OBJECTIVETo study the relativity between La protein and the stability of HBV mRNA and the expression of HBV protein.

METHODSFour specific siRNAs were obtained by transcription in vitro. After transfection with the siRNAs into HepG2.2.15 cells for 3 days, the inhibitive effects of La protein were analyzed by Western blot; the content changes of HBsAg, HBeAg and HBV-DNA were detected by ECL and RT-PCR.

RESULTSIn comparison to normal cells, La protein was less in the cells. There was less La protein in the cells trans-infected with siRNAs. HBsAg, the HBeAg and HBV-DNA secreted by the cells transfected with siRNA were also less than that in the normal cells.

CONCLUSIONThere is a correlation between La protein and HBV mRNA and the expression of HBV protein.

Autoantigens ; metabolism ; Cell Line, Tumor ; DNA, Viral ; Hepatitis B Surface Antigens ; Hepatitis B virus ; genetics ; metabolism ; Humans ; RNA, Messenger ; genetics ; metabolism ; RNA, Small Interfering ; RNA, Viral ; Ribonucleoproteins ; metabolism

To develop a fed-batch fementation process of E. coli TOP10 containing a recombinant plasmid pBAD/HBs Fab. Cells were grown in semi-defined medium at 37 degrees C, and the feed operation using glycerol as carbon source was performed when dissolved oxygen increased. When the target cell concentration reached to 64g/L, arabinose was added to a final concentration of 0.02%. Cells were grown for another 5h with the culture temperature decreased from 37 degrees C to 30 degrees C. In the whole process, cell growth was monitored by measuring OD600 of samples taken at 1/2h intervals and the dissolved oxygen was kept above 30%. After the fementation, E. coli pellets were collected for purification of Fab protein. The specificity of Fab protein was confirmed by Western blot, and binding activity to HBsAg was verified by Dot blot. Cell concentration we got is 96g wet bacteria per liter, the Fab protein is about 6% of total protein of the host, that is 80mg per liter. Stable fermentation parameters were obtained for fermentation to improve productivity of the Fab protein. The Fab protein was produced in the form of soluble biologically active protein, it's better than inclusion bodies from which biologically active protein can only be recovered by complicated and costly denaturation and refolding process.
Blotting, Western ; Escherichia coli ; genetics ; metabolism ; Fermentation ; physiology ; Hepatitis B Surface Antigens ; immunology ; Immunoglobulin Fab Fragments ; biosynthesis ; genetics ; immunology ; Temperature

OBJECTIVETo investigate the association of single nucleotide polymorphisms (SNPs) of the peptidylarginine deiminase IV (PADI4) and HLA-DRB1 shared epitope (SE) alleles with rheumatoid arthritis(RA) in a Chinese population.

METHODSFour exonic SNPs of the PADI4 gene (PADI 4_89*A/G, PADI 4_90*C/T, PADI 4_92*C/G and PADI 4_104*C/T) were genotyped in 67 unrelated patients with RA and 81 healthy controls, using cDNA sequencing and T vector cloning. HLA-DRB 1*01, *04 and *10 subtypes were determined by polymerase chain reaction with sequence specific primers (PCR-SSP).

RESULTSThe distributions of the 4 SNPs were different in the two groups, and increased RA susceptibility was significantly associated with the minor alleles of PADI 4_89*G (P was 0.023), PADI 4_90*T (P was 0.004), PADI 4_104*T (P was 0.003), and the haplotypes carrying the 4 minor alleles (P was 0.008). HLA-DRB1 SE alleles are composed of HLA-DRB 1*0101, *0102, *0401, *0404, *0405, *0408, *0409, *0410 and *1001. Individuals carrying the SE alleles were associated with increased RA susceptibility (P was 0.002). Individuals carrying both the SE alleles and minor alleles of the 4 SNPs were more susceptible to RA than individuals carrying neither the minor SNP alleles nor the SE alleles.

CONCLUSIONThe PADI4 SNPs and haplotypes are associated with RA susceptibility in Chinese. HLA-DRB1 shared epitope is also an important risky factor for RA. There may exist certain synergistic effect between the PADI4 minor alleles and the HLA-DRB1 shared epitope.

