Antonio Atilio Laudanna

Genetic heterogeneity in susceptibility to autoimmune hepatitis types 1 and 2

OBJECTIVES:Susceptibility to autoimmune hepatitis (AIH) type 1 has been associated with DRB1*03, DRB1*04, and DRB3 alleles in European and North-American whites, with DRB1*04 in Japan, and with DRB1*04 and DRB1*13 in Latin America. Very few studies have been performed on AIH type 2. The aim of the present study was to evaluate the association of AIH types 1 and 2 with HLA-DR and DQ loci.METHODS:We performed HLA-DRB and -DQB1 typing by polymerase chain reaction amplification with sequence-specific primers (PCR-SSP) in 139 AIH patients. Most had AIH type 1 associated with circulating anti-smooth muscle antibody with F-actin specificity or antinuclear antibody. Twenty-eight patients presented AIH type 2 with anti-liver/kidney microsome type 1 or anti-liver cytosol type 1 antibodies.RESULTS:We observed a significant increase of DRB1*13 (70%vs 26% of controls, p < 0.00001) and DRB3 (93%vs 69% of controls, p < 0.00001) in AIH type 1 patients. Analysis of patients without DRB1*13 disclosed a secondary association with DRB1*03 (70%vs 30% of controls, p= 0.0001) and either the DRB1*13 or the DRB1*03 alleles were present in the majority of these patients (91%vs 48% of controls, p= 0.001). Comparison of DRB1*13- and DRB1*03-positive subjects revealed that the former alleles conferred susceptibility to younger patients with AIH type 1. DQB1 typing showed a significant increase in DQB1*06 (68%vs 41% of controls, p= 0.00007) in strong linkage disequilibrium with DRB1*13, and a decrease in DQB1*0301 (8%vs 47% of controls, pc= 0.0003). On the other hand, HLA typing of patients with AIH type 2 disclosed a significant increase in the DRB1*07 (68%vs 20% of controls, pc < 0.00014), DRB4 (79%vs 43% of controls, pc= 0.004), and DQB1*02 (86%vs 42%, p= 0.00002) alleles. After exclusion of DRB1*07, a secondary association with HLA-DRB1*03 was further observed in these patients (78%vs 30%, p= 0.007) and most of them had either DRB1*07 or DRB1*03 (93%vs 44% of controls, pc < 0.0001).CONCLUSIONS:Our data indicate that predisposition to AIH types 1 and 2 is associated, respectively, with the DRB1*13 or DRB1*03 and DRB1*07 or DRB1*03 alleles, and suggest that protection against type 1 disease may be conferred by DQB1*0301. In addition, the cluster of DRB1*13 in children with AIH type 1 also supports the concept that different HLA alleles might influence the onset of the disease.

Oxidative stress in the pathogenesis of nonalcoholic fatty liver disease, in rats fed with a choline-deficient diet

Background/Aim: The pathogenesis of Nonalcoholic Fatty Liver Disease remains largely unknown, but oxidative stress seems to be involved. The aim of this study was to evaluate the role of oxidative stress in experimental hepatic steatosis induced by a choline‐deficient diet. Methods: Fatty liver disease was induced in Wistar rats by a choline‐deficient diet. The animals were randomized into three groups: I (G1) and II (G2), n=6 each ‐ fed with a choline‐deficient diet for four and twelve weeks respectively; Group III (control‐G3; n=6) ‐ fed with a standard diet for twelve weeks. Samples of plasma and liver were submitted to biochemical, histological and oxidative stress analysis. Variables measured included serum levels of aminotransferases (AST, ALT), cholesterol and triglycerides. Oxidative stress was measured by lucigenin‐enhanced luminescence and the concentration of hydroperoxides (CE‐OOH‐cholesteryl ester) in the liver tissue. Results: We observed moderate macro‐ and microvesicular fatty change in periportal zones G1 and G2 as compared to controls (G3). In G2, fatty change was more severe. The inflammatory infiltrate was scanty and no fibrosis was seen in any group. There was a significant increase of AST and triglycerides in G1 and G2 as compared to control group G3. The lucigenin‐amplified luminescence (cpm/mg/min × 103) was significantly increased in G1 (1393±790) and G2 (7191±500) as compared to controls (513±170), p<0.05. The concentrations of CE‐OOH were higher in G1 (5.7±0.9 nmol/mg protein) as compared to control (2.6±0.7 nmol/mg protein), p<0.05. Conclusion: 1) Oxidative stress was found to be increased in experimental liver steatosis; 2) The production of reactive oxygen species was accentuated when liver steatosis was more severe; 3) The alterations produced by oxidative stress could be an important step in the pathogenesis of nonalcoholic fatty liver disease.

