B-cell-activating factor affects the occurrence of thyroid autoimmunity in chronic hepatitis C patients treated with interferon alpha.

Chronic hepatitis C (CHC) patients frequently suffer from thyroid disorders during interferon therapy. However, the mechanism remains unclear. In this study, we investigated the association between serum B-cell-activating factor belonging to the TNF family (BAFF) levels and the presence of antithyroid peroxidase antibody (anti-TPO) in CHC patients treated with pegylated interferon alpha and ribavirin combination therapy. Six months after the therapy, anti-TPO antibody was detected in 10 (males, 1; females, 9) of 50 patients. The mean age of these patients was higher than that of the anti-TPO-negative patients (61 yr versus 55 yr). Before treatment, the serum BAFF levels of the anti-TPO-positive patients were higher than those of the anti-TPO-negative patients. After starting therapy, the serum BAFF levels of both the anti-TPO-positive and -negative patient groups were elevated. Our findings suggest that the serum BAFF concentration before therapy can predict the risk of thyroid autoimmunity in elderly female patients with CHC.

Autoantibody IgG subclasses to recombinant antigens and the role of bacterial stimuli in primary biliary cirrhosis.

AIM: Serum antimitochondrial antibody (AMA) of the IgG2 and IgG3 subclasses has been reported to be predominant in patients with primary biliary cirrhosis from developed countries. No data are available as to the significance of AMA subtypes in Japanese primary biliary cirrhosis (PBC) patients who have previously manifested unique serological features, nor it is known whether AMA subclasses are influenced by bacterial stimuli, as suggested by the molecular theory of PBC. We undertook a three-step study to address these questions. METHODS: First, Japanese PBC sera were tested using the established triple recombinant antigen (pML-MIT3) to find AMA subclass distribution. Second, we used the three recombinant mitochondrial antigens in PBC sera of Japanese and USA patients to explore the ethnic difference. Third, we used CpG oligodeoxynucleotides and a B cell mitogen to challenge ex vivo peripheral leukocytes from indirect immunofluorescence (IIF)-AMA-positive patients with Japanese PBC. RESULTS: We detected most frequently IgG2-AMA followed by IgG3-AMA, with the latter being more common in IIF-AMA-positive cases, and demonstrated that the IgG3 reactivity against the dominant antigen was significantly higher in PBC sera from the USA. We determined that the bacterial stimulus was superior to the mitogen at inducing a predominant production of IgG2-AMA and CD20+ B cell activation. CONCLUSION: Our data cumulatively supported the hypothesis that IgG2 AMA subtypes are predominant in PBC and suggest that this might be favored by an innate immune reaction against bacterial particles, such as CpG DNA.

Ursodeoxycholic acid reduces CpG-induced IgM production in patients with primary biliary cirrhosis.

AIM: Ursodeoxycholic acid (UDCA) treatment reduces IgM serum levels in patients with primary biliary cirrhosis (PBC) without affecting serum antimitochondrial antibody (AMA) titers. We previously reported that PBC-associated hyper-IgM is secondary to a disease-specific hyperproduction following bacterial stimulation by B cells. METHODS: We isolated peripheral blood mononuclear cells (PBMC) from patients with PBC and controls and evaluated whether bacterial CpG challenge in the presence of UDCA at concentrations consistent with those achieved in treated patients led to changes in total IgM, IgG-AMA, and IgM-AMA production. Further, p65 phosphorylation and CD38 cell expression were analyzed as measures of activation of the NF-kB signaling pathway and B cell subsets, respectively. RESULTS: UDCA significantly reduced CpG-induced total IgM and IgM-AMA production, but had no impact on IgG-AMA production. UDCA also significantly reduced the activation ofnaïve and IgM memory, but not IgG memory, B cells, as represented by CD38 expression levels. Further, p65 phosphorylation was significantly reduced in the presence of UDCA. CONCLUSION: UDCA reduces total and IgM-AMA production in PBMC from patients with PBC by downregulating B cell activation and NF-kB signaling. These data ultimately suggest novel mechanisms of action for UDCA in chronic autoimmune cholestasis.

Genetic polymorphisms of transforming growth factor beta-1 promoter and primary biliary cirrhosis in Japanese patients.

As suggested by concordance rates in twins, genetic factors are critical to the susceptibility and progression of primary biliary cirrhosis (PBC). Among cytokines, transforming growth factor beta-1 (TGF-beta1) plays an important role in autoimmunity and liver fibrosis and a TGF-beta1 receptor knockout mouse has been recently proposed as a model for PBC. The promoter region of the TGF-beta1 gene has two single nucleotide polymorphisms (SNPs) at positions -800 and -509, which influence serum concentrations of latent and active TGF-beta1. We studied genomic DNA from 65 Japanese patients with PBC and 71 matched healthy controls for the association of TGF-beta1 SNPs analyzed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) with susceptibility and disease progression of PBC. The -800 G to A SNP was not found in the Japanese population and no significant difference in the distribution of TGF-beta1 promoter gene -509 SNP was found between PBC cases and controls. Further, TGF-beta1 genotypes failed to correlate with clinical parameters, including histological stage and prognostic score. In conclusion, the TGF-beta1 promoter gene SNPs are not associated with disease susceptibility or progression in Japanese patients with PBC.

Serum reactivity against bacterial pyruvate dehydrogenase: increasing the specificity of anti-mitochondrial antibodies for the diagnosis of primary biliary cirrhosis.

Antimitochondrial antibodies (AMA) are the serum hallmark of primary biliary cirrhosis (PBC). However, AMA-positivity can be found in non-PBC sera when lower dilutions are used, thus raising issues about the specificity and sensitivity of the test. AMA reacts primarily with the lipoylated domains of pyruvate dehydrogenase-E2 (PDC-E2) which is highly conserved across species, including bacteria. We studied 77 serum samples, including 24 from patients with anti-PDC-E2-positive PBC and 53 controls (16 with autoimmune hepatitis (AIH), 10 with primary sclerosing cholangitis (PSC), and 27 healthy individuals) for their reactivities at serial dilutions (1:10, 1:20 and 1:40) against Escherichia coli DH5 alpha lysate overexpressing human PDC-E2 using immunoblotting (IB). A murine anti-human PDC-E2 monoclonal antibody (mAB) was used as control. We further studied positive sera using adsorption with a synthetic E. coli peptide sharing similarity with human PDC-E2. Finally, we verified whether a unique buffer for E. coli preparation could reduce non-specific serum reactivity. Results demonstrated that 100% of anti-PDC-E2-positive PBC and up to 38% of control sera at 1:10 dilution recognized E. coli PDC-E2 at IB while dilution tests indicated that the overall potency of PBC reactivity was 100-fold higher compared to controls. In fact, a subgroup (20-38%) of non-PBC sera were positive at low titers but lost the reactivity when absorbed with the synthetic E. coli peptide. Finally, our unique buffer reduced the reactivity of non-PBC sera as measured by ELISA. In conclusion, we demonstrated that weak cross-reactivity with E. coli PDC-E2 occurs in non-PBC sera at lower dilutions and that such reactivity is not due to AMA-positivity. The use of a specific buffer might avoid the risk of false positive AMA determinations when E. coli-expressed recombinant antigens are used.