Georgios Germanidis

Human Serum Facilitates Hepatitis C Virus Infection, and Neutralizing Responses Inversely Correlate with Viral Replication Kinetics at the Acute Phase of Hepatitis C Virus Infection

ABSTRACT The factors leading to spontaneous clearance of hepatitis C virus (HCV) or to viral persistence are elusive. Understanding virus-host interactions that enable acute HCV clearance is key to the development of more effective therapeutic and prophylactic strategies. Here, using a sensitive neutralization assay based on infectious HCV pseudoparticles (HCVpp), we have studied the kinetics of humoral responses in a cohort of acute-phase patients infected during a single nosocomial outbreak in a hemodialysis center. The 17 patients were monitored for the spontaneous outcome of HCV infection for 6 months before a treatment decision was made. Blood samples were taken frequently (15 ± 4 per patient). Phylogenetic analysis of the predominant virus(es) revealed infection by only one of two genotype 1b strains. While all patients seroconverted, their sera induced two opposing effects in HCVpp infection assays: inhibition and facilitation. Furthermore, the ability of sera to facilitate or inhibit infection correlated with the presence of either infecting HCV strain and divided the patients into two groups. In group 1, the progressive emergence of a relatively strong neutralizing response correlated with a fluctuating decrease in high initial viremia, leading to control of viral replication. Patients in group 2 failed to reduce viremia within the acute phase, and no neutralizing responses were detected despite seroconversion. Strikingly, sera of group 2, as well as naïve sera, facilitated infection by HCVpp displaying HCV glycoproteins from different genotypes and strains, including those retrieved from patients. These results provide new insights into the mechanisms of viral persistence and immune control of viremia.

Predicting response to peginterferon α-2a, lamivudine and the two combined for HBeAg-negative chronic hepatitis B

Objective: In a trial of patients with hepatitis B e antigen (HBeAg)-negative chronic hepatitis B, 24 week post-treatment biochemical and virological response rates with peginterferon α-2a with or without lamivudine were significantly higher than with lamivudine alone. The effect of pre-treatment factors on post-treatment responses was investigated. Methods: Multivariate analyses were performed using available data from 518 patients treated with peginterferon α-2a with or without lamivudine, or with lamivudine alone. A post-treatment response was defined as alanine aminotransferase (ALT) normalisation and hepatitis B virus (HBV) DNA level of <20 000 copies/ml. Results: In logistic regression analyses across all treatment arms, peginterferon α-2a (with or without lamivudine) therapy, younger age, female gender, high baseline ALT, low baseline HBV DNA and HBV genotype were identified as significant predictors of combined response at 24 weeks post-treatment. In the peginterferon α-2a and lamivudine monotherapy arms, patients with genotypes B or C had a higher chance of response than genotype D infected patients (p<0.001), the latter responding better to the combination than to peginterferon α-2a monotherapy (p = 0.015). At 1 year post-treatment, response rates by intention-to-treat analysis were 19.2% for the peginterferon α-2a, 19.0% for the combination, and 10.0% for the lamivudine groups, with genotypes B or C associated with a sustained combined response to peginterferon α-2a with or without lamivudine therapy. Conclusions: Baseline ALT and HBV DNA levels, patient age, gender, and infecting HBV genotype significantly influenced combined response at 24 weeks post-treatment, in patients treated with peginterferon α-2a and/or lamivudine. At 1 year post-treatment HBV genotype was significantly predictive of efficacy for patients treated with peginterferon α-2a with or without lamivudine.