Unexpected clinical remission of cholestasis after rifampicin therapy in patients with normal or slightly increased levels of γ-glutamyl transpeptidase

Objective:Rifampicin is an effective drug against pruritus in intrahepatic cholestasis. However, there is no specific hepatic disease in which its use could cause undoubtedly biochemical improvement. The aim of this study was to describe patients with complete remission of cholestatic symptoms after rifampicin therapy.Methods:We reported three female patients with intrahepatic cholestasis with no evidence of viral, metabolic, or autoimmune liver diseases. Total bilirubin levels ranged from 13.2 to 27.2 mg/dl (before the first treatment with rifampicin), and in all of them γ-glutamyl transpeptidase values were within the normal range or slightly increased. Rifampicin therapy was administered orally, without any concomitant drug, with an effective dosage of 5–17 mg/kg/day.Results:In all patients, pruritus ceased completely and bilirubin returned to normal values. The symptoms recurred after rifampicin withdrawal on, at least, three occasions in each patient, and these symptoms were always eliminated after its reintroduction. The patients had a total of 16 cholestatic episodes during a follow-up of 8 yr, with a complete clinical recovery in all of them. Undergoing therapy with a suitable dosage of rifampicin, none of the patients had a cholestatic crisis even during a period for as long as 12 months. The diagnosis of two patients was consistent with benign recurrent intrahepatic cholestasis, and it was not well defined in the remaining.Conclusion:Rifampicin may induce clinical remission, and perhaps prevent clinical relapses of intrahepatic cholestasis with normal or slightly increased levels of γ-glutamyl transpeptidase.

Prognostic significance of intraperitoneal free cancer cells obtained by laparoscopic peritoneal lavage in patients with gastric cancer

Laparoscopy is a safe and useful method for examining the local extent and regional spread of disease in patients with gastric cancer. Peritoneal dissemination remains a frequent type of recurrence after surgical treatment. The aim of this study was to determine the prognostic value of intraperitoneal free cancer cells (IFCCs) detected by laparoscopic peritoneal lavage. Forty-nine patients with advanced gastric cancer underwent laparoscopy with cytologic examination for staging. Peritoneal lavage was performed when ascites was not present. Aspirated fluid from the peritoneal cavity was centrifuged and subjected to cytologic examination using Giemsa and Papanicolaou staining methods. Patients were surgically treated and followed for a minimum of 5 years. IFCCs were detected in 41% of the patients. In eight cases (16.3 %) laparoscopy revealed carcinomatosis and/or multiple liver metastases, so laparotomy was not performed. Patterns of recurrence after curative resection included the following: peritoneal (n = 3), local (n = 4), liver (n = 1), and other (n = 1). All patients who tested positive for IFCCs had peritoneal recurrence. The absence of IFCCs was associated with improved overall survival (2 1 months for a 95% confidence interval of 7.4 to 34.6 vs 4 months for a 95% confidence interval of 2.4 to 5.6). Overall survival adjusted for type of resection also demonstrated a favorable outcome for patients who were negative for IFCCs. The following conclusions were drawn: (1) laparoscopic peritoneal lavage cytology may be useful in identifying patients at high risk for peritoneal relapses and may alter treatment, and (2) IFCCs provide additional prognostic information in patients with gastric cancer.

