Agnieszka Adamek

Role of hepatitis C virus proteins (C, NS3, NS5A) in hepatic oncogenesis.

In recent years, the effects of hepatitis C virus (HCV) proteins on hepatocarcinogenesis have undergone intense investigations. The potentially oncogenic proteins include at least three HCV proteins: core (C) protein, NS3, and NS5A. Several authors indicated relationships between subcellular localization, concentration, a specific molecular form of the proteins (full length, truncated, phosphorylated), the presence of specific domains (the nuclear localization signal homologous to e.g. Bcl‐2) and their effects on the mechanisms linked to oncogenesis. The involvement of all the proteins has been described as being in control of the cell cycle, through interactions with key proteins of the process (p53, p21, cyclins, proliferating cell nuclear antigen), transcription factors, proto‐oncogenes, growth factors/cytokines and their receptors, and proteins linked to the apoptotic process. Untilnow, the involvement of the core protein of HCV in liver carcinogenesis is the most recognized. One of the most common proteins affected by HCV proteins is the p53 tumor‐suppressor protein. The p21/WAF1 gene is a major target of p53, and the effect of HCV proteins on the gene is frequently considered in parallel. The results of studies on the effects of HCV proteins on the apoptotic process are controversial. This work summarizes the information collected thus far in the field of HCV molecular virology and principal intracellular signaling pathways in which HCV oncogenic proteins are involved.

Expression of Cytokines (TNF-α, IL-1α, and IL-2) in Chronic Hepatitis C: Comparative Hybridocytochemical and Immunocytochemical Study in Children and Adult Patients

Hepatitis C virus (HCV) is one of the principal causes of hepatitis, which in more than 80% of cases leads to chronic lesions in the liver and involvement of extrahepatic organs. It remains unknown why the infection so frequently turns chronic, independently of patient age. Using immunocytochemistry (IHC) and in situ hybridization (ISH) (both linked to the ImmunoMax technique) we examined cell sources of TNF-α, IL-1α, and IL-2 in control and HCV-infected children and adults. We demonstrated augmented expression of all the cytokines in HCV-infected patients compared to controls. No differences were detected in amounts of studied transcripts or cytokine proteins between biopsies taken from HCV-infected children and adults. Expression of TNF-α was localized mainly in liver sinusoidal cells (macrophages, endothelial cells). A high proportion of hepatocytes demonstrated expression of TNF-α, IL-1α, and IL-2. In both groups of patients, higher amounts of cytokine proteins than studied transcripts were demonstrated. The augmented expression of TNF-α, IL-1α, and IL-2 in liver with a similar proportion of involved cells (mainly hepatocytes) in children and in adults points to participation of the cytokines in the pathogenesis of chronic hepatitis C. The expression is insufficient to terminate the infection and may be linked with the comparably frequent chronic transformation of HCV infection noted in children and adults.

Insulin-like growth factor (IGF) axis in cancerogenesis

Determination of the role of insulin-like growth factor (IGF) family components in carcinogenesis of several human tumors is based on numerous epidemiological and pre-clinical studies, experiments in vivo and in vitro and on attempts at application of drugs affecting the IGF axis. Investigative hypotheses in original studies were based on biological functions manifested by the entire family of IGF (ligands, receptors, linking proteins, adaptor molecules). In the context of carcinogenesis the most important functions of IGF family involve intensification of proliferation and inhibition of cell apoptosis and effect on cell transformation through synthesis of several regulatory proteins. IGF axis controls survival and influences on metastases of cells. Interactions of IGF axis components may be of a direct or indirect nature. The direct effects are linked to activation of PI3K/Akt signaling pathway, in which the initiating role is first of all played by IGF-1 and IGF-1R. Activity of this signaling pathway leads to an increased mitogenesis, cell cycle progression, and protection against different apoptotic stresses. Indirect effects of the axis depend on interactions between IGF and other molecules important for cancer etiology (e.g. sex hormones, products of suppressor genes, viruses, and other GFs) and the style of life (nutrition, physical activity). From the clinical point of view, components of IGF system are first of all considered as diagnostic serous and/or tissue biomarkers of a given cancer, prognostic factors and attractive target of modern anti-tumor therapies. Several mechanisms in which IGF system components act in the process of carcinogenesis need to be clarified, mainly due to multifactorial etiology of the neoplasms. Pin-pointing of the role played in carcinogenesis by any single signaling pathway remains particularly difficult. The aim of this review is to summarize the current data of several epidemiological studies, experiments in vitro and on animal models, to increase our understanding of the complex role of IGF family components in the most common human cancers.

p21/Wafl/Cipl cellular expression in chronic long-lasting hepatitis C: correlation with HCV proteins (C, NS3, NS5A), other cell-cycle related proteins and selected clinical data.

Studies indicate that proteins of hepatitis C virus (HCV) disturb expression of cell-cycle-related proteins. A disturbed cell-cycle control is a hepatocellular carcinoma (HCC) risk factor in patients with HCV-related liver damage. The present study aimed to analyse the cellular expression of p21/Wafl/Cipl (p21) in long-lasting chronic hepatitis C (CH-C), its correlation with the key oncogenic HCV proteins (C, NS3, NS5A), other cell-cycle-related proteins (PCNA, Ki-67, cyclin D1, p53) and selected clinical data. Archival liver biopsies, obtained from patients with CH-C, normal livers, and hepatocellular carcinoma (HCC) specimens were analysed by immunocytochemistry and ImmunoMax technique. In CH-C overexpression of p21 protein was demonstrated. Positive correlations of p21 protein expression in CH-C involved age of the patients, grading, and liver steatosis. Moreover, expression of p21 correlated significantly with expression of p53 protein, of D1 cyclin and Ki-67. Although Ki-67 antigen was related to p21 expression, only Ki-67 expression proved to be directly related to liver staging. Expression of the NS3 protein, which prevailed in CH-C patients, manifested correlation with p21 expression, and that of cyclin D1. In presence of preserved potential for regeneration, overexpression of p21 indicates inhibition of cell cycle in hepatocytes, which probably plays a protective role for the chronically damaged cells. Out of the three HCV proteins only NS3 seems to affect control of p21 protein expression in in vivo infection. Nevertheless, the studies indicate that neither expression of p21 protein nor that of viral NS3 protein can serve as a marker of progression of CH-C to HCC in vivo.