Conservation of the Conformation and Positive Charges of Hepatitis C Virus E2 Envelope Glycoprotein Hypervariable Region 1 Points to a Role in Cell Attachment

ABSTRACT Chronic hepatitis C virus (HCV) infection is a major cause of liver disease. The HCV polyprotein contains a hypervariable region (HVR1) located at the N terminus of the second envelope glycoprotein E2. The strong variability of this 27-amino-acid region is due to its apparent tolerance of amino acid substitutions together with strong selection pressures exerted by anti-HCV immune responses. No specific function has so far been attributed to HVR1. However, its presence at the surface of the viral particle suggests that it might be involved in viral entry. This would imply that HVR1 is not randomly variable. We sequenced 460 HVR1 clones isolated at various times from six HCV-infected patients receiving alpha interferon therapy (which exerts strong pressure towards quasispecies genetic evolution) and analyzed their amino acid sequences together with those of 1,382 nonredundant HVR1 sequences collected from the EMBL database. We found that (i) despite strong amino acid sequence variability related to strong pressures towards change, the chemicophysical properties and conformation of HVR1 were highly conserved, and (ii) HVR1 is a globally basic stretch, with the basic residues located at specific sequence positions. This conservation of positively charged residues indicates that HVR1 is involved in interactions with negatively charged molecules such as lipids, proteins, or glycosaminoglycans (GAGs). As with many other viruses, possible interaction with GAGs probably plays a role in host cell recognition and attachment.

The use of statins alone, or in combination with pioglitazone and other drugs, for the treatment of non-alcoholic fatty liver disease/non-alcoholic steatohepatitis and related cardiovascular risk. An Expert Panel Statement

Non-alcoholic fatty liver disease (NAFLD), the most common liver disease, is characterized by accumulation of fat (>5% of the liver tissue), in the absence of alcohol abuse or other chronic liver diseases. It is closely related to the epidemic of obesity, metabolic syndrome or type 2 diabetes mellitus (T2DM). NAFLD can cause liver inflammation and progress to non-alcoholic steatohepatitis (NASH), fibrosis, cirrhosis or hepatocellular cancer (HCC). Nevertheless, cardiovascular disease (CVD) is the most common cause of death in NAFLD/NASH patients. Current guidelines suggest the use of pioglitazone both in patients with T2DM and in those without. The use of statins, though considered safe by the guidelines, have very limited use; only 10% in high CVD risk patients are on statins by tertiary centers in the US. There are data from several animal studies, 5 post hoc analyses of prospective long-term survival studies, and 5 rather small biopsy proven NASH studies, one at baseline and on at the end of the study. All these studies provide data for biochemical and histological improvement of NAFLD/NASH with statins and in the clinical studies large reductions in CVD events in comparison with those also on statins and normal liver. Ezetimibe was also reported to improve NAFLD. Drugs currently in clinical trials seem to have potential for slowing down the evolution of NAFLD and for reducing liver- and CVD-related morbidity and mortality, but it will take time before they are ready to be used in everyday clinical practice. The suggestion of this Expert Panel is that, pending forthcoming randomized clinical trials, physicians should consider using a PPARgamma agonist, such as pioglitazone, or, statin use in those with NAFLD/NASH at high CVD or HCC risk, alone and/or preferably in combination with each other or with ezetimibe, for the primary or secondary prevention of CVD, and the avoidance of cirrhosis, liver transplantation or HCC, bearing in mind that CVD is the main cause of death in NAFLD/NASH patients.

Foxp3 Expression in Liver Correlates with the Degree but Not the Cause of Inflammation

Patients with chronic viral hepatitis display increased expression of Foxp3 in liver, suggesting that Tregs expansion contributes to persistent infection. The purpose of this study was to elucidate whether the expression of Foxp3 relates not to the viral infection but to the resulting liver inflammation. Liver biopsies obtained from 69 individuals (26 chronic HBV hepatitis, 14 chronic HCV hepatitis, 11 nonalcoholic fatty liver disease, 8 autoimmune diseases, 2 methotrexate-related toxicity, and 8 controls) were examined, by qRT-PCR, for the mRNA expression of Foxp3, IL-10, TGF-β1, Fas, FasL, TRAIL, caspase-3, TNF-α, IFN-γ, and IL-1β. Significant increase of Foxp3 was observed in all disease groups compared to controls, which was positively correlated with the intensity of inflammation. The expression of the apoptosis mediators Fas, FasL, and TRAIL, but not of IL-10 and TGF-β1, was also significantly elevated. Our findings indicate that, independently of the initial inducer, liver inflammation is correlated with elevated expression of apoptosis mediators and is followed by local Treg accumulation. Further research towards the elucidation of the underlying casual relationships is required, in order to clarify whether our results signify the existence of a uniform Treg-mediated regulatory mechanism of apoptosis-induced inflammation.