Different degrees of malnutrition and immunological alterations according to the aetiology of cirrhosis: a prospective and sequential study

ObjectivesIn this work we investigated how immunological dysfunction and malnutrition interact in alcoholic and viral aetiologies of cirrhosis.MethodsTo investigate the matter, 77 cirrhotic patients divided in three aetiologies [Alcohol, HCV and Alcohol + HCV) and 32 controls were prospectivelly and sequentially studied. Parameters of humoral immunity (Components 3 and 4 of seric complement and immunoglobulins A M, G and E) and of cellular immunity (total leukocytes and lymphocytes in peripheral blood, T lymphocytes subpopulations, CD4+ and CD8+, CD4+/CD8+ ratio and intradermic tests of delayed hypersensitivity), as well as nutrititional parameters: anthropometric measures, serum albumin and transferrin were evaluated.ResultsMultiple statistical comparisons showed that IgM was higher in HCV group; IgG was significantly elevated in both HCV and Alcohol + HCV, whereas for the Alcohol group, IgE was found at higher titles. The analysis of T- lymphocytes subpopulations showed no aetiologic differences, but intradermic tests of delayed hypersensitivity did show greater frequency of anergy in the Alcohol group. For anthropometric parameters, the Alcohol +HCV group displayed the lowest triceps skinfold whereas creatinine – height index evaluation was more preserved in the HCV group. Body mass index, arm muscle area and arm fat area showed that differently from alcohol group, the HCV group was similar to control.ConclusionSignificant differences were found among the main aetiologies of cirrhosis concerning immunological alterations and nutritional status: better nutrition and worse immunology for HCV and vice-versa for alcohol.

Cytotoxic T lymphocyte antigen–4 gene polymorphisms do not confer susceptibility to autoimmune hepatitis types 1 and 2 in Brazil

OBJECTIVES:Susceptibility to autoimmune hepatitis (AIH) has been linked to different HLA-DR antigens in distinct populations. Recently, an A–G polymorphism in exon 1 of the cytotoxic T lymphocyte antigen-4 (CTLA-4) gene was associated with predisposition to AIH type 1 (AIH-1) in white individuals in North America. This polymorphism has been associated with several other autoimmune diseases, presumably because of its effect in the expression of CTLA-4, an adhesion molecule that downregulates peripheral T cell responses. The aims of this study were to assess the frequency of CTLA-4 genotypes in Brazilian patients with AIH-1 and AIH type 2 (AIH-1), as well as to investigate the influence of these genotypes in disease expression.METHODS:Determination of CTLA-4 genotypes was carried out in 106 patients with AIH-1, 26 subjects with AIH-2, and 67 healthy control subjects by polymerase chain reaction (PCR)–based techniques.RESULTS:No difference in the distribution of CTLA-4 genotypes was observed in subjects with AIH-1 and AIH-2 as compared to healthy controls. Patients with AIH-1 and AIH-2 with the GG genotype exhibited lower γ-globulin and ALT levels, respectively.CONCLUSIONS:Susceptibility to AIH-1 and AIH-2 in Brazilian patients is not influenced by exon 1 CTLA-4 gene polymorphisms at position 49.

Preoperative serum levels of ca 72-4, cea, ca 19-9, and Alpha-fetoprotein in patients with gastric cancer

INTRODUCTION The clinical importance of preoperative serum levels of CA 72-4, carcinoembryonic antigen (CEA), CA 19-9, and alpha-fetoprotein (AFP) was prospectively evaluated in 44 patients with gastric cancer. METHOD The serum tumor marker levels were determined by commercial radioimmunoassay kits. Positivity for CA 72-4 (>4 U/mL), CEA (>5 ng/mL), CA 19-9 (>37 U/mL), and AFP (>10 ng/mL) were correlated according to the stage, histology, and lymph node metastasis. RESULTS AND DISCUSSION CA 72-4 showed a higher positivity rate for gastric cancer (47.7%) than CEA (25%), CA 19-9 (25%), and AFP (0%). The combination of CA 72-4 with CEA and CA 19-9 increased the sensitivity to 61.4%. The positivity rates of CA 72-4 in patients at stages I and II (initial disease) and in patients at stages III and IV (advanced disease) were 9% and 60.6%, respectively (P < 0.005). No correlation was found between CEA and CA 19-9 levels and the stage of gastric cancer. There was a tendency of positivity for CA 72-4 to suggest lymph node involvement, but it was not significant (P = 0.075). Serum levels of tumor markers did not show a correlation with the histological types of gastric cancer. CONCLUSION Preoperative serum levels of CA 72-4 provided a predictive value in indicating advanced gastric cancer.

