Myxovirus resistance, osteopontin and suppressor of cytokine signaling 3 polymorphisms predict hepatitis C virus therapy response in an admixed patient population: comparison with IL28B

OBJECTIVES: Suppressor of cytokine signaling 3, myxovirus resistance protein and osteopontin gene polymorphisms may influence the therapeutic response in patients with chronic hepatitis C, and an association with IL28 might increase the power to predict sustained virologic response. Our aims were to evaluate the association between myxovirus resistance protein, osteopontin and suppressor of cytokine signaling 3 gene polymorphisms in combination with IL28B and to assess the therapy response in hepatitis C patients treated with pegylated-interferon plus ribavirin. METHOD: Myxovirus resistance protein, osteopontin, suppressor of cytokine signaling 3 and IL28B polymorphisms were analyzed by PCR-restriction fragment length polymorphism, direct sequencing and real-time PCR. Ancestry was determined using genetic markers. RESULTS: We analyzed 181 individuals, including 52 who were sustained virologic responders. The protective genotype frequencies among the sustained virologic response group were as follows: the G/G suppressor of cytokine signaling 3 (rs4969170) (62.2%); T/T osteopontin (rs2853744) (60%); T/T osteopontin (rs11730582) (64.3%); and the G/T myxovirus resistance protein (rs2071430) genotype (54%). The patients who had ≥3 of the protective genotypes from the myxovirus resistance protein, the suppressor of cytokine signaling 3 and osteopontin had a greater than 90% probability of achieving a sustained response (p<0.0001). The C/C IL28B genotype was present in 58.8% of the subjects in this group. The sustained virological response rates increased to 85.7% and 91.7% by analyzing C/C IL28B with the T/T osteopontin genotype at rs11730582 and the G/G suppressor of cytokine signaling 3 genotype, respectively. Genetic ancestry analysis revealed an admixed population. CONCLUSION: Hepatitis C genotype 1 patients who were responders to interferon-based therapy had a high frequency of multiple protective polymorphisms in the myxovirus ...

Antibodies to Interferon beta in Patients with Multiple Sclerosis Receiving CinnoVex, Rebif, and Betaferon

Treatment with interferon beta (IFN-beta) induces the production of binding antibodies (BAbs) and neutralizing antibodies (NAbs) in patients with multiple sclerosis (MS). NAbs against IFN-beta are associated with a loss of IFN-beta bioactivity and decreased clinical efficacy of the drug. The objective of this study was to evaluate the incidence and the prevalence of binding antibodies (BAbs) and neutralizing antibodies (NAbs) to IFN-beta in MS patients receiving CinnoVex, Rebif, or Betaferon. The presence of BAbs was studied in serum samples from 124 MS patients using one of these IFN-beta medications by ELISA. The NAbs against IFN-beta were measured in BAb-positive MS patients receiving IFN-beta using an MxA gene expression assay (real-time RT-PCR). Of the 124 patients, 36 (29.03%) had BAbs after at least 12 months of IFN-beta treatment. The proportion of BAb+ was 38.1% for Betaferon, 21.9% for Rebif, and 26.8% for CinnoVex. Five BAb-positive MS patients were lost to follow-up; thus 31 BAb-positive MS patients were studied for NAbs. NAbs were present in 25 (80.6%) of BAb-positive MS patients receiving IFN-beta. In conclusion, the three IFN-beta preparations have different degrees of immunogenicity.

Interferon-Stimulated Genes Response in Endothelial Cells Following Hantaan Virus Infection

The regulation mechanism of interferon (IFN) and IFN-stimulated genes is a very complex procedure and is dependent on cell types and virus species. We observed molecular changes related to anti-viral responses in endothelial cells during Hantaan virus (HTNV) infection. We found that there are two patterns of gene expression, the first pattern of gene expression being characterized by early induction and short action, as in that of type I IFNs,' and the other being characterized by delayed induction and long duration, as those of IRF-7, MxA, and TAP-1/2. Even though there are significant differences in their induction folds, we found that all of IFN-alpha/beta , IRF- 3/7, MxA, and TAP-1/2 mRNA expressions reached the peak when the viral replication was most active, which took place 3 days of post infection (d.p.i.). In addition, an interesting phenomenon was observed; only one gene was highly expressed in paired genes such as IFN-alpha/beta??(3/277-folds), IRF-3/7 (2.2/29.4-folds), and TAP- 1/2 (26.2/6.1-folds). Therefore, IFN-beta, IRF-7, and TAP-1 seem to be more important for the anti-viral response in HTNV infection. MxA was increased to 296-folds at 3 d.p.i. and kept continuing 207-folds until 7 d.p.i.. The above results indicate that IFN-beta works for an early anti-viral response, while IRF7, MxA, and TAP-1 work for prolonged anti-viral response in HTNV infection.

