Original Research Article Humoral and cellular immune responses to modified hepatitis B plasmid DNA vaccine in mice

Purpose: To evaluate the immunogenicity and types of immune response of a quality-controlled modified recombinant hepatitis B surface antigen (HBsAg) plasmid encoding HBsAg in mice. Methods: The characterized plasmid DNA was used in the immunization of Balb/c mice. Three groups of mice were intramuscularly injected with three different concentrations (50, 25 and 10 μg/100 μL) of the modified plasmid. Humoral immune response was monitored by enzyme-linked immunosorbent assay (ELISA), while cellular immune response was investigated by analysis of spleen cytokine profile (TNFα, IFN γ and IL2) as well as CD69 expression level in CD4 and CD8 positive cells. Results: In general, the activated CD4 cells showing intracellular cytokines were higher than CD8 positive population of cells (p < 0.05). These findings indicate that the vaccine induced both a humoral and cellular immunity. Cytokine profile also showed high levels of TNFα, IFN γ and IL2 and CD69 expression in the group of animals immunized at a dose of 10 μg when compared to control group (p < 0.05). Conclusion: A 10 μg dose intramuscular injection of the modified DNA-based vaccine encoding HBsAg in mice induces both high humoral and cellular immune responses.

Diseño de un vector no viral basado en nanopartículas lipídicas para el tratamiento de la hepatitis C mediante ARN de interferencia

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A role for mammalian target of rapamycin (mTOR) pathway in non alcoholic steatohepatitis related-cirrhosis

Non-alcoholic fatty liver disease (NAFLD) encompasses the whole spectrum of steatosis, nonalcoholic steatohepatitis (NASH), and NASH-related cirrhosis (NASH/Cir). Although molecular advances have been made in this field, the pathogenesis of NAFLD is not completely understood. The gene expression profiling associated to NASH/Cir was assessed, in an attempt to better characterize the pathways involved in its etiopathogenesis. Methods: In the first step, we used cDNA microarray to evaluate the gene expression profiles in normal liver (n=3) and NASH/Cir samples (n=3) by GeneSifter (TM) analysis to identify differentially expressed genes and biological pathways. Second, tissue microarray was used to determine immunohistochemical expression of phosphorylated mTOR and 4E-BP1 in 11 normal liver samples, 10 NASH/Cir samples and in 37 samples of cirrhosis of other etiologies to further explore the involvement of the mTOR pathway evidenced by the gene expression analysis. Results: 138 and 106 genes were, respectively, up and down regulated in NASH/Cir in comparison to normal liver. Among the 9 pathways identified as significantly modulated in NASH/Cir, the participation of the mTOR pathway was confirmed, since expression of cytoplasmic and membrane phospho-mTOR were higher in NASH/Cir in comparison to cirrhosis of other etiologies and to normal liver. Conclusions: Recent findings have suggested a role for the cellular ""nutrient sensor"" mTOR in NAFLD and the present study corroborates the participation of this pathway in NASH/Cir. Phospho-mTOR evaluation might be of clinical utility as a potential marker for identification of NASH/Cir in cases mistakenly considered as cryptogenic cirrhosis owing to paucity of clinical data.

Management of Bell's Palsy: A Report of 2 Cases

Bell's palsy is a neuropathy of the peripheral seventh cranial nerve, resulting from traumatic, compressive, infective, inflammatory or metabolic abnormalities or it can be idiopathic. HIV, Epstein-Barr virus and hepatitis B virus have been suspected as initiating organisms, but herpes simplex virus is the most frequently implicated. This report describes 2 cases of Bell's palsy in children that were managed with antiviral agents. Both patients experienced complete recovery within 28 days; after 1 year follow-up, no recurrence was observed and both patients have normal facial movement. Differential diagnosis is essential to guide the treatment plan in Bell's palsy. Special attention should be given to children with respect to prescription of medications that can cause important side effects.