Adult ; Aged ; Aged, 80 and over ; Alleles ; Arthritis, Rheumatoid ; genetics ; Asian Continental Ancestry Group ; genetics ; Case-Control Studies ; Epitopes ; genetics ; Female ; Gene Frequency ; Genetic Predisposition to Disease ; HLA-DR Antigens ; genetics ; HLA-DRB1 Chains ; Humans ; Hydrolases ; genetics ; Male ; Middle Aged ; Phenotype ; Polymorphism, Single Nucleotide ; Protein-Arginine Deiminases

OBJECTIVETo identify autoepitopes of E2 subunit of pyruvate dehydrogenase complex (PDC-E2) specific CD8+ CTL in primary biliary cirrhosis (PBC) patients.

METHODSAn online database SYFPEITHI was applied to predict HLA-A*0201 restricted epitopes which located in PDC-E2 30-50 aa and 150-190 aa where B-cell epitopes clustered with CD4+ T-cell epitopes. T2 cell line reconstitution and stabilization assay, induction of specific CTL lines from peripheral blood mononuclear cells (PBMCs) of patients with PBC and cytotoxicity of peptides-induced CTL were performed to screen the epitopes from those candidates.

RESULTSFive potential epitopes were predicted by database. Of the 5 candidates, two peptides 159-167 aa and 165-174 aa, with highly binding activity to HLA-A*0201 molecules, could stimulate PBMCs from most HLA-A*0201 positive PBC patients to proliferate and peptide-induced CTL lines showed specific cytotoxicity.

CONCLUSIONPeptides of KLSEGDLLA (159-167 aa) and LLAEIETDKA (165-174 aa) in the inner lipoyl domain of PDC-E2 are HLA-A*0201 restricted CD8+ CTL immunodominant epitopes in PBC.

Antibody-Producing Cells ; cytology ; Autoantigens ; immunology ; Autoimmunity ; CD8-Positive T-Lymphocytes ; cytology ; immunology ; metabolism ; Cell Line ; Dihydrolipoyllysine-Residue Acetyltransferase ; Epitope Mapping ; Epitopes, T-Lymphocyte ; immunology ; HLA-A Antigens ; immunology ; HLA-A2 Antigen ; Humans ; Liver Cirrhosis, Biliary ; enzymology ; genetics ; immunology ; Phenotype ; Protein Binding ; Pyruvate Dehydrogenase Complex ; genetics ; immunology ; metabolism ; T-Lymphocytes, Cytotoxic ; immunology

OBJECTIVETo investigate the association between Chinese patients with autoimmune hepatitis (AIH), primary biliary cirrhosis (PBC) and the polymorphisms of cytotoxic T lymphocyte -associated antigen-4 (CTLA-4) gene promoter (-318) and exon 1 (+49).

METHODSThe CTLA-4 promoter (-318 T/C) and exon 1 (+49A/G) polymorphisms were genotyped via restriction fragment length polymorphism methods in 62 Chinese AIH patients, 77 Chinese PBC patients and 160 healthy controls.

RESULTSThere was no difference in the distribution of CTLA-4 promoter -318 T/C polymorphisms between AIH patients and controls, but the C allele frequency was significantly increased in patients with AIH, compared to controls (P=0.02, OR=2.43). The distribution of CTLA-4 gene exon 1 49 A/G genotypes exhibited significant difference between PBC patients and controls (P=0.006), and the frequency of G allele showed a significant increase in PBC group as compared with controls (P=0.0046, OR=1.8). Although the genotype distribution of the CTLA-4 exon 1-promoter gene displayed no significant difference between AIH and PBC patients and controls, the occurrence of GG-CC was increased in the patients of the two groups (AIH: 32.3%, PBC: 37.7%; control: 22.5%).

CONCLUSIONThe above findings suggest that the polymorphisms of CTLA-4 gene probably confer susceptibility to AIH and PBC in the Chinese population.

Adolescent ; Adult ; Aged ; Antigens, CD ; genetics ; Asian Continental Ancestry Group ; genetics ; CTLA-4 Antigen ; China ; Exons ; genetics ; Female ; Genetic Predisposition to Disease ; genetics ; Genotype ; Hepatitis, Autoimmune ; ethnology ; genetics ; Humans ; Liver Cirrhosis, Biliary ; ethnology ; genetics ; Male ; Middle Aged ; Polymerase Chain Reaction ; Polymorphism, Genetic ; Polymorphism, Restriction Fragment Length ; Promoter Regions, Genetic ; genetics

OBJECTIVETo investigate the frequencies of human leuckocyte antigens (HLA) -A, B and DRB1 alleles in Chinese patients with primary biliary cirrhosis (PBC) using polymerase chain reaction-based techniques, and to assess the correlation of HLA molecules with other clinical and laboratory profiles.

