Edward Penner

The relation of iron status and hemochromatosis gene mutations in patients with chronic hepatitis C

BACKGROUND & AIMS Elevated hepatic iron concentration may affect the response to antiviral therapy in chronic hepatitis C. This study explored the contribution of genetic hemochromatosis to iron accumulation in chronic hepatitis C. METHODS HFE mutations (C282Y and H63D) were assessed in 184 patients with chronic hepatitis C virus and 487 controls. Liver biopsy specimens were available in 149 patients. Hepatic iron content was measured in 114 patients by atom-absorption spectrophotometry. RESULTS The C282Y and H63D allele frequencies were 7.06 and 11.6 in patients and 4.83 and 11.09 in controls, respectively. Eight patients were homozygotes (5 C282Y [2.7%] and 3 H63D [1.6%]), 2 compound heterozygotes (1%), and 49 heterozygotes (14 C282Y [7.6%] and 35 H63D [19%]). Biochemical evidence of iron overload was more common in patients with HFE mutations (28 of 47) than in those without (34 of 102; P = 0.0045). Histological iron grading and hepatic iron content overlapped among patients with or without mutations. A hepatic iron index of >1.9 was observed only in 1 of the 4 C282Y homozygotes and 1 of the 3 H63D homozygotes. CONCLUSIONS HFE mutations contribute to but do not fully explain hepatic iron accumulation in chronic hepatitis C. Furthermore, C282Y or H63D homozygosity in chronic hepatitis C is not necessarily associated with a high hepatic iron content.

Correlation of initial autoantibody profile and clinical outcome in primary biliary cirrhosis

Although there have been significant advances in understanding the clinical and biochemical features of primary biliary cirrhosis (PBC), there is still a paucity of data on the usefulness of biomarkers as prognostic indicators. This is particularly important at the time of initial diagnosis. Indeed, the widespread use of antimitochondrial antibody testing has led to an earlier diagnosis of asymptomatic PBC and it is difficult to predict which patients will experience a benign versus a rapidly progressive course. To address this issue, we examined a unique population of 127 newly diagnosed patients with PBC during a 15‐year period of observation that began in January 1990. Sera from these patients were analyzed for antimitochondrial, antinuclear, and anti–smooth muscle antibodies, and immunoblotting was performed for nuclear pore complex (NPC). The patients were then followed up longitudinally using biochemical liver function tests. No patient was under any medical therapy for PBC at the time of the initial sera collection. Data were analyzed based not only on the clinical features, but also the Mayo score and specific outcome measures, including time to death, need for liver transplantation, and complication free survival. Among patients with early disease, bilirubin increased to >2 mg/dL in the anti‐NPC(+) patients (26% vs. 5%, P = .019). Anti‐NPC antibodies remained stable or slightly increased over the period of observation. In conclusion, anti‐NPC identifies patients likely to experience an unfavorable clinical course and more rapid disease progression. (HEPATOLOGY 2006;43:1135–1144.)

Autoantibodies against nuclear pore complexes are associated with more active and severe liver disease in primary biliary cirrhosis

BACKGROUND/AIMS Antibodies against nuclear pore complexes (NPCs) have been detected in primary biliary cirrhosis (PBC), but their clinical relevance is still unsettled. METHODS We tested sera from 171 consecutive PBC patients and 230 control subjects (149 with autoimmune or viral liver diseases, 28 with systemic lupus erythematosus, and 53 healthy) by immunoblotting for antibodies against purified human NPCs. RESULTS Antibodies to NPCs were detected in 27% of the patients with PBC, were highly specific (97%), and were not associated with antimitochondrial antibodies. Their prevalence was higher in symptomatic patients (36 vs. 16%, P < 0.01) and was associated (P < 0.001) with more severe disease, as assessed by the presence of cirrhosis or its complications (13% prevalence in patients without cirrhosis, 31% in uncomplicated, and 54% in complicated cirrhosis), or by the application of the Mayo prognostic model (12% in the lowest, 21% in the median, 47% in the highest score tertile). Positive patients had higher levels of serum bilirubin (2.2 +/- 3.7 vs. 1.0 +/- 1.1 mg/dl, P < 0.01) and more marked inflammatory infiltrates on liver biopsy (P < 0.05). CONCLUSIONS Autoantibodies to NPCs are more prevalent in PBC patients than in controls and are strongly associated with more active and severe disease.