Analysis of major histocompatibility complex and CTLA-4 alleles in Brazilian patients with primary biliary cirrhosis

Background and Aims:  Predisposition to primary biliary cirrhosis (PBC) has been classically linked to HLA‐DRB1 locus. However, the presence of the HLA‐DRB1*08 antigen has been reported in less than one‐third of PBC patients from Northern Europe and Japan. Recently, polymorphisms in the tumor necrosis factor alpha (TNFA) gene promoter at position −308 and in exon 1 of the cytotoxic T lymphocyte antigen‐4 (CTLA‐4) gene at position 49 have been associated with susceptibility to PBC in Caucasians. In addition, the presence of HLA‐DRB1*08 and the TNFA*1 allele was also linked to progression to end‐stage liver disease. The aims of the present study were to investigate the frequencies of HLA‐DR and DQ antigens and TNFA and CTLA‐4 alleles in PBC patients from a different genetic background, as well as to assess the role of TNFA alleles and HLA‐DR antigens in disease progression.

Autoimmune hepatitis in Brazilian patients is not linked to tumor necrosis factor α polymorphisms at position −308

BACKGROUND/AIMS Susceptibility to autoimmune hepatitis (AIH) has been linked to different HLA-DR antigens. Recently, AIH type 1 was associated with polymorphisms in the tumor necrosis factor alpha gene promoter (TNFA) at position -308. In this respect, the frequency of the TNFA*2 allele, in linkage disequilibrium with HLA-DRB1*0301, was shown to be significantly increased in whites with AIH type 1. The aim of this study was to assess the role of TNFA alleles in conferring susceptibility to AIH, studying a population where the disease is not primarily associated with HLA-DRB1*03. METHODS The determination of HLA-DRB1 and TNFA alleles was performed in 92 patients with AIH type 1, 29 subjects with AIH type 2 and 83 healthy controls by polymerase chain reaction-based techniques. RESULTS The distribution of TNFA alleles was similar in patients with AIH types 1 and 2, when compared with controls. In addition, the TNFA*2 allele was identified in patients carrying HLA-DR antigens other than HLA-DRB1*03. Interestingly, higher gammaglobulin levels were observed in TNFA*2 positive patients. CONCLUSIONS Our data indicate that susceptibility to AIH remains primarily linked to the HLA-DRB1 locus, and suggest that the association of AIH with TNFA*2 previously observed in whites might be secondary to a linkage disequilibrium with HLA-DRB1*0301.

Lack of evidence for the pathogenic role of iron and HFE gene mutations in Brazilian patients with nonalcoholic steatohepatitis

The hypothesis of the role of iron overload associated with HFE gene mutations in the pathogenesis of nonalcoholic steatohepatitis (NASH) has been raised in recent years. In the present study, biochemical and histopathological evidence of iron overload and HFE mutations was investigated in NASH patients. Thirty-two NASH patients, 19 females (59%), average 49.2 years, 72% Caucasians, 12% Mulattoes and 12% Asians, were submitted to serum aminotransferase and iron profile determinations. Liver biopsies were analyzed for necroinflammatory activity, architectural damage and iron deposition. In 31 of the patients, C282Y and H63D mutations were tested by PCR-RFLP. Alanine aminotransferase levels were increased in 30 patients, 2.42 1.12 times the upper normal limit on average. Serum iron concentration, transferrin saturation and ferritin averages were 99.4 31.3 g/dl, 33.1 12.7% and 219.8 163.8 g/dl, respectively, corresponding to normal values in 93.5, 68.7 and 78.1% of the patients. Hepatic siderosis was observed in three patients and was not associated with architectural damage (P = 0.53) or with necroinflammatory activity (P = 0.27). The allelic frequencies (N = 31) found were 1.6 and 14.1% for C282Y and H63D, respectively, which were compatible with those described for the local population. In conclusion, no evidence of an association of hepatic iron overload and HFE mutations with NASH was found. Brazilian NASH patients comprise a heterogeneous group with many associated conditions such as hyperinsulinism, environmental hepatotoxin exposure and drugs, but not hepatic iron overload, and their disease susceptibility could be related to genetic and environmental features other than HFE mutations.