Hepatocellular carcinoma

Hepatitis C infection is associated with the development of hepatocellular carcinoma, and progress has been made in a number of areas. Transgenic mice lines expressing the hepatitis C core protein develop hepatic steatosis, adenomas, and hepatocellular carcinomas, with no significant hepatitis or fibrosis. This implies that hepatitis C can lead directly to malignant transformation, A new lesion, irregular regeneration, has been described in chronic hepatitis C infection and is associated with a 15-fold increase in the relative risk of developing hepatocellular carcinoma. A minority of patients with hepatitis C-related hepatocellular carcinoma have intense lymphocytic infiltration of the cancer, a feature associated with a better prognosis, Several studies have confirmed the association between large cell dysplasia and hepatocellular carcinoma, However, large cell dysplasia may not be a premalignant lesion and instead may be a marker for premalignant alterations elsewhere in the liver. Oral contraceptives previously have been linked to an increased risk of hepatocellular carcinoma. A large multicenter European case-control study has shown minimal increased risk of hepatocellular carcinoma with use of steroidal contraception. Tamoxifen had shown promise in the management of advanced hepatocellular carcinoma. However, a randomized placebo-controlled study of 477 patients with hepatocellular carcinoma found no benefit from tamoxifen, In a preliminary study, however, octreotide has shown improved survival and quality of life in patients with advanced hepatocellular carcinoma, Finally, interferon treatment continues to be linked to a reduced risk of hepatocellular carcinoma in patients with hepatitis C, These studies generally are not randomized, and a randomized prospective study is required to address this issue. (C) 1999 Lippincott Williams & Wilkins, Inc.

Expression of the iron regulatory peptide hepcidin is reduced in patients with chronic liver disease

Disturbances in iron metabolism often accompany liver disease in humans and hepatic iron deposition is a frequent finding. Since the peptide hepcidin, a major regulator of body iron homeostasis, is synthesised in the liver, alterations in hepcidin expression could be responsible for these effects. To investigate this possibility, we studied hepcidin expression in liver biopsies from patients with hepatitis C virus (HCV) infection, non-alcoholic fatty liver disease (NAFLD) and hemochromatosis (HC). Total RNA was extracted from the liver tissue of 24 HCV, 17 NASH and 5 HC patients, and 17 liver transplant donors (controls). The levels of mRNA for hepcidin and several other molecules involved in iron metabolism (DMT1, Dcytb, hephaestin, ferroportin, TfR1, TfR2, HFE and HJV) were examined by ribonuclease protection assay and expressed relative to the housekeeping gene GAPDH. The expression of hepcidin was significantly decreased in HCV and NASH patients relative to control liver (109±16 and 200±44 versus 325±26 respectively; P=0.008 and 0.02). We have previously reported similar findings in patients with HC, and this was confirmed in the current analysis (176±21; P=0.003). In both HCV and NAFLD patients the expression of the iron reductase Dcytb and the transferrin binding regulatory molecule TfR2 was also decreased, while the cellular iron exporter ferroportin showed a significant increase. Levels of the mRNA for the iron oxidase hephaestin were lower in HCV patients alone, while expression of the major transferrin binding molecule TfR1 was decreased only in NAFLD patients. Of particular interest was the finding that the expression of HJV (which is mutated in patients with juvenile HC) was significantly increased in NAFLD patients. No changes were seen in the expression of the iron importer DMT1 or the regulatory molecule HFE. Decreased expression of hepcidin in patients with HCV and NAFLD provides an explanation why iron homeostasis could be perturbed in these disorders. Reduced hepcidin levels would increase intestinal iron absorption and iron release from macrophages, which could contribute to hepatic iron accumulation. This in turn could lead to alterations in the expression of various proteins involved in iron transport and its regulation. Indeed most of the changes in the expression of such molecules observed in this study are consistent with this. However, the mechanisms leading to changes in the expression of hepcidin in these diseases remain to be elucidated.

