Arleta Kowala-Piaskowska

NK Cells Prevalence, Subsets and Function in Viral Hepatitis C

Innate immunity appears to play an important role in the pathogenesis of viral hepatitis C. Among various cell subsets of this immunity natural killer (NK) cells raised particular interest. These cells are abundant in liver, possess significant cytotoxic potential and show links with adaptive immunity. They play important role, particularly in the acute phase of viral infections, including hepatitis C. They exhibit various types of receptors, either inhibitory or activating, that are able to react with distinct ligands on infected cells. Homozygosity of some receptors, namely KIR2DL3 reacting with recipient HLA-C1 antigens is a herald of good prognosis in hepatitis C virus (HCV) infection. In the early stage of the latter, both the prevalence and the cytotoxicity of NK cells are increased. Their inhibitory receptors are down regulated whereas activating ones are up regulated. Interferon-γ secreted by NK56+bright NK cells has a direct cytotoxic effect on infected hepatocytes. In contrast, in the chronic phase of HCV liver disease both, the prevalence and function of NK cells are impaired. Nevertheless, their cytotoxicity contributes to liver injury. Cells show change in the polarization profile from NK1 to NK2, manifested by secretion of immunosuppressive cytokines. Some HCV peptides are inhibitory for NK cells leading to the reduction of their antiviral activity. The unwanted effects of HCV peptides can be at least partly reversed by the antiviral therapy.

Hepatitis C virus load and expression of a unique subset of cellular genes in circulating lymphoid cells differentiate non‐responders from responders to pegylated interferon alpha–ribavirin treatment

Based on investigations of liver biopsy material, certain cellular genes have been implicated as correlates of success or failure to interferon alpha–ribavirin (IFN/RBV) therapy against hepatitis C. The current study aimed at determining whether expression of host genes thought to be relevant to HCV replication in the liver would be correlated with HCV infection status in peripheral blood mononuclear cells (PBMCs) and also with patient responsiveness to IFN/RBV treatment. Therefore, PBMCs from patients with chronic hepatitis C responding (n = 35) or not (n = 49) to IFN/RBV and from healthy controls (n = 15) were evaluated for HCV RNA load and cellular gene expression. Non‐responders had 3‐ to 10‐fold higher basal levels of interleukin (IL)‐8, IFN‐stimulated gene 15 (ISG15), 2′,5′‐oligoadenylate synthetase (OAS), and Toll‐like receptors (TLR)‐4, ‐5, and ‐7 compared to responders. Non‐responders with similar post‐treatment follow‐ups as responders persistently expressed 6‐ to 20‐fold greater levels of IL‐8, ISG15, and OAS after therapy. Higher expression of IFN‐α, IFN‐γ, and IFN‐λ was found in PBMCs of individuals achieving sustained virological response, either before or after therapy. Pre‐treatment HCV RNA loads in PBMCs of non‐responders were significantly higher (P = 0.016) than those of responders. In conclusion, the data indicate that immune cells of responders and non‐responders to IFN/RBV therapy exhibited significantly different virological and host gene expression profiles. Elevated baseline HCV loads and TLR‐4, ‐5, and ‐7 levels, and persistently high levels of IL‐8, ISG15, and OAS were correlated with IFN non‐responsiveness. The results warrant further investigations on the utilization of PBMCs for predicting success or failure to IFN‐based therapies. J. Med. Virol. 85:441–448, 2013.

The incidence and significance of pattern-recognition receptors in chronic viral hepatitis types B and C in man.