Scl 70 autoantibodies from scleroderma patients recognize a 95 kDa protein identified as DNA topoisomerase I

Sera of patients suffering from the autoimmune disease progressive systemic sclerosis (PSS) are known to contain autoantibodies which have been reported to recognize a 70 kDa antigenic protein, designated the Scl 70 antigen. By immunoblotting of nuclear extracts from HeLa cells with sera from scleroderma patients we observed that the size of the antigen present in such cells depends on the conditions of antigen isolation. When protease inhibitors were included in the extraction buffer, a 95 kDa protein was identified instead of a 70 kDa protein. When protease inhibitors were omitted, a number of polypeptides in the size range 66 to 95 kDa was found. Furthermore, antibodies which had been affinity purified on the 95 kDa antigen, crossreacted with the 66 to 95 kDa polypeptides. These results suggest that the smaller proteins were degradation products of the 95 kDa antigen. Immunofluorescence studies on PtK-2 cells with the antibody specific for the 95 kDa protein gave staining of nuclei, nucleoli and of chromosomes and the nucleolar organizer region in mitotic cells. Since this distribution of antigens within the nucleus was reminiscent of the intranuclear distribution of DNA topoisomerase I found by others we probed purified DNA topoisomerase I from calf thymus directly with the autoantibodies from PSS patients, and also the 95 kDa antigens of HeLa cell nuclei with antibodies raised against the bovine DNA topoisomerase I. From the crossreaction pattern observed with the different antigens and antibodies we conclude that DNA topoisomerase I is one of the antigenic components against which autoantibodies are formed in scleroderma patients.

Treatment of pruritus in chronic liver disease with the 5-hydroxytryptamine receptor type 3 antagonist ondansetron: a randomized, placebo-controlled, double-blind cross-over trial.

Background Recently, the serotonin antagonist ondansetron has been reported to have a positive effect on cholestasis-associated pruritus. Objectives To study the effect of orally administered ondansetron on pruritus in chronic liver disease in a randomized, placebo-controlled, double-blind, cross-over study. Methods Subjective severity of pruritus was assessed using a visual analogue scale (VAS) recorded four times daily by the patients. After a one week pretreatment baseline period the patients were randomized to receive ondansetron tablets 8 mg tds or placebo tablets tds for one week. Following a one week wash-out period patients were switched to the other treatment for one week. The study was ended by an additional follow-up week without medication. For each day peak VAS values were determined and the mean value of the last five days of each week was calculated and referred to as the composite peak VAS score. Results We observed a significant but moderate reduction of the composite peak VAS score of 1.34 points (Cl(95%): 0.12–2.56; P= 0.033) during treatment with ondansetron as compared to placebo (treatment effect). In addition, a period effect was observed: a reduction of composite peak VAS score by 1.26 points (Cl(95%): 0.04–2.48; P = 0.044) was seen in the second treatment period as compared to the first period, irrespective of the kind of treatment. Although under treatment with ondansetron a significant improvement of itching as assessed by the VAS score was demonstrated, this treatment was not preferred over placebo by the patients. Conclusions The 5-hydroxytryptamine receptor type 3 antagonist ondansetron has a small, but significant positive effect on pruritus in chronic liver disease as compared to placebo.

Prevalence and clinical importance of hypertransaminasaemia in coeliac disease.

OBJECTIVE To assess the prevalence and potential pathogenetic factors of hypertransaminasaemia in patients with coeliac disease prior to initiation of a gluten-free diet (GFD) and to assess the course of transaminases on a GFD. PATIENTS A retrospective study was made of 178 patients with coeliac disease (130 women, 48 men; median age 36 years; range 17-84 years) at the gastroenterological department of a university hospital. METHODS Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured prior to initiation of a GFD and at 3, 6 and 12 months of GFD. Intestinal permeability, a test for functional integrity of the small bowel, was investigated before starting a GFD in 116 patients by an oral test using lactulose and mannitol. RESULTS In 72 patients (40.4%) AST and/or ALT were increased prior to initiation of a GFD. Within 1 year on a GFD ALT and AST normalized except in eight cases (4.6%). The intestinal permeability index (% lactulose/% mannitol in 5 h urine) was higher in patients with elevated (median 0.34; range 0.03-1.43) than in patients with normal transaminases (0.11; 0.02-1.28) (P < 0.0001) and correlated with AST (tau = 0.34; P < 0.0001) and ALT (tau = 0.32; P < 0.0001). In five cases with hypertransaminasaemia a liver biopsy was performed prior to initiation of a GFD. Two patients had mild to moderate hepatitis with septal fibrosis. The other three had minimal lymphocytic infiltrates of the portal tracts. Inflammatory alterations of the bile ducts were not found. CONCLUSION Hypertransaminasaemia before GFD is frequent in coeliac patients, correlates with intestinal permeability and normalizes on a GFD in most patients. In cases of persistently elevated liver function tests of unknown origin underlying coeliac disease should be considered.

Predominance of the HLA-H Cys282Tyr mutation in Austrian patients with genetic haemochromatosis