METHODSGenotyping of HLA-A, B, and DRB1 were performed in 65 well-characterized patients with primary biliary cirrhosis and 431 healthy controls with PCR amplification with sequence-specific primers (PCR-SSP).

RESULTSThe frequency of DRB1*0701 was increased to 29.2% compared with 13.9% in the controls (PC < 0.05, OR = 2.55, 95% CI: 1.4 approximately 4.6). No association was found with HLA-DRB1*08 which had been constantly reported. The A*2 allele (53.8%) was more frequent in the PBC patient group but without a significant statistical difference. The frequencies for the other A, B and DRB1 alleles were similar between patients and healthy controls. There was no difference between patients with or without DRB1*0701 in some clinical and laboratory profiles.

CONCLUSIONSusceptibility to primary biliary cirrhosis in Chinese is associated with DRB1*0701 allele and differs from people in North America, South America, North Europe and even in Japan, but the association is not restricted to any particular subgroup of patients. Valine at position 78 of HLA DRbeta1 may play an important role in the pathogenesis of primary biliary cirrhosis.

Adult ; Aged ; Aged, 80 and over ; Alleles ; Female ; HLA Antigens ; genetics ; HLA-A Antigens ; genetics ; HLA-B Antigens ; genetics ; HLA-DR Antigens ; genetics ; Humans ; Liver Cirrhosis, Biliary ; genetics ; immunology ; Male ; Middle Aged ; Polymorphism, Genetic

OBJECTIVEThe aim of this study was to evaluate the association between Chlamydia pneumoniae (CP) infection and primary biliary cirrhosis (PBC).

METHODSChlamydia pneumoniae IgG and IgM were determined by enzyme-linked immunosorbent assay (ELISA) in 41 well-established PBC patients and two race-matched control groups, PHC, n = 70; healthy controls, HC, n =57).

RESULTSThe mean levels and seroprevalence of CP IgG in PBC group and PHC group were significantly higher than in the HC [(46.8 +/- 43.4) RU/ml, (49.5 +/- 45.2) RU/ml vs (28.3 +/- 32.7) RU/ml, P = 0.042 and P < 0.001 respectively; 68.3%, 71.4% vs 42.1%, chi2 values were 5.389 and 11.110 respectively]. There was a markedly elevated seroprevalence of CP IgM in patients with PBC (22.0%) compared with the PHC and HC groups. The odds ratios (ORs) for the presence of CP IgG and IgM for the PBC patients versus the HC were 2.7 (95% CI 0.9 to 6.1) and 5.1 (95% CI 1.4 to 18.5). Though there was no correlation in the level of CP IgG with total IgG in sera of patients with PBC (r=-0.857, p=0.344), CP IgM was related with the abnormally high concentrations of total IgM in the PBC group.

CONCLUSIONSThe results of this study do not support the hypothesis that infection with Chlamydia pneumoniae may be a triggering agent for PBC, but suggest that Chlamydia pneumoniae infection probably contributes to the high level of IgM presented in most of the patients with PBC

Adult ; Aged ; Aged, 80 and over ; Antibodies, Bacterial ; blood ; Chlamydophila Infections ; complications ; Chlamydophila pneumoniae ; isolation & purification ; Female ; Humans ; Immunoglobulin M ; blood ; Liver Cirrhosis, Biliary ; complications ; microbiology ; Male ; Middle Aged

OBJECTIVETo determine the relationship between polymorphisms in the genes encoding IL-1, IL-6, and IL-10 with primary biliary cirrhosis (PBC) in Chinese population.

METHODSWhole-blood samples were taken from 77 patients with PBC and 160 healthy controls. DNA was extracted and the polymorphisms at positions IL-1 +3953, IL-1RN intron 2, IL-6 -174, and IL-10 -1082, -819, and -592 were determined by using sequence-specific polymerase chain reaction (SSP) or polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP).

RESULTSThe frequency of IL-1RN1,1 allele in PBC group was significantly higher than in control group (90.9% vs 79.4%, P=0.026), and the frequency of IL-1RN1,2 in PBC group was significantly lower than in control group (6.5% vs 18.8%, P=0.013). There was no significant difference in the frequence of IL-1RN*2 allele between PBC group and control group (P=0.06). Of the 77 patients with PBC, 4 patients were IL-6 -174GC, 73 were IL-6 174GG. All the 160 health controls are IL-6 -174GG (P=0.0036). The frequence of IL-6 -174C allele in PBC group was significantly higher than that in control group (P=0.0038). No significant differences of polymorphisms for IL-1 +3953 and IL-10 (-1082, -819 and -592) were found between PBC group and control group.

