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.

Values of Doppler sonography predicts high risk variceal bleeding in patients with viral cirrhosis.

BACKGROUND/AIMS: Gastrointestinal bleeding such as rupture of esophagogastric varices remains one of the leading causes of death in patients with liver cirrhosis. As a critical issue, assessment of the bleeding risk of esophageal varices is extremely important. In the present study, by determining the relationship between several parameters measured by pulsed Doppler sonography and the bleeding risk of esophageal varices assessed by upper endoscopy, we investigated what is the most valuable parameter as a supplement to the bleeding risk. METHODOLOGY: A total of 158 patients with hepatitis virus-infected liver cirrhosis (56 positive for HBs antigen and 102 positive for HCV antibody) were studied. As controls, 171 normal subjects were used. The flow volumes of the portal trunk and the splenic vein, the Congestion Index, and the S/P ratio were measured by pulsed Doppler sonography. Based on upper endoscopic findings, we classified the patients into two groups based on bleeding risk of esophageal varices: high-risk and low-risk. Logistic regression analysis was employed to identify the most valuable parameter as a supplement to the bleeding risk. RESULTS: The flow volume of the splenic vein, the Congestion Index, and the S/P ratio in cirrhotic patients with esophageal varices were significantly higher than those in normal subjects (P = 0.000). The mean flow volumes of the portal trunk and splenic vein and the mean of the S/P ratio in the high-risk group for bleeding of esophageal varices were significantly higher than those in the low-risk group (P = 0.000-0.005). Based on logistic regression analysis, the flow volume of the splenic vein was found to be the most valuable parameter for bleeding risk (P < 0.001). CONCLUSIONS: The flow volume of splenic vein with pulsed Doppler sonography was the most valuable parameter for the bleeding risk of esophageal varices.

Spontaneous scratching behavior in MRL/lpr mice, a possible model for pruritus in autoimmune diseases, and antipruritic activity of a novel kappa-opioid receptor agonist nalfurafine hydrochloride.

Pruritus is a common, distressing and difficult to manage complication of many autoimmune diseases. A suitable animal model of autoimmune disease associated pruritus would contribute to a better understanding of the pathophysiology of this symptom and lead to the development of safe and effective antipruritic agents. We noticed spontaneous scratching behavior in aged MRL/lpr mice, a model of autoimmune disease. This scratching behavior was observed in a specific pathogen-free environment and was more frequent in female mice. In contrast to animal models of dermatitis; NC/Nga mice, the serum IgE and IgG1 levels in MRL/lpr mice were not elevated. These features indicate that this scratching behavior is similar to human autoimmune disease associated pruritus. The antipruritic effects of an antihistamine (chlorpheniramine), an opioid receptor antagonist (naltrexone), and a novel kappa-opioid receptor agonist (nalfurafine hydrochloride [TRK-820]) were evaluated. The frequency of scratching was not reduced by oral administration of chlorpheniramine, suggesting that the behavior is antihistamine-resistant. The oral administration of nalfurafine and subcutaneously administered naltrexone inhibited the scratching behavior without causing gross behavioral changes. In conclusion, MRL/lpr mice scratching behavior is a suitable model of pruritus that occurs in autoimmune diseases, and nalfurafine was shown to be efficacious against this behavior suggesting that it may be beneficial in patients with autoimmune disease associated pruritus.

Association of single nucleotide polymorphisms of the interleukin-10 promoter gene and susceptibility to primary biliary cirrhosis: immunogenetic differences in Italian and Japanese patients.

Several lines of data suggest that genetic factors play an important role in the onset and/or progression of primary biliary cirrhosis (PBC). Since PBC is an autoimmune disease, it is reasoned to assume that genes encoding cytokines may confer susceptibility to disease. Amongst these factors, interleukin-10 (IL-10) has received significant attention. The promoter region of IL-10 gene has three single nucleotide polymorphisms (SNPs) at positions -1082, -819 and -592. To elucidate the association of the three SNPs of IL-10 promoter region with susceptibility of PBC in two different genetic populations, 159 unrelated patients with PBC (94 Italian and 65 Japanese) and 143 local controls (72 Italian and 71 Japanese) were enrolled. SNPs were determined using allele-specific PCR/RFLP. In Italian PBC patients, the frequency of homozygosity for G/G at position -1082 was significantly higher than that of local controls (p < 0.041, OR = 2.44, 95% C.I.; 1.02-5.86). The frequencies of haplotype GCC in PBC patients, possibly linked to higher IL-10 production, were also significant higher than local controls (p < 0.033). However, in Japanese population, there were no significant differences in the three SNPs and haplotypes between PBC patients and controls. Excessive production of IL-10 may play an important role in some populations in modulating the onset of PBC. Further, immunogenetic studies of PBC should take into account ethnic and geographic variations; this makes such studies in heterogeneous population, like the USA, more difficult.

Direct refolding of inclusion bodies using reversed micelles.