Review of genetic and epigenetic alterations in hepatocarcinogenesis

Hepatocellular carcinoma (HCC) is associated with multiple risk factors and is believed to arise from pre-neoplastic lesions, usually in the background of cirrhosis. However, the genetic and epigenetic events of hepatocarcinogenesis are relatively poorly understood. HCC display gross genomic alterations, including chromosomal instability (CIN), CpG island methylation, DNA rearrangements associated with hepatitis B virus (HBV) DNA integration, DNA hypomethylation and, to a lesser degree, microsatellite instability. Various studies have reported CIN at chromosomal regions, 1p, 4q, 5q, 6q, 8p, 10q, 11p, 16p, 16q, 17p and 22q. Frequent promoter hypermethylation and subsequent loss of protein expression has also been demonstrated in HCC at tumor suppressor gene (TSG), p16, p14, p15, SOCS1, RIZ1, E-cadherin and 14-3-3 sigma. An interesting observation emerging from these studies is the presence of a methylator phenotype in hepatocarcinogenesis, although it does not seem advantageous to have high levels of microsatellite instability. Methylation also appears to be an early event, suggesting that this may precede cirrhosis. However, these genes have been studied in isolation and global studies of methylator phenotype are required to assess the significance of epigenetic silencing in hepatocarcinogenesis. Based on previous data there are obvious fundamental differences in the mechanisms of hepatic carcinogenesis, with at least two distinct mechanisms of malignant transformation in the liver, related to CIN and CpG island methylation. The reason for these differences and the relative importance of these mechanisms are not clear but likely relate to the etiopathogenesis of HCC. Defining these broad mechanisms is a necessary prelude to determine the timing of events in malignant transformation of the liver and to investigate the role of known risk factors for HCC.

Efficacy and safety of entecavir for chronic HBV in HIV/HBV coinfected patients receiving lamivudine as part of antiretroviral therapy

Background: Prolonged use of lamivudine in patients coinfected with HIV and hepatitis B virus (HBV) leads to an increasing risk of lamivudine resistance in both diseases. We investigated the addition of entecavir, a potent inhibitor of HBV polymerase, to lamivudine-containing highly active antiretroviral therapy (HAART) in patients who experienced rebound in HBV viremia while maintaining Suppression of plasma HIV RNA less than 400 copies/ml. Methods: Sixty-eight patients were randomized to entecavir 1 mg (n = 51) or placebo (n = 17) once daily for 24 weeks; 65 patients continued the study with entecavir for an additional 24 weeks. Lamivudine-containing HAART was continued throughout. Results: At week 24, the mean HBV DNA in entecavir-treated patients was 5.52 log(10) - copies/ml versus 9.27 log(10) copies/ml for placebo, and at week 48, it was 4.79log(10) copies/ml versus 5.63log(10) copies/ml, respectively. The mean HBV DNA change from baseline for entecavir was -3.65 log(10) copies/ml (versus + 0.11 for placebo, P < 0.0001) and alanine aminotransferase normalization in 34%. of patients (versus 8% for placebo, P=0.08)At 48 weeks, mean change in HBV DNA reached -4.20log(10) copies/ml inpatients who received entecavir for the entire 48 weeks. The frequency of adverse events with entecavir and placebo was comparable. Through 48 weeks, no clinically relevant changes in HIV viremia or CD4 cell Counts were identified. Conclusion: In this study, entecavir was associated with rapid, clinically significant reductions in HBV DNA, with maintenance of HIV viremia suppression, in HIV/HBV coinfected patients with HBV viremia while on lamivudine treatment. (C) 2008 Wolters Kluwer Health vertical bar Lippincott Williams & Wilkins.

Very early prediction of response to HCV treatment with PEG-IFN-alfa-2a and ribavirin in HIV/HCV-coinfected patients

