Objective To investigate whether the expression of sterile alpha motif and histidine/aspartic acid domain containing protein 1 ( SAMHD1 ) could be induced by IFN-α and mediated by microRNA-181a (miR-181a). Methods THP-1 and Jurkat cells were treated with different doses of IFN-α(200 IU/ml and 1 000 IU/ml) for 24 h. The expression of miR-181a and SAMHD1 at mRNA level were de-tected by quantitative PCR. Western blot assay was performed to measure the expression of SAMHD1 at pro-tein level. THP-1 and Jurkat cells were transfected with p-181a, an over-expression vector of miR-181a, and then treated with 200 IU/ml of IFN-α. Changes in the expression of SAMHD1 in those cells were analyzed. Results IFN-α significantly enhanced the expression of SAMHD1 at mRNA and protein levels, but inhibi-ted the expression of miR-181a, especially in Jurkat cells. The expression of SAMHD induced by IFN-αwas inhibited in cells over-expressing miR-181a. Conclusion This study suggests that IFN-α could induce the expression of SAMHD1 by down-regulating the level of miR-181a.

Objective To explore the mechanism of latent human immunodeficiency ciency virus type 1 (HIV-1) infection is unclear, especially in dendritic cells (DC).We hypothesized that DC-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) binds with HIV-1 may activate HIV-1 provirus.Methods We generated a model by transfecting 293T cells with a DC-SIGN expression plasmid and a HIV-1 5'long terminal repeat (LTR) reporter plasmid, and then stimulated the 293T cells with HIV-1 gp120 protein, wild-type HIV-1 and VSV-G-pNL4.3 pseudotype virus ( without gp120 protein).CEM-Bru cells were transfected with the DC-SIGN expression plasmid and stimulated by HIV-1 gp120 protein.Then HIV-1 replication was detected.The involvement of the ERK, p38 and NF-κB pathways signaling in this response were determined by inhibiting the pathways specifically and detecting the phosphorylation of the signaling kinase.Results The HIV-1 5'LTR was reactivated by HIV-1 gp120 in DC-SIGN-expressing 293T cells.After HIV-1 gp120 protein stimulation of the mold of CEM-Bru cells, the increasing expression of HIV-1 Tat mRNA and HIV-1 p24,which implies early and late HIV-1 provirus replication was reactivated by the HIV-1 gp120/DC-SIGN stimulation.HIV-1 gp120/DC-SIGN stimulation reactivates latent HIV-1 provirus via the NF-κB signal pathway.Conclusion HIV-1 gp120/DC-SIGN stimulation reactivates latent HIV-1 provirus via the NF-κB signal pathway.

Objective To detect the role of AP-1 or ETS-1 transcription factor binding sites (TFBS) in activity of DC-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) pro-moter. Methods Genomic DNA was extracted from peripheral blood. Complete DC-SIGN promoter and those without AP-1 or ETS-1 TFBS were amplified by PCR using designed primers. The PCR products were digested with MLu Ⅰ and Bgl Ⅱ and then ligated into MLu Ⅰ and Bgl Ⅱ digested pGL-3/Basic and pGL-3/En-hancer vectors. Transfection in Hacat and 293 cells was accomplished with Trans Fast liposome. Activity of luciferase was detected after 48 h. Results The DC-SIGN promoters without AP-1 or ETS-1 TFBS and the recombined pGL-3 vectors were correctly constructed. The activity of DC-SIGN promoters without AP-1 was reduced 20% (293) and 10% (Hacat), which was 40%-50% with enhancer. The activity of DC-SIGN pro-moters without ETS-1 was nearly vanished, no matter with or without enhancer. Conclusion ETS-1 TFBS, not AP-1 TFBS, plays an important role in activity of DC-SIGN promoter.

Objective To investigate the effect of different HAART regimens on liver function,viral load and CD4+T cells of HIV infected children with HCV co-infection.Methods A total of 40 patients were divided into 4 groups:13 cases in AZT+3TC+NVP group(group A),16 cases in AZT+3TC+EFV group(group B),5 cases in D4T+3TC+EFV group(group C)and 6 cases in D4T+3TC+NVP group(group D).Liver function,viral load and CD4+T cells of patients in 4 groups were compared between before and after HAART.Results Eight patients(20％)had abnormal liver function before HAART treatment,and all of them had 1 level of liver damage.After receiving HAART,levels of ALT and AST were increased in group A and B(P<0.01);AST levels were increased in group C(P<0.01).CD4+T counts were all increased and HIV viral loads were decreased in 4 groups(P<0.01).There was no significant difference in RNA HCV load before and after HAART treatment(P>0.05).Conclusion Different HAART regimens can increase the liver damage in HIV infected children co-infected with HCV,but have little effect on the treatment efficiency of HAART regimens.