The protein refolding of inclusion bodies was investigated using reversed micelles formed by aerosol OT (AOT). Ribonuclease A (RNase A) was overexpressed in Escherichia coli and used as native inclusion bodies. The enzymatic activity of RNase A was completely regained from the inclusion bodies within 14 h by solubilization in reversed micelles. To further enhance the refolding rate, a molecular chaperone, GroEL, was incorporated into the refolding system. The resultant refolding system including GroEL showed better performance under optimized conditions for the refolding of RNase A inclusion bodies. The refolding rate was considerably improved by the addition of the molecular chaperone, and the refolding step was completed in 1 h. The protein refolding in the GroEL-containing refolding system was strongly dependent on the coexistence of ATP and Mg2+, suggesting that the GroEL hosted in the reversed micelles was biologically active and assisted in the renaturation of the inclusion bodies. The addition of cold acetone to the reversed micellar solution allowed over 90% recovery of the renatured RNase A.

Eta-1/osteopontin genetic polymorphism and primary biliary cirrhosis.

Early T-lymphocyte activation 1 (Eta-1)/osteopontin is a soluble ligand with pleomorphic immunologic activities including activation of macrophage chemotaxis, promotion of Th1 responses, and activation of B1 B-cells. A recent study suggested that a single-nucleotide polymorphism (SNP) at position nt 9250 (C to T) in exon 7 was highly associated with systemic lupus erythematosus (SLE). Eta-1/osteopontin was reported to be highly expressed in the MRL/lpr mouse, which is recognized as one of the spontaneous autoimmune models of SLE. In the present study, we first investigated the association with this SNP and susceptibility to primary biliary cirrhosis (PBC). The allele frequencies of C/C, C/T, and T/T at position nt 9250 on the Eta-1/osteopontin gene in 50 PBC patients were 20, 32, and 48%, respectively, compared with 9, 47, and 44% in 34 healthy controls (P<0.16-0.72). The gene frequencies of C and T at this position in such PBC patients were 0.36 and 0.64, whereas those in the healthy controls were 0.32 and 0.68 (P<0.91), respectively. Moreover, clinical findings and pathologic stages were not correlated with the variation of SNP. Those findings suggest no associations with Eta-1/osteopontin genetic polymorphism and susceptibility to PBC.

Acute liver dysfunction complicated with uncontrollable glycemia due to insulin antibody: successful treatment with glucocorticoid and lispro insulin.

Here, we report a case of acute liver dysfunction complicated with uncontrollable glycemia due to insulin antibody. The patient was admitted to our hospital due to diabetic ketoacidosis. He was administered insulin immediately, however, his fasting plasma glucose level remained unstable despite the insulin treatment. Blood biochemistry revealed severe liver dysfunction, although no markers including hepatitis virus or autoantibodies associated with autoimmune liver diseases were detected. The 125I-insulin binding rate was high (54%). The characteristics of insulin antibody in this patient were similar to the antibodies of IAS patients, therefore we administered oral glucocorticoid against insulin antibody. The reduction in the 125I-insulin binding rate and the binding capacity of the high affinity site of insulin antibodies were balanced after oral glucocorticoid therapy. In addition, preprandial subcutaneous regular insulin was switched to lispro insulin. Postprandial plasma glucose levels were relatively improved by lispro insulin. The etiology of acute liver dysfunction was unknown, however, we believe that the combination of oral glucocorticoid and lispro insulin was suitable and useful for preventing recurrent liver dysfunction in this patient.

Low frequency of anti-SLA/LP autoantibody in Japanese adult patients with autoimmune liver diseases: analysis with recombinant antigen assay.

Anti-soluble liver antigen/liver pancreas (SLA/LP) autoantibody has been proposed to be one of the autoantibodies characterizing autoimmune hepatitis (AIH). Recently, one of the autoantigens to anti-SLA/LP was identified as a UGA suppressor tRNA-associated protein. Although the function of this protein remains unknown, the recombinant protein has been prokaryotically expressed. Using this protein as an antigen, a recombinant immunoassay for anti-SLA/LP autoantibody has been established and the frequency and significance of this autoantibody have been discussed in European countries. So, in the present study, we investigated anti-SLA/LP autoantibodies in Japanese patients with autoimmune liver diseases using the recombinant antigen ELISA and Western blot assay. Seventy-five patients with AIH type 1, 5 with AIH type 2, 46 with primary biliary cirrhosis, 10 with primary sclerosing cholangitis, 47 with chronic hepatitis C, 48 with systemic lupus erythematosus, 3 with cryptogenic hepatitis, and 40 normal controls were the subjects of the present study. Anti-SLA/LP autoantibodies were detected in only 5 of 75 (6.7%) patients with AIH type 1, but in none of the other 159 patients or 40 normal controls. The clinicopathologic features of anti-SLA/LP-positive AIH type 1, including carriers of HLA DR locus variations, were not significantly different from anti-SLA/LP-negative patients except for the mortality rate. Anti-SLA/LP autoantibody was detected at a low frequency in Japanese patients with AIH type 1 and did not significantly influence clinical features. However, since it has high disease-specificity to AIH type 1, further analysis of SLA/LP may contribute to help clarify the pathogenesis of AIH type 1.