The objective of this study was to find very early viral kinetic markers to predict nonresponse to hepatitis C virus (HCV) therapy in a group of human immunodeficiency virus (HIV)/HCV-coinfected patients. Twenty-six patients (15 HCV genotype-1 and 11 genotype-3) were treated with a 48-week regimen of peginterferon-alfa-2a (PEG-IFN) (180 mu g/week) and weight-based ribavirin (11 mg/kg/day). Samples were collected at baseline; 4, 8, 12, 18, 24, 30, 36 and 42 h; days 2, 3, 4, 7, 8, 15, 22, 29, 43 and 57 then weekly and monthly. Five patients discontinued treatment. Seven patients (27%) achieved a sustained virological response (SVR). Nadir HCV RNA levels were observed 1.6 +/- 0.3 days after initiation of therapy, followed by a 0.3- to 12.9-fold viral rebound until the administration of the second dose of PEG-IFN, which were not associated with SVR or HCV genotype. A viral decline < 1.19 log for genotype-1 and < 0.97 log for genotype-3, 2 days after starting therapy, had a negative predictive value (NPV) of 100% for SVR. The day 2 virological response had a similar positive predictive value for SVR as a rapid virological response at week 4. In addition, a second-phase viral decline slope (i.e., measured from day 2 to 29) < 0.3 log/week had a NPV = 100% for SVR. We conclude that first-phase viral decline at day 2 and second-phase viral decline slope (< 0.3 log/week) are excellent predictors of nonresponse. Further studies are needed to validate these viral kinetic parameters as early on-treatment prognosticators of nonresponse in patients with HCV and HIV.

Pharmacodynamics of PEG-IFN-alpha-2a and HCV Response as a Function of IL28B Polymorphism in HIV/HCV-Coinfected Patients

We examined the association between IL28B single-nucleotide polymorphism rs12979860, hepatitis C virus (HCV) kinetic, and pegylated interferon alpha-2a pharmacodynamic parameters in HIV/HCV-coinfected patients from South America. Twenty-six subjects received pegylated interferon alpha-2a + ribavirin. Serum HCV-RNA and interferon concentrations were measured frequently during the first 12 weeks of therapy and analyzed using mathematical models. African Americans and whites had a similar distribution of IL28B genotypes (P = 0.5). The IL28B CC genotype was overrepresented (P = 0.015) in patients infected with HCV genotype-3 compared with genotype-1. In both genotype-1 and genotype-3, the first-phase viral decline and the average pegylated interferon-alpha-2a effectiveness during the first week of therapy were larger (trend P <= 0.12) in genotype-CC compared with genotypes-TC/TT. In genotype-1 patients, the second slower phase of viral decline (days 2-29) and infected cells loss rate, delta, were larger (P = 0.02 and 0.11, respectively) in genotype-CC than in genotypes-TC/TT. These associations were not observed in genotype-3 patients.

Prevalence and distribution of the GBV-C/HGV among HIV-1-infected patients under anti-retroviral therapy

Infection with GB virus C (GBV-C) or hepatitis G virus (HGV) is highly prevalent among HIV/AIDS patients. GBV-C/HGV viremia has not been associated with liver disease and seems to slow HIV disease progression. To study the GBV-C/HGV genotypes prevalence among HIV/AIDS patients and its association with HIV viral load (VL) and CD4+ lymphocyte counts. From February 2003 to February 2004, we analyzed 210 HIV-1-infected subjects who were on anti-retroviral therapy (ART). For 63 of them a PCR-nested to the non-coding 5` (5`NCR) region of the GBV-C/HGV was done, and for 49 a DNA direct sequencing was done. A phylogenetic analysis was performed by PHYLIP program. 63(30%) of the HIV-1-infected patients were co-infected with GBV-C/HGV. The phylogenetic analysis revealed the following genotypes (and respective relative frequencies): 1(10%), 2a (41%), 2b (43%), and 3 (6%). Co-infected patients presented lower HIV-1 VL and higher T CD4+ lymphocyte cells counts as compared with patients negative for GBV-C/HGV sequences (log = 4.52 vs. 4.71, p = 0.036), and T CD4+ lymphocyte counts (cells/mm(3) = 322.6 vs. 273.5, p = 0.081, respectively). T CD4+ cells counts equal to, or higher than, 200/mm(3) were significantly more common among co-infected patients than among HIV-infected-only patients (p = 0.042). The lowest T CD4+ cells counts were associated with genotype 1 and the highest with genotype 2b (p = 0.05). The GBV-C/HGV infection prevalence was 30% among HIV-1-infected subjects, and was associated with lower VL and higher CD4+ lymphocyte counts. GBV-C/HGV genotype 2b may be associated with better immunological response. Published by Elsevier B.V.