Reactivation of occult hepatitis B virus infection, following treatment of refractory rheumatoid arthritis with abatacept

To the Editor: with many apoptotic and ballooned hepatocytes (Fig. 1A). Moderate steatosis was also present. Portal inflammatory cell infiltration was mild. Immunohistochemical stain for HBcAg showed extensive nuclear and cytoplasmic expression in hepatocytes, indicative of active viral replication (Fig. 1B). Immunohistochemical stain for HBsAg was negative. The basic medical treatments and laboratory data pertaining to HBV infection over time are depicted in Fig. 2. Abatacept is a soluble fusion protein which links the cytotoxic T-lymphocyte antigen-4 (CTLA-4) extracellular domain to the Fc region of the IgG molecule. CTLA-4 is an inhibitory T cell receptor, similar to CD28 in structure, expressed by activated and regulatory T cells (Tregs). CTLA-4 is constitutively expressed on CD4 CD25 Tregs, and such expression is important for Treg-mediated

Hepatitis B virus kinetics under antiviral therapy sheds light on differences in hepatitis B e antigen positive and negative infections.

BACKGROUND Hepatitis B e antigen (HBeAg)–negative chronic hepatitis B infection has a presentation and clinical course that is divergent from that of HBeAg‐positive infection. The former usually presents with lower viral levels but faster progression to liver disease. We sought to better understand the balance between replication and the immune response against hepatitis B virus (HBV). METHODS Viral kinetics in 50 HBeAg‐negative patients under various treatment protocols with interferon α and/or nucleoside or nucleotide analogs was analyzed. HBV DNA level was measured frequently and the data fitted to a viral dynamic model. A meta‐analysis of all published studies of viral kinetics in HBeAg‐positive and HBeAg‐negative infection was also conducted. RESULTS We found that the clearance of both HBV virions and infected cells was significantly faster in HBeAg‐negative infection than in HBeAg‐positive infection. In HBeAg‐negative infection, there was also a negative correlation between baseline HBV DNA levels and infected cell half‐life, suggesting that the higher the viral load the faster the turnover of infected cells. CONCLUSIONS These results reveal the dual role played by the immune response in maintaining lower viral levels and inducing faster turnover of infected cells, the latter of which may be responsible for the more aggressive nature of HBeAg‐negative infection.

Acute Hepatitis C in Patients Receiving Hemodialysis

Hepatitis C virus (HCV) infection is frequent in patients with end-stage renal disease treated by chronic dialysis, with a prevalence varying from 10–65% according to the geographical data. The prevalence is significantly associated with the duration of dialysis and the number of transfused blood products12 and has dramatically declined with efficient blood screening.3 We studied patients with acute HCV infection in a dialysis unit. The diagnosis was based on both anti-HCV detection and HCV-RNA detection. Other virological tools including HCV genotype determination was also used to tailor treatment to the individual patient and determine its efficacy for a one-year follow-up period. Seventeen patients (7 male and 10 female, mean age: 63.7 ± 11.6 SD) with acute hepatitis C were enrolled to our study. All of them were followed up for a period of one year after the diagnosis was established. Phylogenetic analysis distinguished two separate HCV subtypes 1b, which were both responsible for this acute infection (see ). These types did not differ in their behavior on the clinical situation of our patients, as confirmed by the fact that in both groups of patients, there was only one patient who presented with acute illness. Six patients of our study group, three months after the acute infection, received pegylated interferon (Peg-IFNa2a) 135 μg for a six-month period. Four of them responded very well to therapy and at the first determination HCV RNA was below the cutoff point. One of our patients with very high HCV levels (HCV RNA > 50,000,000 IU/mL), despite receiving the same therapy, did not respond well and developed cirrhosis. In conclusion, it is clear from our experience that better information is needed about the current incidence, prevalence, and risk factors for HCV infection in dialysis patients. Algorithms for the diagnosis and management of hepatitis C should be developed by academic societies. Routine screening for hepatitis C also would allow for better definition of the natural history of hepatitis C in patients with end stage renal disease. Figure 1. NS 5B gene phylogenetic tree analysis of the acute hepatitis C epidemic.