Cloning and expression of MxA gene from chickens in E.coli and identification of biological activity of MxA / 中国免疫学杂志

Objective:To clone chickens MxA gene, construct its recombinant expression plasmid and induce the expression of fusion protein using a prokaryotic expression system.Methods:The MxA gene fragment was amplified by RT-PCR from CEF cells and subcloned into the pMD18-T vector, filtrated the positive clone and reclaimed the MxA.Subcloned the MxA into the prokaryotic expression plasmid pGEX-6p-1. After recombinant plasmid was induced by IPTG, the expressed proteins were analyzed by SDS-PAGE, the NDV intervence experiment and VSV-CEF restrain experiment.Results:The sequence of MxA gene amplified by RT-PCR was the same as the sequence in gene map of Genebank; SDS-PAGE, the NDV intervence experiment and VSV-CEF restrain experiment showed that a protein was expressed, the molecular weight of this protein was 45 000, which was the same as the fusion protein GST-MxA.Conclusion:The MxA is cloned and its recombinant expression plasmid is constructed successfully.The fusion protein GST-MxA is successfully expressed in the prokaryotic expression system E.coli DH5? induced by IPTG. This research lay a foundation for further studying on ints antiviral effects and exploring new way of antiviral medication.

Study of the MxA protein induced by adenovirus type 3 and it' s antiviral effect against Ad3 / 中国免疫学杂志

Objective: This study aimed to investigate the induction of MxA protein in PBMC treated with various doses of adenovirus type 3(Ad3) and to test the antiviral effect of MxA protein against Ad3 in vitro.Methods:The content of MxA protein in cytoplasm of the peripheral blood mononuclear cells( PBMC) , which were treated with various dose of Ad3 was detected by flow cytometry. The antiviral effect of MxA protein against Ad3 in Hela cells was studied by the microdose cytopathogenic effect inhibition assay. Results: MxA protein that in all the cell groups that were treated with various dose of Ad3 was higher than that of control. 10 ng/ml MxA protein can resist 20TCID50 Aad3. Conclusion: It was suggest that MxA protein that can be induced by Ad3 in PBMC and recombinant MxA can resist Ad3.

Construction and identification of the recombinant adenovirus AdMxA / 重庆医科大学学报

Objective: To clone the Chinese MxA gene and to construct the recombinant adenovirus plasmid carring human MxA gene for further study.Methods: The peripheral blood mononuclear cells(PBMCs) were isolated by gradient fractionation,then total RNA was extracted from the PBMCs.The MxA gene was amplified by RT-PCR using the primers based on the published sequence of MxA.The PCR product,double-digested with restrictive endonucleases NotI and XbaI,was cloned into pAdTrack-CMV by molecular cloning technique.The plasimid pAdTrack-MxA was linearized with Pme I,and subsequently co-transformed into electrocompetent BJ5183 cells with an adenoviral backbone plasmid(pAdEasy-1).Recombinants were selected for kanamycin resistence and confirmed by PacI.Finally,the linearized recombinant plasmid was transfected into HEK 293 cells.Recombinant adenoviruses were generated within 7 to 12 days,then viral tite was checked by GFP.Results:Sequencing analysis revealed that the amino acid sequence of MxA gene had only one amino acid residue displace from isoleucine to valine(compared with that of published sequence) although there were five variations in nucleotide sequence.The restrictive endonuclease analysis confirmed that correct recombinant adenovirus plasmid was constructed.24 hours after transfection,the fluorescence was observed in 293 cells,and viral title checked by GFP was 1.59?108(pfu/mL).Conclusion: The MxA gene is cloned from a Chinese donor and a recombinant adenovirus vector containing human MxA gene is constructed successfully.It lays the foundation for further study of gene therapy for HBV.

Construction and identification of the recombinant eucaryotic plasmid PcDNA3.1-MxA / 重庆医科大学学报

Objective:To construct and identify the recombinant eukaryotic plasmid PcDNA3.1-MxA encoding MxA with the eukaryotic plasmid PcDNA3.1.Methods:Recombinant plasmid PAdTrack-MxA constructed before and PcDNA3.1 were amplified in Escherchia coli JM109.Plasmids were double-digested with restrictive endonucleases NotⅠ and XbaⅠ after extracted,and then ligated to construct recombinant eukaryotic plasmid PcDNA3.1-MxA.The recombinant plasmid was selected for Ampicillin resistance and then confirmed by digestion with NotⅠ and XbaⅠ,PCR and sequencing.The sequence was homology-analysed compared with the sequence of MxA gene in PAdTrack-MxA and the sequence of MxA gene published in Genebank(NM_002462) with sofeware DNAssist 2.0.Results:The restrictive endonuclease and PCR analysis confirmed that the recombinant eukaryotic plasmid PcDNA3.1-MxA was constructed successfully.Sequencing analysis revealed that the cloned segment was 2012bp including MxA gene sequence,and the nucleotide sequence of MxA gene was same to that in PAdTrack-MxA exactly.There were five variations in nucleotide sequence compared with published sequence in Genebank,which caused only one amino acid residue replaced from isoleucine to valine.This replaced amino acid residue was located in the non functional area of MxA protein.Conclusions:The recombinant eukaryotic plasmid PcDNA3.1-MxA is constructed successfully,which lays foundation for the further study on anti-HBV effects of MxA.