A Neuropsychological Study Comparing Patients Infected With HCV and HBV Without Psychiatric Comorbidities

Hepatitis C is one of the most common chronic infectious diseases worldwide, with well-documented extra-hepatic manifestations, such as a broad number of cognitive deficits. These impairments may be explained by psychiatric comorbidities, which have not been investigated properly in the literature. In order to elucidate a specific hepatitis C virus (HCV) induced cognitive impairment not related to mental disorders, neuropsychological performance of patients infected with HCV was compared with that of patients infected with hepatitis B virus cognitive impairment, especially psychiatric comorbidities. A total of 33 patients infected with HCV and 22 patients infected with HBV were included in the study. There were no significant differences between the two groups with regard to age or years of education. The group of patients infected with HCV performed significantly worse on visuo-spatial memory tasks after adjusting for years of education and age. There were no significant differences between patients infected with HCV and patients infected with HBV with regards to other neuropsychological functions. The data indicate that patients infected with HCV patients have poorer visuo-spacial memory performance than patients infected with HBV, suggesting that the cognitive deficit may be specific to HCV infection and not to secondary comorbid psychiatric disorders. J. Med. Virol. 81: 1184-1188, 2009. (C) 2009 Wiley-Liss, Inc.

The effect of early virological response in health-related quality of life in HCV-infected patients

Twenty-nine HCV-infected patients were treated with pegylated interferon alpha. Diagnosis was based on serum HCV RNA-PCR positive results and liver biopsy. All patients had elevated serum levels of alanine aminotransferase at the time of the study, but liver disease was compensated. Patients were evaluated at baseline treatment and after 4 and 12 weeks of antiviral treatment with the Medical Outcomes Study 36-item Short-Form Health Survey. The Mini-International Neuropsychiatric Interview was used to exclude previous or current psychiatric diagnoses. Both patients and psychiatrists were blind to the HCV RNA status, and serum HCV RNA test results only became available after the visit at week 12. After antiviral treatment, 16 patients (55.2%) were classified as nonresponders and 13 (44.8%) were classified as responders. When compared to nonresponders, responders had a greater improvement in the HRQOL scores for the mental health domain (P<.019). Differences in other domains were not significant. The present study confirms that active viral infection is one possible reason for the poor Health-Related Quality of Life in this population.

Magnetic purification of biotinylated cDNA removes false priming and ensures strand-specificity of RT-PCR for enteroviral RNAs

The detection of replicative intermediate RNAs as markers of active replication of RNA viruses is an essential tool to investigate pathogenesis in acute viral infections, as well as in their long-term sequelae. In this regard, strand-specific PCR has been used widely to distinguish (-) and (+) enteroviral RNAs in pathogenesis studies of diseases such as dilated cardiomyopathy. It has been generally assumed that oligonucleotide-primed reverse transcription of a given RNA generates only the corresponding specific cDNA, thus assuring the specificity of a PCR product amplified from it. Nevertheless, such assumed strand-specificity is a fallacy, because falsely primed cDNAs can be produced by RNA reverse transcription in the absence of exogenously added primers, (cDNA(primer)(-)), and such falsely primed cDNAs are amplifiable by PCR in the same way as the correctly primed cDNAs. Using as a prototype the coxsackievirus B5 (CVB5), a (+) strand RNA virus, it was shown that cDNA(primer)(-) renders the differential detection of viral (-) and (+) RNAs by conventional PCR virtually impossible, due to gross non-specificity. Using in vitro transcribed CVB5 RNAs (+) and (-), it was shown that cDNA(primer)(-) could be removed effectively by magnetic physical separation of correctly primed biotinylated cDNA. Such strategy enabled truly strand-specific detection of RNA (-) and (+), not only for CVB5, but also for other non-polio enteroviruses. These findings indicate that previous conclusions supporting a role for the persistence of actively replicating enterovirus in the pathogenesis of chronic myocarditis should be regarded with strong skepticism and purification of correctly primed cDNA should be used for strand-specific PCR of viral RNA in order to obtain reliable information on this important subject. (C) 2009 Elsevier B.V. All rights reserved.