Betulin from Hedyotis hedyotidea ameliorates concanavalin A-induced and T cell-mediated autoimmune hepatitis in mice.

Hedyotis hedyotidea has been used in traditional Chinese medicine for the treatment of autoimmune diseases. However, the mechanisms underlying for the effect remain unknown. We previously showed that, among 11 compounds extracted from H hedyotidea, betulin produced the strongest suppressive effect on T cell activation. Here, we examined the hepatoprotective effects of betulin against acute autoimmune hepatitis in mice and the mechanisms underlying the effects. Freshly isolated mouse splenocytes were stimulated with concanavalin A (Con A, 5 µg/mL) in the presence of betulin, the cell proliferation was assessed with CSFE-dilution assay. Mice were injected with betulin (10, 20 mg·kg-1·d-1, ip) for 3 d. One hour after the last injection, the mice were injected with Con A (15 mg/kg, iv) to induce acute hepatitis. Blood samples and liver tissues were harvested at 10 h after Con A injection, and serum transaminase levels and liver histopathology were detected; serum levels of proinflammatory cytokines, hepatic T lymphocyte ratios, and functional statuses of conventional T and NKT cells were also analyzed. Betulin (16 and 32 µmol/L) dose-dependently suppressed the proliferation of Con A-stimulated mouse splenocytes in vitro. In Con A-challenged mice, preinjection with betulin (20 mg·kg-1·d-1) significantly decreased the levels of proinflammatory cytokines IFN-γ, TNF-α and IL-6, and ameliorated liver injury. Furthermore, pretreatment with betulin (20 mg·kg-1·d-1) significantly inhibited the Con A-induced activation of NKT and conventional T cells, and decreased production of proinflammatory cytokines IFN-γ, TNF-α and IL-6 in these two cell populations. Betulin has immunomodulatory effect on overly activated conventional T and NKT cells and exerts hepatoprotective action in mouse autoimmune hepatitis. The findings provide evidence for the use of H hedyotidea and its constituent betulin in the treatment of autoimmune diseases.

Serum DKK-1 level in the development of ankylosing spondylitis and rheumatic arthritis: a meta-analysis.

To explore the association of serum Dickkopf-1 (DKK-1) levels with the development of ankylosing spondylitis (AS) and rheumatic arthritis (RA) in humans, databases including PubMed, EBSCO, Springerlink, Ovid, WANFANG and China National Knowledge Infrastructure (CNKI) were searched to identify relevant studies. On the basis of rigorous inclusion and exclusion criteria, case-control studies of the relationships between serum DKK-1 levels and AS and RA published before December 2014 were enrolled. Statistical analyses were performed using Comprehensive Meta-analysis 2.0 (CMA 2.0). Seven case-control trials with a total of 300 AS patients, 136 RA patients and 232 healthy controls were included in this study. Meta-analysis results revealed that DKK-1 serum levels were significantly higher in AS patients than in normal controls (standard mean differences (s.m.d.)=0.301, 95% confidence interval (CI)=0.094-0.507, P=0.004), whereas no significant difference in DKK-1 serum levels was observed between RA patients and healthy controls (s.m.d.=0.798, 95% CI=-2.166-3.763, P=0.598). Serum DKK-1 level may be closely related to the development of AS but not of RA.

Hepatitis B virus protein X-induced expression of the CXC chemokine IP-10 is mediated through activation of NF-kappaB and increases migration of leukocytes.

Interferon-gamma inducible protein 10 (IP-10) involves inflammatory cell recruitment and cellular immune damage during virus infection. Although an increase of the peripheral IP-10 level is known in HBV-infected patients, the molecular basis of HBV infection inducing IP-10 expression has remained elusive. In the present study, we demonstrate that hepatitis B virus protein X (HBx) increases IP-10 expression in a dose-dependent manner. Transfection of the HBx-expressing vector into HepG2 cells results in nuclear translocation of NF-kappaB, which directly binds the promoter of IP-10 at positions from -122 to -113, thus facilitating transcription. The addition of the NF-kappaB inhibitor blocks the effect of HBx on IP-10 induction. In parallel, increase of NF-kappaB subunits p65 and p50 in HepG2 cells also augments IP-10 expression. Furthermore, we show that HBx induces activation of NF-kappaB through the TRAF2/TAK1 signaling pathway, leading to up-regulation of IP-10 expression. As a consequence, up-regulation of IP-10 may mediate the migration of peripheral blood leukocytes in a NF-kappaB-dependent manner. In conclusion, we report a novel molecular mechanism of HBV infection inducing IP-10 expression, which involves viral protein HBx affecting NF-kappaB pathway, leading to transactivation of the IP-10 promoter. Our study provides insight into the migration of leukocytes in response to HBV infection, thus causing immune pathological injury of liver.

[Study on the distribution of hepatitis C virus genotypes in patients visiting one methadone maintenance clinic in Wuhan].