Objective To evaluate the immune reconstitution of HIV-1 infected individuals after long-term highly antiretroviral therapy (HAART). Methods Twenty-five HIV-1 infected individuals receiving HAART, 17 without HAART and 15 healthy controls were included in the study. CD4 +T, CD8 +T,CD8/human leukocyte antigen DR (HLADR) + T, CD8/CD38 +T cells, and the expression of CD127 on CD3 +T cells from peripheral blood samples were measured by flow cytometry. IL-7 in peripheral blood was measured by enzyme-linked immunosorbort assay (ELISA) in HAART group. t test was performed to compare the measurement data among the groups. Results Before HAART, the count of CD4 + T cells in HIV-1 infected group was lower than that of the healthy control (t =9. 12, P ＜0. 01 ), while the counts of CD8 + T,CD8/HLADR+T, and CD8/CD38 +T cells were higher than those of the healthy control (t = 4.48, 4.89 and 3.88, P＜0. 01 ). Ater 7 years' antiviral therapy, CD4 +T cells increased, CD8 +T cells decreased, but both of them didn' t reach to the normal levels ( t = 2.66 and 2.43, P ＜ 0.05 ). While the counts of CD8/HLADR+T cells and CD8/CD38 +T cells almost reached to the normal levels (t = 0. 86 and 1.39, P ＞0.05). Before HAART, the concentration of IL-7 in HIV-1 infected group was higher than that in healthy controls (t =5.31, P ＜0.01 ). It decreased with HAART, but was still higher than the normal level (t =2. 81, P ＜ 0. 05 ). The expression of CD127 on CD3 + CD8 + T cells in non-HAART group was significantly lower than that in healthy control ( t =- 6.01, P ＜ 0.01 ), while that in HAART group was higher ( t = 2.32,P ＜0.05), but still not reached to the normal level ( t = 4.49, P ＜ 0. 05 ). CD127 expression on ( CD45RA + ) CD3 + CD8 + T cells almost increased to the normal level ( t= 0. 28, P ＞ 0. 05 ), while that on ( CD45RO + ) CD3 + CD8 + T cells was still remarkably lower than the normal ( t = 4. 86, P ＜ 0. 05 ). Conclusion Long-term HAART can partially restore the count and function of lymphocyte subsets in HIV-1 infected individuals, and the abnormal immune activation can be inhibited.

Objective To detect the signal pathways through which IL-4 regulates expression of DC-SIGN in THP-1 cells.Methods We used phorbol 12-myristate 13-acetate(PMA) differentiated THP-1 cells as the in vitro model of monocyte/macrophage cells to study the signal pathways involved in IL-4 regulated expression of DC-SIGN.DC-SIGN mRNA expression was detected by RT-PCR.Cytoplasmic DC-SIGN protein was tested by Western blot.Flow cytometry was used to detect cell surface expression of DC-SIGN.Cytoplasm and nuclear protein of PMA stimulated THP-1 cells induced by IL-4 for 0,10,20,30,60 and 120 min was extracted and detected by Western blot for signal pathway signaling protein and phosphoprotein.Results We found that a high expression of DC-SIGN could be induced by IL-4 at the levels of mRNA and cell surface protein.Up-regulated expression of DC-SIGN was almost completely blocked by the specific inhibitor of ERK pathway,and partly reduced by the specific inhibitors of JAK-STAT and NF-κB pathways.The activation of the three signaling pathways was directly confirmed by testing the phosphorylation of protein kinase within the cytoplasm and nucleus over time.Conclusion Multiple signaling pathways are involved in IL-4 induced high expression of DC-SIGN on THP-1 cells,in which ERK pathway is the main signal pathway.

Objective To explore the effects of signaling pathways inducing activation of DC-SIGN promoter on the activity of HIV-1 5'LTR.Methods The sequences of DC-SIGN promoter and HIV-1 5'LTR were amplified by PCR and then cloned into pGL-3/Basic plasmid to constructluciferase reporter plasmids for DC-SIGN promoter and HIV-1 5'LTR.Differentiated THP-1 cells stimulated by PMA (phorbol myristate acetate) were used as the in vitro model of DCs.The activaties of DC-SIGN promoter and HIV-1 5'LTR induced by IL-4 in differentiated THP-1 cells were studied using luciferase reporter plasmids.The signaling pathways were identified by using specific inhibitors.Results IL-4 induced signaling pathways could increase the activities of HIV-1 5'LTR and DC-SIGN promoter for more than two times in THP-1 cells transfected with luciferase reporter plasmids.However,the activity of HIV-1 5'LTR was weaker than that of DCSIGN promoter.ERK/JAK-STAT/NF-κB signal pathway blockers could inhibit the luciferase activity driven by DC-SIGN promoter,of which ERKI/2 blocker showed the strongest inhibitory effect that almost completely blocked IL-4 induction.NF-κB blocker had a significant inhibitory effect on HIV-1 5'LTR activity at a rate of 52.32％,followed by the ERK blocker at a rate of 43.31％.Conclusion This study suggested that IL-4-induced signaling pathways mediate the activation of DC-SIGN promoter and HIV-1 5'LTR through NFκB and ERK.

Autoimmune hepatitis (AIH) affects all age group and occurs mainly in women. Pyroptosis is a novel programmed cell death featured with cell bursting and release of proinflammatory cytokines. A deeper understanding of AIH pathogenesis will contribute to novel therapy for AIH patients. Here, we aimed to investigate the role of IL-17 in immune-mediated liver injury.The levels of cytokines were measured by ELISA, and mRNA levels of STAT3 and IFN gammainducible protein 16 (IFI16) were detected by PCR. Expressions of STAT3, IFI16, gasdermin D and cleaved caspase-1 were measured by western-blotting. Immunohistochemical staining and transmission electron microscopy were applied to evaluate liver histopathological changes of the treated mice. Our results showed that the levels of IFI16 was increased in hepatocytes treated with IL-17 protein, and further elevated after STAT3-overexpressed (STAT3-OE) lentivirus treatment. The levels of IFI16 were reduced in hepatocytes treated with IL-17 neutralizing Ab (nAb), but were significantly increased after STAT3-OE treatment. Pyroptosis was observed in hepatocytes treated with IL-17 protein, and further cell damage was observed after STAT3-OE lentivirus treatment. Liver damage was alleviated in mice treated with IL-17 nAb, however sever damage was experienced after STAT3-OE lentivirus treatment. A binding interaction between IFI16 and STAT3 was detected in IL-17 treated hepatocytes. Glutathione transaminase activity was enhanced in concanavalin A-induced AIH mice compared to the control group (p<0.01). IL-17 plays an important role in activating STAT3 and up-regulating IFI16, which may promote the pyroptosis in AIH-related liver injury through STAT3-IFI16 axis.