Mannan-binding lectin MBL2 gene polymorphism in chronic hepatitis C: association with the severity of liver fibrosis and response to interferon therapy

Hepatitis C virus (HCV) is a major cause of hepatic disease and of liver transplantation worldwide. Mannan-binding lectin (MBL), encoded by the MBL2 gene, can have an important role as an opsonin and complement activating molecule in HCV persistence and liver injury. We assessed the MBL2 polymorphism in 102 Euro-Brazilian patients with moderate and severe chronic hepatitis C, paired for gender and age with 102 HCV seronegative healthy individuals. Six common single nucleotide polymorphisms in the MBL2 gene, three in the promoter (H/L, X/Y and P/Q) and three in exon 1 (A, the wild-type, and B, C or D also known as O) were evaluated using real-time polymerase chain reaction with fluorescent hybridization probes. The concentration of MBL in plasma was measured by enzyme-linked immunosorbent assay. The frequency of the YA/YO genotype was significantly higher in the HCV patients compared with the controls (P = 0.022). On the other hand, the genotypes associated with low levels of MBL (XA/XA, XA/YO and YO/YO) were decreased significantly in the patients with severe fibrosis (stage F4), when compared with the patients with moderate fibrosis (stage F2) (P = 0.04) and to the control group (P = 0.011). Furthermore, MBL2 genotypes containing X or O mutations were found to be associated with non-responsiveness to pginterferon and ribavirin treatment (P = 0.023). MBL2 polymorphisms may therefore be associated not only with the development of chronic hepatitis C, but also with its clinical evolution and response to treatment.

Incidence and predictors of severe liver fibrosis in HIV-infected patients with chronic hepatitis C in Brazil

The aim of this study was to examine the incidence and factors associated with the severity of liver fibrosis in 234 coinfected patients in Brazil. Patients were cared for in our clinic, from 1996 to 2004. Eligible patients were defined as patients with documented HIV and hepatitis C virus (HCV) infections and had previously undergone a liver biopsy. Patients with persistently normal alanine aminotransferase (ALT) were also included. The variables selected for study were age, gender, risk category, history of high alcohol consumption, CD4(+) T cell count, antiretroviral therapy usage, HCV genotype and duration of HCV infection. Stage of fibrosis was scored as follows: F0, no fibrosis; F1, portal fibrosis with no septa; F2, portal fibrosis with few septa; F3, bridging fibrosis with many septa; and F4, cirrhosis. The liver fibrosis stage was F3 in 39 (16.6%) and F4 in 20(8.5%) patients. Among patients with normal ALT, the liver fibrosis stage was F3-F4 in three patients (5.6%). Predictors of severe liver fibrosis (17344) by multivariate analysis were age (older patients) and genotype 3 (genotype I odds ratio [OR], 0.28; 95% confidence interval [0], 0.12 0.65). In summary, in the present study severe liver fibrosis was found in 25% of our patients and was associated with an age of more than 38 years at the time of liver biopsy as well as, HCV genotype 3. No differences were found with respect to CD4(+) T cell counts although patients with a CD4(+) T cell count greater than 50 were excluded.

Homocysteine induces oxidative stress, inflammatory infiltration, fibrosis and reduces glycogen/glycoprotein content in liver of rats

Hyperhomocysteinemia has been related to various diseases, including homocystinuria, neurodegenerative and hepatic diseases. In the present study we initially investigated the effect of chronic homocysteine administration on some parameters of oxidative stress, named total radical-trapping antioxidant potential, total antioxidant reactivity, catalase activity, chemiluminescence, thiobarbituric acid-reactive substances, and total thiol content in liver of rats. We also performed histological analysis, evaluating steatosis, inflammatory infiltration, fibrosis, and glycogen/glycoprotein content in liver tissue sections from hyperhomocysteinemic rats. Finally, we evaluated the activities of aminotransferases in liver and plasma of hyperhomocysteinemic rats. Wistar rats received daily subcutaneous injection of Hcy from their 6th to their 28th day of life. Twelve hours after the last injection the rats were sacrificed, liver and plasma were collected. Hyperhomocysteinemia decreased antioxidant defenses and total thiol content, and increased lipid peroxidation in liver of rats, characterizing a reliable oxidative stress. Histological analysis indicated the presence of inflammatory infiltrate, fibrosis and reduced content of glycogen/glycoprotein in liver tissue sections from hyperhomocysteinemic rats. Aminotransferases activities were not altered by homocysteine. Our data showed a consistent profile of liver injury elicited by homocysteine, which could contribute to explain, at least in part, the mechanisms involved in human liver diseases associated to hyperhomocysteinemia. (C) 2009 ISDN. Published by Elsevier Ltd. All rights reserved.