Older HIV-infected patients--an underestimated population in northern Greece: epidemiology, risk of disease progression and death.

OBJECTIVES HIV prevalence among older people is on the increase. The aim of this study was to evaluate the epidemiological and clinical features at diagnosis and survival of older patients. METHODS This was a retrospective analysis of the data of 558 newly diagnosed antiretroviral-naïve patients between January 1998 and December 2008. Patients were divided into two groups according to their age at diagnosis: ≥50 years (n=103) and 18-49 years (n=455). RESULTS The most common risk factor for older patients was heterosexual contact (p<0.013). Older patients were more likely to suffer from hypertension (33.0% vs. 5.1%, p<0.0005), cardiovascular disease (20.4% vs. 2.9%, p<0.0005), neurological disorders (11.7% vs. 5.5%, p=0.02), renal dysfunction (12.6% vs. 5.3%, p=0.01), and infections (66.0% vs. 49.7%, p=0.003) than their younger counterparts, and to have more hospital admissions during follow-up (47.5% vs. 19.6%, p<0.0005). Older patients had a shorter survival time (p<0.0005). A statistically significant increase in CD4+ cell number through time was observed in both groups (p<0.0005). Younger patients reached higher magnitudes of absolute numbers of CD4+ cells during follow-up (p<0.0005) after the initiation of antiretroviral therapy. The total number of patients with clinical AIDS from baseline throughout the study period was also higher in the older age group (35.9% vs. 25.0%). CONCLUSIONS HIV-infected people aged ≥50 years differ in epidemiological and clinical features to younger HIV-infected people. The issue of increasing prevalence of HIV infection is a matter of concern due to existing comorbidities, which probably lead to higher mortality rates and faster progression to clinical AIDS.

Liver FOXP3 and PD1/PDL1 expression is down-regulated in chronic HBV hepatitis on maintained remission related to the degree of inflammation

Background and Aim: T cell expression of PD1 and inhibition of T effector cells by Foxp3+-T regulatory cells are among the most powerful mechanisms for achieving a balanced immune response. Our aim was to investigate, how liver FOXP3 and PD1/PDL1 expression is regulated in chronic HBV hepatitis (CHB) on maintained long-term remission in comparison with active disease, and whether they are correlated to the expression of pro- and anti-inflammatory cytokines and apoptosis mediators, along with the degree of histological inflammation and markers of T cell effector restoration. Methods: Fifty-three HBeAg-negative CHB patients with both active (30) and completely remitted disease on long-term antiviral treatment (23) and four controls (submitted to liver biopsy due to a mild increase of aminotransferases but without liver necroinflammatory and architecture changes) were enrolled in the study. Liver mRNA levels of immunoregulatory genes (FOXP3, IL10, TGFB1, and those of PD1/PDL1/PDL2 pathway), major apoptosis mediators (FAS, FASL, TNFA, TRAIL), cytokines of effector T cell restoration (IL2, IFNG), and those of IL1B, CD4, and CD8, were evaluated by quantitative real-time reverse-transcriptase PCR and were correlated with each other, along with the intensity of liver inflammation and fibrosis staging. The expression and localization of FOXP3, PD1, PDL1, CD4, and CD8 were also assessed by immunohistochemistry. Results: The expression of FOXP3, IL10, TGFB1, PD1, PDL1, FASL, and CD8 was significantly down-regulated in the remission state. In contrast, liver expression of IL2 and IFNG, along with CD4, IL1B, TNFA, and FAS did not change significantly. Moreover, FOXP3, PD1, PDL1, and CD8 transcripts were positively correlated to the intensity of liver inflammation. Conclusion: Our data indicate that in the CHB disease model, the immunosuppressive liver environment is down-regulated in the maintained on-treatment long-term remission state and correlates with the intensity of liver inflammation, but not liver T cell restoration.