhibited by the freshly-cultured supernatant from stably-transfected CHO cells.Furthermore,the supernatant from stably-transfected CHO cells dose-dependently inhibited the level of IL-2.The inhibitory effect could be reversed by Tim-3-Fc blocking.Administration of Galectin-9 significantly prolonged the survival of allogeneic cardiac transplants[(22.7±1.2)d vs(7.2±0.4)d)].Conclusion Galectin-9 may be secreted in physical situation to exert its immunomodulatory function on allogeneic immune response.Furthermore.Galectin-9 may be a novel therapeutic drug in transplant medicine.

Transfer of regulatory T cells induced by TGF-β1prolonged the skin-graft survival in mice / 中华微生物学和免疫学杂志

Objective To study the probability of transferring the regulatory T cells induced by TGF-β1 to prolong the allograft survival and the mechanisms involved.Methods According to the different culture conditions.three experimental groups were established:control group(T cells from C57 BL/6 mice cultured with II-2),MLR group(T cells from C57BL/6 mice activated by alloantigen)and TGF-βgroup(T cells from C57BL/6 mice activated by alloantigen and cultured with 5.0 ng/ml TGF-β1).After the culture,the ratio of CI4+CD25+T and the Foxq3 expression were measured by FACS and RT-PCR,respectively.On 9th day,the pathologic analysis was performed and the ratios of TH1,TH2 and Treg and the proliferation of lymphocytes were measured.Results The ratio of CD4+CD25+T in TGF-β group was higher than that in control group and MLR group(P<0.05),and Foxp3 was expressed in CD4+CD25+T cell from TGF-βgroup.After transferring ofthe cells,the allografi survival time in TGF-β group was prolonged and its mean survival time(MST)was(22.8±1.9)d,which was longer than that in MLR group and control group (P<O.05),but MST of MLR group was(9.4 ±1.3)d,which was shorter than that of control group(P<0.05).In TGF-β group,the ratio of THl was lower than that of control group and MLR group(P<O.05), but the ratio of TH2 was similar with MLR group(P>0.05),lower than that of control group(P<0.05). And the ratio of CD4+CD25+T in TGF-β group was higher than that in control and MLR group obviously(P<O.05).In addition,the proliferation of lymphocytes in TGF-β group was impaired.Conclusion TGF-β1 could induce na(i)ve T cens to difierentiate into regulatory T cells.After transferring these cells in mice skin transplant model,the ratio of CD4+CD25+Treg waS increased,and the differentiation of TH1 and TH2 and the proliferation of lymphocytes were inhibited.Finally.the allograft survival time was prolonged successfully.