Mate attenuates DNA damage and carcinogenesis induced by diethylnitrosamine and thermal injury in rat esophagus

Drinking hot mate has been associated with risk for esophageal cancer in South America. Thus. the aims of this study were to evaluate the modifying effects of mate intake on DNA damage and esophageal carcinogenesis induced by diethylnitrosamine (DEN) and thermal injury (TI) in male Wistar rats. At the initiation phase of carcinogenesis, rats were treated with DEN (8 x 80 mg/kg) and submitted to TI (water at 65 degrees C, 1 ml/rat, instilled into the esophagus). Concomitantly, the animals received mate (2.0% w/v) for 8 weeks. Samples of peripheral blood were collected 4 h after the last DEN application for DNA damage analysis. At weeks 8 and 20, samples from esophagus and liver were also collected for histological and immunohistochemical analysis. Mate significantly decreased DNA damage in leukocytes, cell proliferation rates in both esophagus and liver and the number of preneoplastic liver lesions from DEN/TI-treated animals at week 8. A significant lower incidence of esophageal papillomas and liver adenomas and tumor multiplicity was observed in the animals previously treated with mate at week 20. Thus, mate presented protective effects against DNA damage and esophageal and liver carcinogenesis induced by DEN. (C) 2009 Elsevier Ltd. All rights reserved.

Effect of a thrombin receptor (protease-activated receptor 1, PAR-1) gene polymorphism in chronic hepatitis C liver fibrosis

Background and Aim: Tissue injury leads to activation of coagulation and generation of thrombin. Inhibition of thrombin receptor protease-activated receptor 1 (PAR-1) has been shown to reduce liver fibrosis in animals. This study aimed to evaluate the effect of PAR-1 gene polymorphism on rate of liver fibrosis (RF) in chronic hepatitis C. Methods: Polymorphisms studied: C > T transition 1426 bp upstream of translation start site (-1426C/T), 13 bp repeat of preceding -506 5`-CGGCCGCGGGAAG-3` sequence (-506I/D), and A > T transversion in intervening sequence (IVS) 14 bp upstream of exon-2 start site (IVS-14A/T). A total of 287 European and 90 Brazilian patients were studied. Results: 1426C/T polymorphism: There was a trend to higher RF in patients with the TT genotype (P = 0.06) and an association between genotype CC and slow fibrosis (P = 0.03) in Europeans. In males, RF was significantly higher in those with the TT genotype compared to CT (P = 0.003) and CC (P = 0.007). There was a significant association between TT and fast fibrosis (P = 0.04). This was confirmed in an independent cohort of Brazilians where RF was higher in TT than in CC (P = 0.03). Analysis of -506I/D showed no difference in RF and distribution of slow/fast fibrosis among different genotypes in both populations. Analysis of IVS-14A/T showed no difference between genotypes. Conclusion: In conclusion, these findings suggest that PAR-1 receptor polymorphisms influence the progression of liver fibrosis.

Liver HLA-G expression is associated with multiple clinical and histopathological forms of chronic hepatitis B virus infection

As the mechanisms leading to the persistence of hepatitis B virus (HBV) infection are poorly understood and as the histocompatibility leucocyte antigen (HLA)-G is well described as a tolerogenic molecule, we evaluated HLA-G expression in 74 specimens of HBV liver biopsies and in 10 specimens obtained from previously healthy cadaver liver donors. HBV specimens were reviewed and classified by the METAVIR score, and HLA-G expression was assessed by immunohistochemistry. No HLA-G expression was observed in control hepatocytes. In contrast, 57 (77%) of 74 HBV specimens showed soluble and membrane-bound HLA-G expression in hepatocytes, biliary epithelial cells or both. No associations between the intensity of HLA-G expression and patient age or gender, HBeAg status, severity of liver fibrosis, and grade of histological findings were observed. Although significance was not reached (P = 0.180), patients exhibiting HLA-G expression presented a higher median HBV DNA viral load (105 copies/mL) than those who did not express HLA-G (103.7 copies/mL). These results indicate that HLA-G is expressed in most cases of chronic HBV infection in all stages and may play a role in the persistency of HBV infection.