Chronic viral hepatitis B and C are among the most common and devastating liver diseases worldwide. Immune response plays a crucial role in the course of both diseases. In spite of the importance of the adaptive arm of the immune response, there is a growing role of innate immunity, the earliest confronted with viral attack. Pattern-recognition receptors (PRRs) and, in particular, Toll-like receptors (TLRs) are molecules which are able not only to recognize foreign invaders, but also quickly mount an antiviral defense. Activation of PRRs has been demonstrated in both hepatitis types, i.e. in situ in the liver and on while blood cells. Both viruses, HCV and HBV, are able to subvert the PRR-mediated antiviral response by means of various proteins and enzymes. HCV acts via the non-structural proteins NS2 and NS3/4A, while HBV HBeAg is inversely correlated with TLR activity. Viral counterattack is particularly directed toward dendritic cells, those creating the link with the adaptive immune response. Apart from TLRs, other PRRs such as RIG-1 and MDA-5 are also able to recognize viral infection and participate in the activation of type I interferon synthesis. TLRs manifest gene polymorphism, which was shown to affect several consequences associated with chronic viral hepatitis such as liver cirrhosis and the outcome of liver allotransplantation. There have been numerous attempts to take advantage of the existence and activity of PRRs for the patients’ benefit. Several authors examined the role of TLR synthetic agonists as inducers of TLR activation. In hepatitis C the most promising agonists appear to be TLR3, 7, and 9 for potential antiviral therapy. PRRs may also act as potent adjuvants in HBV vaccines. Their baseline mRNA levels may have predictive value in the course of antiviral therapy.

Seroprevalence of anti-HEV IgG in 182 Polish patients.

INTRODUCTION Hepatitis E virus (HEV) infection is an emergent disease in developed countries. HEV seroprevalence in such areas significantly exceeds values expected when one considers infection with this virus only as a problem restricted to classical endemic regions. To date, no related data are available in Poland. In this study we aimed to obtain HEV seroprevalence data and compare them with similar data for hepatitis A virus (HAV) in Polish patients. MATERIAL/METHODS From February 1st, 2013, to October 15th, 2013, we performed anti-HEV IgG (anti-HEV) tests (EIAgen HEV IgG Kit; Adaltis, Milano, Italy) in 182 patients (101 men and 81 women; 61 patients were HIV-positive) of one center in Poland, aged 19-85 (47.2 ± 14.2 years). RESULTS We found a 15.9% seropositivity rate for anti-HEV (16.3% of the study population with an unequivocal test result) and 38.5% for anti-HAV. In 6 cases (3.4%), anti-HEV-positive persons had never travelled abroad. In contrast to HAV seroprevalence data, there was no significant difference in HEV seroprevalence between young adults (18-40 years) and older patients (p<0.0001 and p=0.0967, respectively). Anti-HEV were found in 21.3% of HIV-infected individuals. CONCLUSIONS HEV infection may occur in Poland. Anti-HAV seropositivity among Polish patients is significantly higher than anti-HEV. In contrast to HAV, HEV seroprevalence is similar in younger and older patients. The clinical course of HEV infection in Polish citizens seems to be largely asymptomatic. Polish HIV patients may be more commonly exposed to HEV than similar individuals from other countries.

Contribution of genes for killer cell immunoglobulin-like receptors (KIR) to the susceptibility to chronic hepatitis C virus infection and to viremia.

BACKGROUND Natural killer (NK) cells are an important element of innate immunity against viruses, although their numbers decrease in the liver during chronic HCV infection. NK cells express a large panel of inhibitory and activating receptors. The most polymorphic of these are killer cell immunoglobulin-like receptors (KIRs) which are encoded by multiple genes that may be present or absent in given individuals depending on their genotype. This variability results in differential susceptibility to viral infections and diseases, including HCV infection and its consequences. AIMS AND METHODS The aim of this study was to test whether chronical infection with HCV and the viremia levels are associated with any KIR gene in the Polish population. We typed 301 chronically HCV-infected patients and 425 non-infected healthy individuals for the presence or absence of KIR genes and their ligands, HLA-C C1 and C2 groups as well as HLA-B and HLA-A Bw4-positive alleles. RESULTS We found that males, but not females, possessing KIR2DS2 and KIR2DL2 genes had a 1.7 higher probability to become chronically HCV-infected than males negative for these genes (p=0.0213). In accord with this, centromeric B region, containing KIR2DS2 and KIR2DL2 genes, was also associated with chronic HCV infection in males. In addition, patients of both genders possessing KIR2DS3 but not KIR2DS5 gene exhibited, on average, 2.6 lower level of viremia than HCV-infected individuals with other genotypes (p=0.00282). This was evident in those infected at a young age. KIR2DS3-positive patients also had lower mean levels of bilirubin than KIR2DS3-negative ones (p=0.02862). CONCLUSION Our results suggest a contribution of the KIR2DS2 and KIR2DL2 genes (cenB haplotype) to the susceptibility to chronic HCV infection, and an association of the KIR2DS3 gene in the absence of KIR2DS5 with low viremia levels.