OBJECTIVE: To study the prevalence of hepatitis C virus (HCV) infection and characteristics on molecular biology related to HCV among patients who were enrolled in a Methadone maintenance clinic in Wuhan. METHODS: Serum samples from 332 injection drug users (IDUs) were obtained and anti-HCV IgG was detected by enzyme linked immunosorbrent assay(ELISA), together with 86 anti-HCV positive specimens genotyped. A reverse transcriptase-polymerase chain reaction (RT-nPCR) assay using conserved primers deduced from the core-envelopel (C-E1) region of the HCV genome was employed to amplify a 474 bp fragment. Phylogenetic analysis of the C-E1 sequences was conducted by direct sequencing of the RT-nPCR products and alignment with determined by nucleotide sequencing followed by composition of a phylogenetic tree. RESULTS: There were 313 cases (94.3%) appeared positive anti-HCV IgG in the 332 patients from a Methadone maintenance clinic in Wuhan. It was demonstrated that there were four different subtypes of HCV in that clinic in Wuhan, including 6a--71 cases (82.5%), 3b--7 cases (8.2%), 1a--5 cases (5.8%) and 1b--3 cases (3.5%). CONCLUSION: Infection of 6a genotype HCV was predominant in patients from the Methadone maintenance clinic in Wuhan, followed by HCV 3b, 1a and 1b.

Induction of the Epstein-Barr virus latent membrane protein 2 antigen-specific cytotoxic T lymphocytes using human leukocyte antigen tetramer-based artificial antigen-presenting cells.

Cytotoxic T lymphocytes (CTLs) specific for the Epstein-Barr virus (EBV) latent membrane protein 2 (LMP2) antigen are important reagents for the treatment of some EBV-associated malignancies, such as EBV-positive Hodgkin's disease and nasopharyngeal carcinoma. However, the therapeutic amount of CTLs is often hampered by the limited supply of antigen-presenting cells. To address this issue, an artificial antigen-presenting cell (aAPC) was made by coating a human leukocyte antigen (HLA)-pLMP2 tetrameric complex, anti-CD28 antibody and CD54 molecule to a cell-sized latex bead, which provided the dual signals required for T cell activation. By co-culture of the HLA-A2-LMP2 bearing aAPC and peripheral blood mononuclear cells from HLA-A2 positive healthy donors, LMP2 antigen-specific CTLs were induced and expanded in vitro. The specificity of the aAPC-induced CTLs was demonstrated by both HLA-A2-LMP2 tetramer staining and cytotoxicity against HLA-A2-LMP2 bearing T2 cell, the cytotoxicity was inhibited by the anti-HLA class I antibody (W6/32). These results showed that LMP2 antigen-specific CTLs could be induced and expanded in vitro by the HLA-A2-LMP2-bearing aAPC. Thus, aAPCs coated with an HLA-pLMP2 complex, anti-CD28 and CD54 might be promising tools for the enrichment of LMP2-specific CTLs for adoptive immunotherapy.

[HLA-G1 molecule expressed by ECV304 cells inhibit cytotoxic activity of allogeneic NK cells].

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.

[Prokaryotic expression of BirA enzyme and identification of the bioactivity of expressed product].

AIM: To construct the expression vector of biotin-protein ligase (BirA enzyme) gene and express the BirA enzyme with bioactivity in E.coli BL-21 (DE3). METHODS: The BirA gene was amplified from E.coli genome by PCR and cloned into pGEX-4T-2 to construct the recombinant plasmid pGEX-BirA. After being verified by DNA sequencing, the fusion protein was expressed under IPTG induction in the E.coli BL-21 (DE3). The expressed product was purified through Glutathione-agarose chromatography column. The enzyme activity of the expressed product was identified by ELISA and Western blot. RESULTS: The recombinant prokaryotic expression vector pGEX-BirA was constructed and the fusion protein GST-BirA was expressed successfully. The results of ELISA and Western blot showed that the purified BirA enzyme biotinylated HLA-A2 peptide complex. CONCLUSION: BirA enzyme with bioactivity is prepared successfully, which can be used for studying the interaction between protein and protein.

[In vitro refolding and functional study of soluble HLA-G1-peptide complex].

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.

[Improvement of refolding efficiency of HLA-A2-peptide complex in vitro by preformed disulfide bonds in heavy chain].

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.

[In vitro induction and expansion of dendritic cells from rat bone marrow and their characterization].

AIM: To induce and expand dendritic cells (DC) from rat bone marrow in vitro and identify their biological characterization. METHODS: The rat bone marrow cells were collected and cultured for 48 hours and the floating cells were removed. Then IL-4 and GM-CSF were added into the fresh medium. After 2 weeks, the morphological character of the cultured DCs was observed under light microscope and transmission electron microscope. Expressions of MHC class II molecule, B7-1 and B7-2 were detected by flow cytometry. The cultured DCs were co-cultured with allogenic T cells derived from rat spleen. T cell proliferation was measured by MTT colorimetry. RESULTS: The cultured DCs had the typical morphological characterization of DC, and the expression rates of MHC class II molecule, B7-1 and B7-2 were 74.2%, 81% and 76% respectively. The cultured DCs could notably stimulate the proliferation of allogeneic T cells. CONCLUSION: The adherent culture of rat bone marrow cells, and co-culture with IL-4 and GM-CSF can obtain a number of high purity of DCs, which lay the foundation for study on DC's function.

[In-vitro refolding and biotinylation of soluble HLA-A2-peptide complex].

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.

[Immune tolerance inducing effects of soluble human leucocyte antigen G1 on natural killer cells and T cells].

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.