CONCLUSIONThe polymorphisms of IL-1RN and IL-6 -174G/C appear to be associated with PBC, and the polymorphisms of IL-1 +3953 and IL-10 promoter gene are not associated with PBC in a Chinese population.

Adult ; Aged ; Female ; Humans ; Interleukin-1 ; genetics ; Interleukin-10 ; genetics ; Interleukin-6 ; genetics ; Liver Cirrhosis, Biliary ; genetics ; Male ; Middle Aged ; Polymerase Chain Reaction ; methods ; Polymorphism, Restriction Fragment Length

OBJECTIVEAutoimmune hepatitis (AIH) and primary biliary cirrhosis (PBC) are two autoimmune diseases of unknown etiology. Genetic factors appear to be involved in the pathogenesis of both diseases. Tumor necrosis factor (TNF)-alpha is one of the proinflammatory cytokines and immunomodulators, and is implicated in the pathogenesis of AIH and PBC. In this study, we studied the association between Chinese patients with AIH, PBC and the polymorphisms in promoter-region polymorphisms of the TNF-alpha gene at position -308 and -238.

METHODSWe have investigated four candidate gene loci in 49 patients with AIH, 58 patients with PBC, and 160 healthy controls. The polymorphisms were assessed by the PCR specifically for the single-nucleotide polymorphisms.

RESULTSWe found the difference in the TNF-alpha gene at position -308 genotype distributions between Chinese health controls and Caucasian health controls. Although the percent of TNF-alpha*2 was decrease on PBC patient group (10.34% vs. 16.88%), there was no significant difference between PBC patients and health control in the Chinese. There were also no significant differences between AIH and health control on the TNF-alpha gene at position -308 and -238.

CONCLUSIONOur findings suggest that the TNF-alpha promoter-region polymorphisms distribution is different between differe of ethnic groups; there are no genetic links of the TNF-alpha promoter-region polymorphisms to AIH and PBC in Chinese.

Adult ; Aged ; Female ; Genotype ; Hepatitis, Autoimmune ; genetics ; Humans ; Liver Cirrhosis, Biliary ; genetics ; Male ; Middle Aged ; Polymorphism, Genetic ; Tumor Necrosis Factor-alpha ; genetics

OBJECTIVETo investigate whether three biallelic polymorphisms at the position -592, -819 and -1082 in the promoter region of the IL-10 gene were associated with the incidence of autoimmune liver disease.

METHODSThe IL-10 -592 and IL-10-1082 polymorphisms were genotyped by polymerase chain reaction-restriction fragment length polymorphisms analysis (PCR-RFLP), while polymerase chain reaction- sequence specific primer (PCR-SSP) assay was used to detect IL-10 -819 polymorphisms.

RESULTSAmong 54 Chinese patients with AIH or 77 Chinese patients with PBC versus healthy controls, the frequency of AA, GA genotypes at IL-10 gene promoter -1082 position was 87.0% or 83.1% versus 90.0%, 13.0% or 16.9% versus 10.0%, respectively (P > 0.05), the GG genotype in Chinese populations is absent; the frequency of CC, CT, TT genotypes at IL-10 gene promoter -819 position was 11.11% or 9.1% versus 8.1%, 44.4% or 53.3% versus 45.0%, 44.4% or 37.7% versus 46.9%, respectively (P > 0.05); the frequency of CC, CA, AA genotypes at IL-10 gene promoter -592 position was 4.9% or 14.3% versus 10.0%, 51.2% or 53.3% versus 51.9%, 43.9% or 32.5% versus 38.1%, respectively (P > 0.05). No alleles differed significantly in each groups.

CONCLUSIONThere were no association between IL-10 gene polymorphisms and autoimmune liver disease

Adult ; Aged ; Female ; Hepatitis, Autoimmune ; genetics ; immunology ; Humans ; Interleukin-10 ; genetics ; Liver Cirrhosis, Biliary ; genetics ; Male ; Middle Aged ; Polymerase Chain Reaction ; methods ; Polymorphism, Restriction Fragment Length ; Promoter Regions, Genetic ; genetics