PKC beta II inhibition attenuates myocardial infarction induced heart failure and is associated with a reduction of fibrosis and pro-inflammatory responses

Protein kinase C beta II (PKC beta II) levels increase in the myocardium of patients with end-stage heart failure (HF). Also targeted overexpression of PKC beta II in the myocardium of mice leads to dilated cardiomyopathy associated with inflammation, fibrosis and myocardial dysfunction. These reports suggest a deleterious role of PKC beta II in HF development. Using a post-myocardial infarction (MI) model of HF in rats, we determined the benefit of chronic inhibition of PKC beta II on the progression of HF over a period of 6 weeks after the onset of symptoms and the cellular basis for these effects. Four weeks after MI, rats with HF signs that were treated for 6 weeks with the PKC beta II selective inhibitor (beta IIV5-3 conjugated to TAT(47-57) carrier peptide) (3 mg/kg/day) showed improved fractional shortening (from 21% to 35%) compared to control (TAT(47-57) carrier peptide alone). Formalin-fixed mid-ventricle tissue sections stained with picrosirius red, haematoxylin and eosin and toluidine blue dyes exhibited a 150% decrease in collagen deposition, a two-fold decrease in inflammation and a 30% reduction in mast cell degranulation, respectively, in rat hearts treated with the selective PKC beta II inhibitor. Further, a 90% decrease in active TGF beta 1 and a significant reduction in SMAD2/3 phosphorylation indicated that the selective inhibition of PKC beta II attenuates cardiac remodelling mediated by the TGF-SMAD signalling pathway. Therefore, sustained selective inhibition of PKC beta II in a post-MI HF rat model improves cardiac function and is associated with inhibition of pathological myocardial remodelling.

Liver transplantation and quality of life: relevance of a specific liver disease questionnaire

Aim: A positive effect of liver transplantation on health-related quality of life (HRQOL) has been well documented in previous studies using generic instruments. Our aim was to re-evaluate different aspects of HRQOL before and after liver transplantation with a relatively new questionnaire the `liver disease quality of life` (LDQOL). Methods: The LDQOL and the Short Form 36 (SF-36) questionnaires were applied to ambulatory patients, either in the transplant list (n=65) or after 6 months to 5 years of liver transplant (n=61). The aetiology of cirrhosis, comorbidities, model for end-stage liver disease (MELD) Child-Pugh scores and recurrence of liver disease after liver transplantation were analysed using the Mann-Whitney and Kruskall-Wallis tests. Results: In patients awaiting liver transplantation, MELD scores >= 15 and Child-Pugh class C showed statistically significant worse HRQOL, using both the SF-36 and the LDQOL questionnaires. HRQOL in pretransplant patients was found to be significantly worse in those with cirrhosis owing to hepatitis C (n=30) when compared with other aetiologies (n=35) in 2/7 domains of the SF-36 and in 7/12 domains of the LDQOL. Significant deterioration of HRQOL after recurrence of hepatitis C post-transplant was detected with the LDQOL questionnaire although not demonstrated with the SF-36. The statistically significant differences were in the LDQOL domains: symptoms of liver disease, concentration, memory and health distress. Conclusions: The LDQOL, a specific instrument for measuring HRQOL, has shown a greater accuracy in relation to liver symptoms and could demonstrate, with better reliability, impairments before and after liver transplantation.

Peptides containing T cell epitopes, derived from Sm14, but not from paramyosin, induce a Th1 type of immune response, reduction in liver pathology and partial protection against Schistosoma mansoni infection in mice

Sm14 and paramyosin are two major Schistosoma mansoni vaccine candidate antigens. Recently, we have identified Sm14 and paramyosin epitopes that are recognized by T cells of resistant individuals living in endemic areas for schistosomiasis. Herein, mice were immunized with these peptides separately or in association in order to evaluate their vaccine potential. Immunization of mice with Sm14 peptides alone or mixed with paramyosin peptides was able to induce 26%-36.7% or 28%-29.2% of worm burden reduction, 67% or 46% of intestinal eggs reduction and also 54%-61% or 43%-52% of liver pathology reduction, respectively. Protection was associated with a Th1 type of immune response induced by Sm14 peptide immunization. In contrast, paramyosin peptide vaccination did not engender protective immunity or liver pathology reduction and immunization was associated with a Th2 type of immune response. (C) 2008 Elsevier B.V. All rights reserved.

