[Human hnRNP I prokaryotic expression and preliminary application.].

AIM: To express recombinant protein hnRNP I with prokaryotic expression system and assess the presence of autoantibodies against hnRNP I in systemic sclerosis (SSc) as well as other CTD. METHODS: Human hnRNP I gene was amplified by RT-PCR from HeLa cells and cloned into pET-30a vector , then pET-30a-hnRNP I plasmid was transferred into E.coli BL21 (DE3) to express recombinant protein. The sera from patients including SSc, SLE, SS, MCTD, UCTD, RA and controls were detected by ELISA with the purified recombinant hnRNP I protein. RESULTS: The recombinant protein was highly expressed in E.coli BL21(DE3) and specially reacted with clinically diagnosed SSc patients's serum. The result suggested that the autoantibodies against hnRNP I had higher positive ratio(48.72%) in SSc than other serum of AID and control. CONCLUSION: Human hnRNP I protein was successfully expressed in prokaryotic expression system. The purified hnRNP I protein can be used to diagnosis of SSc.

[Expression of recombinant mouse IL-1beta significantly down-regulates NK cell mediated cytolysis [corrected] against H22 hepatoma cells].

AIM: To investigate the effect of the expression of recombinant IL-1beta in H22 hepatoma cells on its response to NK cell mediated cytotoxicity. METHODS: BALB/c mouse was stimulated by 6% of starch. Total RNA was prepared from peripheral blood monocytes (PBMCs). IL-1beta gene (843 bp) was obtained by RT-PCR. The purified PCR product digested by Xho I and EcoR I was cloned into pIRES2-EGFP to construct the recombinant pIRES2-EGFP-mIL-1beta expression vector which was verified by PCR, restriction enzyme assay (Xho I and EcoR I) and DNA sequencing. Then the purified pIRES2-EGFP-mIL-1beta plasmid was transfected into H22 hepatoma cells by jetPEI. The expression level of recombinant IL-1beta was detected by RT-PCR and confocal microscopy. The cytotoxicity of wild-type spleenic NK cells against H22 cells was assessed by MTT assay. RESULTS: After the total RNA isolated from the starch stimulated BALB/c mouse PBMC, 843 bp IL-1beta gene in length was prepared by RT-PCR. The purified PCR product digested by EcoR I and Xho I was ligated by pIRES2-EGFP to create pIRES2-EGFP-mIL-1beta expression plasmid which was verified by PCR, restriction enzyme assay and DNA sequencing. Then pIRES2-EGFP-mIL-1beta was transfected into H22 hepatoma cells by jetPEI. RT-PCR and confocal microscopy assay showed these cells expressed high level of recombinant IL-1beta expression vector. In a 4-hour based MTT assay, IL-1beta in H22 cells was more resistant to NK92 cell mediated cytotoxicity compared with the cells transfected with pIRES2-EGFP. Meanwhile, the cytolytic capacity of the spleenic NK cells separated from wild-type mouse decreased about 10% when the ratio of effector to target was 40:1. CONCLUSION: The expression of proinflammatory cytokine IL-1beta can significantly down-regulate the cytolytic activity of NK cells against H22 hepatoma cells. It plays a crucial role in the immune escape of hepatoma from NK cell mediated innate immunity.

[IL-1beta antisense RNA enhanced sensitivity of HepG2 cells to NK cell mediated cytotoxicity].

AIM: To investigate the inhibitory effect of IL-1beta antisense RNA on the sensitivity of HepG2 cells to the NK cell mediated cytotoxicity. METHODS: Two gene segments of IL-1beta [IL-1beta1(17-331, 315 bp) and IL-1beta2(246-505, 260 bp)] were selected for antisense RNA. Total RNA was extracted from PBMC of a healthy donor treated with LPS. IL-1beta1 and IL-1beta2 were prepared by RT-PCR. PCR products were cloned into pMD18-T-simple vector and then sub-cloned to construct the pcDNA3.0-antiIL-1beta1 and pcDNA3.0-antiIL-1beta2 antisense RNA expression vectors. HepG2 cells were transfected by jetPEI, the expression of antisense RNA in HepG2 cells was assayed by RT-PCR, level of IL-1beta was analyzed by intracellular staining. The response of HepG2 cells to NK-92 cells was assessed by MTT assay. RESULTS: Two gene fragments of 260 bp and 315 bp products were obtained by RT-PCR. The purified gene fragments were cloned to construct pMD18 T-IL-1beta1 and pMD18 T-IL-1beta2 which were verified by PCR, restriction enzyme assay (Xho I) and DNA sequencing. The PCR products using Pfu DNA polymerase from cloning vectors were sub-cloned to create the antisense RNA expression vectors of pcDNA3.0-antiIL-1beta1 and pcDNA3.0-antiIL-1beta2 which were confirmed by PCR, restriction enzyme assay (Pst I) and DNA sequencing. When transfected into HepG2 cells, HepG2 cells expressed high level of antisense RNA, and simultaneously expression of IL-1beta was markedly suppressed which rendered HepG2 cells to be more sensitive to NK-92 cell mediated cytotoxicity compared with the cells transfected by pcDNA3.0. The cytolytic activity of NK-92 cells to HepG2 cells increase about 20% at the effector to target ratio of 10:1. CONCLUSION: Inhibiting of proinflammatory cytokine IL-1beta can reduce the sensitivity of hepatoma cells to the NK cell mediated cytolysis which provide an useful way of rendering NK cell activity against hepatoma.

Efficacy and tolerability of anti-asthma herbal medicine intervention in adult patients with moderate-severe allergic asthma.

BACKGROUND: Chinese herbal medicine has a long history of human use. A novel herbal formula, anti-asthma herbal medicine intervention (ASHMI), has been shown to be an effective therapy in a murine model of allergic asthma. OBJECTIVE: This study was undertaken to compare the efficacy, safety, and immunomodulatory effects of ASHMI treatment in patients with moderate-severe, persistent asthma with prednisone therapy. METHODS: In a double-blind trial, 91 subjects underwent randomization. Forty-five subjects received oral ASHMI capsules and prednisone placebo tablets (ASHMI group) and 46 subjects received oral prednisone tablets and ASHMI placebo capsules (prednisone group) for 4 weeks. Spirometry measurements; symptom scores; side effects; and serum cortisol, cytokine, and IgE levels were evaluated before and after treatment. RESULTS: Posttreatment lung function was significantly improved in both groups as shown by increased FEV(1) and peak expiratory flow findings (P<.001). The improvement was slightly but significantly greater in the prednisone group (P<.05). Clinical symptom scores, use of beta(2)-bronchodilators, and serum IgE levels were reduced significantly, and to a similar degree in both groups (P<.001). T(H)2 cytokine levels were significantly reduced in both treated groups (P<.001) and were lower in the prednisone-treated group (P<.05). Serum IFN-gamma and cortisol levels were significantly decreased in the prednisone group (P<.001) but significantly increased in the ASHMI group (P<.001). No severe side effects were observed in either group. CONCLUSION: Anti-asthma herbal medicine intervention appears to be a safe and effective alternative medicine for treating asthma. In contrast with prednisone, ASHMI had no adverse effect on adrenal function and had a beneficial effect on T(H)1 and T(H)2 balance.