Evolution of hepatitis C virus hypervariable region 1 in chronically infected children.

Hepatitis C virus (HCV) quasispecies diversification plays an essential role in the establishment of chronic infections. Our earlier analysis of HCV population structure in children subjected to interferon-ribavirin treatment demonstrated that viral quasispecies is homogenous in patients who failed to respond to the therapy and heterogeneous in sustained responders. We also showed that certain variants of HCV hypervariable region 1 (HVR1) are conserved in non-responders. To better elucidate the pathways of HCV evolution, here we examined the changes of HVR1 in viral populations isolated from sera of eight treatment-naive pediatric patients. We found that HCV evolution in untreated chronically infected children occurs according to two pathways and results in the formation of either genetically homogenous or variable quasispecies. Variable populations are prone to quasispecies shifts. In contrast, homogenous populations are composed of closely related variants that undergo only minor changes. In addition, we observed that a phenomenon of inter-quasispecies conservation of HVR1 is associated with some of the homogenous HCV populations. The collected data suggest that there exist HVR1 variants with superior fitness, capable of persisting in different hosts.

Pneumocystis pneumonia in children - the relevance of chemoprophylaxis in different groups of immunocompromised and immunocompetent paediatric patients

Introduction Pneumocystis jirovecii is an opportunistic pathogen causing pneumocystis pneumonia (PCP), a life-threatening infection, in immunocompromised patients. In this study, retrospective analysis of the presence of P. jirovecii DNA in different samples collected from children with suspected PCP was carried out. Material and methods Three hundred and six specimens [152 bronchoalveolar lavage (BAL) specimens, 80 blood specimens, 18 bronchial secretions (BS), 34 induced sputum samples, 10 endotracheal aspirates (ETA), and 12 other type samples] obtained from patients with suspected PCP were examined by real-time PCR. Results Forty (13.1%) patients were positive for P. jirovecii: 4 (7.7%) patients with malignancies, 3 (6.8%) transplant recipients, 15 (23.1%) other immunocompromised patients, and 18 (12.4%) immunocompetent patients. Pneumocystis jirovecii DNA was detected in 20.4% of BAL specimens, 11.1% of BS samples, 10% of ETA sample, 8.8% of induced sputum samples, and in 3.7% of blood samples. Comparing the frequency of the presence of P. jirovecii DNA between the group of children treated with PCP chemoprophylaxis (malignancy patients and transplant recipients) and a group of children not receiving this prophylaxis (other immunocompromised and immunocompetent children), we found that the occurrence of PCP was twice as high in the latter group of children (7.3% and 15.7%, respectively). Conclusions Respiratory samples, such as BS, BAL, or ETA specimens, are the material of choice for the diagnosis of PCP. Due to high incidence of PCP in certain groups of immunocompetent and immunocompromised patients, besides cancer patients and transplant recipients, consideration of PCP prophylaxis is required in these groups as well.