Leprosy in transplant recipients: report of a case after liver transplantation and review of the literature

P>Leprosy still is an important public health problem in several parts of the world including Brazil. Unlike the diseases caused by other mycobacteria, the incidence and clinical presentation of leprosy seems little affected in immunosuppressed patients. We report the first case, to our knowledge, of a liver transplant patient who developed multi-bacillary leprosy. The patient presented with papules and infiltrated plaques with loss of sensation suggestive of leprosy 3.5 years after living-related liver transplantation for autoimmune hepatitis. A skin biopsy showing non-caseating macrophagic granulomas, neuritis, and intact acid-fast bacilli on Fite-Faraco stain, confirmed the diagnosis of borderline lepromatous leprosy. The donor of the liver did not show any evidence of leprosy. During follow-up, the patient presented 2 episodes of upgrading leprosy type I reactions, 1 mild before leprosy treatment, and 1 moderate 3 months after receiving standard multi-drug treatment (rifampicin, clofazimine, and dapsone). These reactions were accompanied by increase in liver function tests, especially of canalicular enzymes. This reaction occurred despite the patient`s triple immunosuppression regimen. The moderate reaction was successfully treated with further immunosuppression (prednisone, 0.5 mg/kg). Currently, the patient is asymptomatic, off leprosy medication, with routine liver transplant follow-up. The dilemmas in diagnosis and management of such a case are discussed and the literature on leprosy in transplant recipients is reviewed.

Effects of Lecithin and Vitamin E Supplementation on Liver Steatosis and Oxidative Stress Induced by Chronic Ethanol Consumption in Rats

Oxidative stress and lipid peroxidation, associated with ethanol, are considered important pathogenic mechanisms in the formation of hepatic steatosis. The objective of the present study was to assess the effects of supplementation with lecithin and vitamin E on the oxidatives stress and hepatic steatosis induced in rats by chronic ethanol consumption. Fifty-two Wistar rats were divided into 4 experimental groups: control (AIN-93 diet), ethanol group (control diet plus a 20% hydroalcoholic solution), ethanol + vitamin E group (addition of 0.6% vitamin E to the diet plus a 20% hydroalcoholic solution); ethanol + soy lecithin group (addition of 5 % soy lecithin to the diet plus a 20% hydroalcoholic solution). At the end of 4 weeks the animals were sacrificed. The results showed a significantly smaller number of animals (p < 0.05) classified as having a low degree of steatosis in the ethanol + vitamin E group and ethanol + soy lecithin group compared to the ethanol group. In addition, the ethanol + soy lecithin group had a significantly lower concentration of hepatic fat (p < 0.05) than the ethanol group. A significant reduction of hepatic TBARS concentration (p < 0.05) was detected in the ethanol + vitamin E group compared to the ethanol group. Hepatic carbonyl concentration was significantly lower in the ethanol + soy lecithin group. However, hepatic GSH was significantly lower in the ethanol + vitamin E and ethanol + soy lecithin groups compared to the control group. In conclusion, supplementation with lecithin and vitamin E attenuated the hepatotoxic effects of chronic ethanol intake and contributed to a reduction of the progression of steatosis status.

Expression and distribution of connexin 32 in rat liver with experimentally induced fibrosis

The connexin 32 (Cx32) is a protein that forms the channels that promote the gap junction intercellular communication (GJIC) in the liver, allowing the diffusion of small molecules through cytosol from cell-to-cell. Hepatic fibrosis is characterized by a disruption of normal tissue architeture by cellular lesions, and may alter the GJIC. This work aimed to study the expression and distribution of Cx32 in liver fibrosis induced by the oral administration of dimethylnitrosamine in female Wistar rats. The necropsy of the rats was carried out after five weeks of drug administration. They presented a hepatic fibrosis state. Sections from livers with fibrosis and from control livers were submitted to immunohistochemical, Real Time-PCR and Western-Blot analysis to Cx32. In fibrotic livers the Cxs were diffusely scattered in the cytoplasm, contrasting with the control livers, where the Cx32 formed junction plaques at the cell membrane. Also it was found a decrease in the gene expression of Cx32 without reduction in the protein quantity when compared with controls. These results suggest that there the mechanism of intercellular communication between hepatocytes was reduced by the fibrotic process, which may predispose to the occurrence of a neoplastic process, taken in account that connexins are considered tumor suppressing genes.

Combination of N-acetylcysteine and metformin improves histological steatosis and fibrosis in patients with non-alcoholic steatohepatitis