BACKGROUND/AIMS Genetic haemochromatosis is the most common autosomal recessive disorder in Northern European populations. A major histocompatibility complex class I-like gene, HLA-H, has been proposed to be responsible for genetic haemochromatosis. The prevalence of HLA-H gene mutations 282(TGC; Cys/TAC; Tyr) and 63(CAT; His/GAT; Asp) was determined in patients of Austrian origin. METHODS DNA extracted from the blood of 40 Austrian patients and 271 controls was used to amplify HLA-H gene fragments by the polymerase chain reaction method. The base changes responsible for mutations Cys282Tyr and His63Asp alter recognition sites for restriction enzymes SnaB I and Bcl I, respectively. Digestion products were separated by agarose gel electrophoresis and visualised by ethidium bromide staining. RESULTS Thirty-one (77.5%) genetic haemochromatosis patients were homozygous for mutation Cys282Tyr and three compound heterozygous for mutations Cys282Tyr and His63Asp. One patient was homozygous for mutation His63Asp but normal for mutation Cys282Tyr. Four patients were normal at both genetic loci and one patient was heterozygous for mutation His63Asp. One control subject homozygous for mutation Cys282Tyr was found on investigation to fulfill diagnostic criteria for haemochromatosis. Eight control subjects homozygous for mutation His63Asp showed no biochemical or clinical evidence of haemochromatosis indicating that this variant is not directly responsible for haemochromatosis. Absence of the Cys282Tyr mutation in six genetic haemochromatosis patients with distinct haplotypes indicates mutations within the HLA-H gene or at alternative genetic loci are the cause of genetic haemochromatosis in these patients. CONCLUSIONS The HLA-H Cys282Tyr defect is likely to play a key role in the pathogenesis of haemochromatosis in most patients. Predominance of a single HLA-H gene mutation in haemochromatosis allows presymptomatic screening by genotypic analysis.

Members of the glutathione S-transferase gene family are antigens in autoimmune hepatitis.

BACKGROUND & AIMS Autoimmmune hepatitis (AIH), a chronic liver disorder, can be classified into two subtypes on the basis of the specificities of circulating autoantibodies. Type I AIH is defined by antibodies to nuclear and/or smooth muscle antigens (SMA), and type II is characterized by antibodies to cytochrome P450IID6. There is an additional type of AIH characterized by antibodies to a cytosolic soluble liver antigen (SLA), which can occur alone or in combination with antinuclear antibodies and SMA. The aim of this study was to identify the reactive antigen in SLA, a heterogenous cytosolic fraction consisting of at least 100 extremely soluble proteins. METHODS Sera from 31 patients with AIH reacting with SLA and from 30 disease controls were tested. The immunoreactive antigens were determined using immunoprecipitation and immunoblotting after one- and two-dimensional polyacrylamide gel electrophoresis. The antigens were identified by microsequencing of the corresponding protein spots. RESULTS Twenty-five of 31 anti-SLA-positive sera (80, 7%) reacted with a set of proteins ranging from 25 to 27 kilodaltons that were identified as three subunits of glutathione S-transferases: Ya, Yb1, and Yc. CONCLUSIONS Glutathione S-transferase subunit proteins represent the major autoantigen in anti-SLA-positive AIH. This new finding permits the establishment of standardized immunoassays for routine diagnosis.

Antibodies to nuclear lamins in autoimmune liver disease

Antibodies to nuclear lamins were detected in sera of patients with autoimmune liver disease. In indirect immunofluorescence tests, these sera revealed staining of the nuclear periphery. Using isolated nuclei, nuclear matrices, nuclear lamina-pore complexes, and chromatographically purified lamins as antigen source, the nuclear lamins A, B, and C were identified as reactive antigens in immunoblotting experiments. The lamins were also identified by 2-D gel electrophoresis. Antibodies to nuclear lamins occurred in 12 of 16 cases of active lupoid hepatitis, but not in 35 patients with the disease in remission. However, only 3 of 37 sera of patients with primary biliary cirrhosis contained anti-lamin antibodies. Autoimmune liver disease sera reacted preferentially with lamins A/C and less frequently with lamin B or lamins A/B/C.

The melatonin receptor subtype MT1 is expressed in human gallbladder epithelia

Abstract: Based on the fact that human bile and, particularly gallbladder bile, contains high physiological levels of the antioxidant melatonin, the aim of this study was to investigate whether the melatonin receptor MT1 is present in human gallbladder. Expression and localization of MT1 was assessed by RT‐PCR, Western blotting and immunofluorescence analysis in gallbladder samples from patients with cholelithiasis and with advanced gallbladder carcinoma. Additionally, we monitored mRNA expression of the two key enzymes of melatonin synthesis, i.e. arylalkylamine‐N‐acetyltransferase (AANAT) and hydroxyindole‐O‐methyltransferase (HIOMT). MT1 mRNA and protein were present in all cholelithiasis (n = 10) and gallbladder carcinoma (n = 5) samples. As indicated from RT‐PCR and Western blot studies, MT1 is located in gallbladder epithelia. Epithelial expression was further proven by immunofluorescence staining of MT1 in paraffin‐embedded cholelithiasis and gallbladder carcinoma sections. Analysis of AANAT and HIOMT mRNA expression showed that HIOMT mRNA is present in gallbladder. Surprisingly, AANAT was not detectable under conditions where it was found in a human colon specimen. The absence of AANAT suggests that in human gallbladder, HIOMT might be involved in the formation of 5‐hydroxytryptamine products other than melatonin. In summary, our results provide the first evidence for the presence of MT1 in human gallbladder epithelia. Therefore, in addition to its profound antioxidative effects in the biliary system, melatonin might also act through MT1‐mediated signal transduction pathways. Thereby, it might be involved in the regulation of gallbladder function.