Expression of pattern recognition receptors in liver biopsy specimens of children chronically infected with HBV and HCV

Pattern recognition receptors (PRRs) constitute a pivotal arm of innate immunity. Their distribution is widespread and not limited to cells of the immune system. Following our previous findings concerning the expression of Toll-like receptors (TLRs) 2, 3 and 4 in chronic viral hepatitis C of children, we wished to search for other PRRs, including other TLRs, NOD-like receptors (NLRs) and RIG-1-like helicase receptors (RLR) in infected hepatocytes. Liver biopsy fragments from ten children with chronic hepatitis B and C were used and two others in which hepatotropic virus infection was excluded. Frozen sections of liver samples were subjected to ABC immunohistochemistry (IHC) following incubation with a set of antibodies. Results of IHC findings were screened for correlation with clinical/laboratory data of patients. It was found that several PRRs could be shown in affected hepatocytes, but the incidence was higher in hepatitis C than in B. In hepatitis C, TLR1, 2, 4, NALP and RIG-1 helicase showed the most marked expression. In hepatitis B, TLR1, 3, 9, NOD1 and NALP expression were the most conspicuous. Expression PRRs in liver from hepatitis of unknown origin was much lower. It was also the case in cytospins from human hepatoma cell line. Several correlations between PRRs expression and clinical findings in patients could be shown by statistical exploration. In conclusion, this data suggests some role for PRRs in the pathogenesis of chronic viral hepatitis.

Interleukin 6 and 12, alanine aminotransferase activity, and HCV viral load in children with chronic hepatitis C treated with interferon and ribavirin.

The response to viral infections is mediated through the co-operation of cellular and humoral mechanisms. The aim of this study was to seek the correlation between IL-6 and IL-12 level, HCV viral load, ALT activity during the 48-week treatment with interferon-α-2b (IFN-α-2b) combined with ribavirin in children with diagnosed CHC and to search their influence on positive response to treatment. The group of 27 children with CHC was enrolled into this study. The children were treated with interferon-α and ribavirin in the course of 48-week therapy. The results show that both ALT activity and the viral load at the time of implementation of treatment with IFN-α and ribavirin is an important prognostic tool when treating children. It has been shown that the levels of IL-6 do not bear any significant prognostic importance to the implemented therapy, yet the increase of IL-12 levels in the 24th week of the treatment may be of prognostic value and may point out the possible elimination of HCV-RNA.

Results of antiviral treatment of patients with chronic hepatitis C: experience of Poznan centre.

INTRODUCTION Hepatitis C virus (HCV) infection in Poland affects approximately 750 thousand persons. The prevention of cirrhosis and hepatocellular carcinoma, of which approximately 20% of patients with chronic hepatitis C virus are at risk, aims at eradication of the virus by applying antiviral treatment with pegylated interferon alpha with ribavirin. MATERIAL/METHODS In this paper the results of the standard treatment of chronic hepatitis C in a population of 169 adult patients in whom it was started in the period of 01.01.2007-30.06.2008 are analyzed. Moreover, the influence of various clinical, biochemical and viral factors on achieving therapeutic success in the form of the sustained virological response (SVR) was studied. RESULTS In the group of 128 patients who received the full course of antiviral treatment, the SVR was achieved by 67.2% of patients (86 persons), whereas regarding all 169 patients who started the therapy, the sustained disappearance of viremia was found in 53.2% of patients (90 persons). Regarding 155 persons in whom the treatment was not interrupted for reasons others than virology, this value was 55.5%. For the sustained disappearance of viremia the following was favorable: genotype 3 virus, age under 40 years, body mass up to 75 kg, correct value of body mass index (BMI), low gamma-glutamyl transpeptidase (GGTP) activity before the treatment, minimum advancement of liver fibrosis in a liver biopsy (S1), complete early biochemical response (cEBR), and moreover, the achievement of negation of viremia after 12 weeks of the treatment in a group of patients infected with genotype 1 (complete early virological response, cEVR). These factors were strongly correlated with each other and that is why an analysis by the method of logistic multiple regression was impossible. Adverse reactions to the treatment and other health problems were the reasons for earlier discontinuation of the standard therapeutic scheme in 14 patients, whereby the lack of an SVR occurred in 10 of them (71.5% which is 5.9% of the studied population).