Aim: There is no proven medical therapy for the treatment of non-alcoholic steatohepatitis (NASH). Oxidative stress and insulin resistance are the mechanisms that seem to be mostly involved in its pathogenesis. The aim of our study was to evaluate the efficacy of N-acetylcysteine (NAC) in combination with metformin (MTF) in improving the aminotransferases and histological parameters (steatosis, inflammation, hepatocellular ballooning, and fibrosis) after 12 months of treatment. Methods: Twenty consecutive patients (mean age 53 +/- 2 years [36-68] and body mass index [BMI] 29 [25-35]) with biopsy-proven NASH were enrolled in the study. NAC (1.2 g/day) and MTF (850-1000 mg/day) were given orally for 12 months. All patients underwent evaluation of serum aminotransferases, fasting lipid profile and serum glucose, anthropometric parameters, and nutritional status at 0 and 12 months. A low calorie diet was prescribed for all patients. Results: Serum alanine aminotransferase, high-density lipoprotein, insulin, and glucose concentrations and thehomeostasis model assessment-insulin resistance (HOMA-IR) index were reduced significantly at the end of study (P < 0.05). The BMI declined, but without statistical significance. Aspartate aminotransferase, gamma-glutamyl transferase, alkaline phosphatase, cholesterol, and triglycerides levels were not altered with the treatment. Liver steatosis and fibrosis decreased (P < 0.05), but no improvement was noted in lobular inflammation or hepatocellular ballooning. The NASH activity score was significantly improved after treatment. Conclusion: Based on the biochemical and histological evidence in this pilot study, NAC in combination with MTF appears to ameliorate several aspects of NASH, including fibrosis. Further studies of this form of combination therapy are warranted to assess its potential efficacy.

Morphological and Molecular Pathology of CCl(4)-Induced Hepatic Fibrosis in Connexin43-Deficient Mice

Gap junction channels, formed by connexins (Cx), are involved in the maintenance of tissue homeostasis, cell growth, differentiation, and development. Several studies have shown that Cx43 is involved in the control of wound healing in dermal tissue. However, it remains unknown whether Cx43 plays a role in the control of liver fibrogenesis. Our study investigated the roles of Cx43 heterologous deletion on carbon tetrachloride (CCl(4))-induced hepatic fibrosis in mice. We administered CCl(4) to both Cx43-deficient (Cx43(+/-)) and wild-type mice and examined hepatocellular injury and collagen deposition by histological and ultrastructural analyses. Serum biochemical analysis was performed to quantify liver injury. Hepatocyte proliferation was analyzed immunohistochemically. Protein and messenger RNA (mRNA) expression of liver connexins were evaluated using immunohistochemistry as well as immunoblotting analysis and quantitative real-time PCR. We demonstrated that Cx43(+/-) mice developed excessive liver fibrosis compared with wild-type mice after CCl(4)-induced chronic hepatic injury, with thick and irregular collagen fibers. Histopathological evaluation showed that Cx43(+/-) mice present less necroinflammatory lesions in liver parenchyma and consequent reduction of serum aminotransferase activity. Hepatocyte cell proliferation was reduced in Cx43(+/-) mice. There was no difference in Cx32 and Cx26 protein or mRNA expression in fibrotic mice. Protein expression of Cx43 increased in CCl(4)-treated mice, although with aberrant protein location on cytoplasm of perisinusoidal cells. Our results demonstrate that Cx43 plays an important role in the control and regulation of hepatic fibrogenesis. Microsc. Res. Tech. 74:421-429, 2011. (C) 2010 Wiley-Liss, Inc.

Hepatotrophic factors reduce hepatic fibrosis in rats

CONTEXT: Hepatic fibrosis occurs in response to several aggressive agents and is a predisposing factor in cirrhosis. Hepatotrophic factors were shown to stimulate liver growth and to restore the histological architecture of the liver. They also cause an improvement in liver function and accelerate the reversion of fibrosis before it progresses to cirrhosis. OBJECTIVE: To test the effects of hepatic fibrosis solution composed by amino acids, vitamins, glucose, insulin, glucagon and triiodothyronine on hepatic fibrosis in rats. METHODS: Fibrosis was induced in rats by gastric administration of dimethylnitrosamine (10 mg/kg) for 5 weeks. After liver biopsy, the rats received either hepatotrophic factors solution (40 mg/kg/day) or saline solution for 10 days by intraperitoneal injection. Blood samples and liver fragments were collected for hepatic function analysis, standard histopathology evaluation, and morphometric collagen quantification. RESULTS: Rats in the hepatotrophic factors group showed a decrease of the histopathological components of fibrosis and an increase of their hepatic mass (12.2%). There was no development of neoplasic lesions in both groups. Compared with the saline group, the hepatotrophic factors group also had a decrease of blood levels of hepatic-lesion markers (AST, ALT) and a decrease of collagen content in the portal spaces (31.6%) and perisinusoidal spaces (42.3%), as well as around the hepatic terminal vein (57.7%). Thus, hepatotrophic factors administration in the portal blood promoted a regenerative hepatic response, with an overall reduction of the volumetric density of collagen, improved hepatic function, and a general improvement in the histopathological aspects of fibrosis. CONCLUSION: Taken together, these results suggest the potential therapeutic use of this hepatotrophic factors solution to treat chronic liver diseases.