The insulin-like growth factor-1 and expression of its binding protein‑3 in chronic hepatitis C and hepatocellular carcinoma

The role of growth factors produced by the liver, including insulin-like growth factor-1 (IGF-1) and its main binding protein, IGF binding protein-3 (IGFBP-3), in hepatitis C virus (HCV)-associated carcinogenesis has only partially been recognized and there is not much data available on the local expression of IGF-1 and IGFBP-3 in chronic hepatitis C (CH‑C). Therefore, the aim of the present study was to evaluate the IGF‑1 and IGFBP‑3 serum levels and tissue expression in liver biopsies of CH‑C patients (n=37) and hepatocellular carcinoma (HCC) samples (n=61) as related to age- and gender-matched control serum samples (n=15) and healthy liver samples (n=10). Serum concentrations of IGF-1 (S-IGF-1) and IGFBP‑3 (S-IGFBP‑3) were measured by the ELISA method. Tissue expression of proteins was detected using ABC immunocytochemistry and evaluated applying a spatial visualization technique. Concentrations of S-IGF-1 and hepatic expression of IGF-1 (H-IGF-1) proved to be lower in CH-C compared to the controls. No significant differences were detected in the concentration of S-IGFBP-3 between the studied groups but the S-IGF-1/IGFBP-3 ratio in the CH-C group was significantly lower compared to the control. H-IGFBP-3 was higher in CH-C compared to those in the control and HCC. In HCC, lower expression of H-IGF-1 was detected compared to the control and a higher H-IGF-1/IGFBP-3 ratio compared to CH-C. A negative correlation was detected between S-IGF-1 and S-IGF-1/IGFBP-3 ratio, on the one hand, and age, grading and concentration of α-fetoprotein (AFP) on the other, while H-IGFBP-3 was negatively correlated with BMI in the CH‑C group. In patients with CH‑C, the H‑IGF‑1/IGFBP‑3 ratio was higher compared to that of the S‑IGF‑1/IGFBP‑3 ratio. The studies documented a disturbed H‑IGF‑1 and H‑IGFBP‑3 in CH‑C, which may be of significance in carcinogenesis. Examination of serum concentration and tissue expression of the two proteins and, first of all, estimation of the IGF‑1/IGFBP‑3 ratio may provide additional (to the estimation of IGF‑1 and AFP) non-invasive markers in HCV‑related liver injury.

p53 immunocytochemistry and TP53 gene mutations in patients with chronic hepatitis C virus (HCV) infection.

Chronic infection with hepatitis C virus (HCV) is regarded as a risk factor for hepatocellular carcinoma (HCC), mostly in patients with liver cirrhosis. Present study aimed at evaluation of cellular expression of p53 protein, genetic TP53 changes in liver samples and anti-p53 in serum of patients with chronic hepatitis C virus infection. The expression of p53 protein were analysed by immunocytochemistry in liver biopsies from adult patients with chronic, long-lasting hepatitis C. In order to detect TP53 mutations, PCR/SSCP and sequencing were performed. Antibodies against p53 in serum were determined using enzyme immunoassay (ELISA).In two out of 14 examined patients TP53 point mutations were detected in the liver samples. In the first patient, a substitution of C to T was demonstrated in position 1 of the codon 250, resulting in substitution of proline by serine. The other patient carried a substitution of C to G in position 13274 of the intron 6. The patient carrying mutation in the codon 250 demonstrated morphological traits of liver cirrhosis and had high number of p53-immunoreactive cell nuclei in tissue. None of the patients manifested elevated titres of serum anti-p53. In the liver, significant positive correlations were disclosed between expression of p53 on one hand and grading and staging on the other. A negative correlation was disclosed between cellular expression of p53 and duration time of infection. In conclusions, genetic changes in TP53 can be detected also in non-neoplastic lesions linked to chronic HCV infection.

Polysaccharides and mucin 5AC (MUC5AC) expression in gallbladder mucosa of young patients with gallstones as evaluated by spatial visualization and quantification.

The study aimed at examination of tissue expression of polysaccharides and secretory mucin 5AC (MUC5AC) in young patients (up to 25 years of age) with a symptomatic gallstones. For comparison, patients most frequently subjected to cholecystectomy were studied, i.e. patients of approximately 50 years of age with the same diagnosis. In quantitative studies on tissue expression of both mucus components, the modern technique of spatial visualization was applied for the first time. Application of the technique permitted to demonstrate significant positive relationships between expression of glycoproteins (immunocytochemical ABC technique for detection of MUC5AC) and expression of sugar components in mucus (PAS technique) and to confirm suitability of the technique for quantitative appraisal of both histochemical and immunocytochemical reactions. An even higher expression of polysaccharides in the entire mucosa and of MUC5AC was detected in gallbladder epithelium of 50-year-old patients, as compared to young patients with symptomatic gallstones. In the young patients, expression of polysaccharides correlated with inflammatory activity (grading), width of gallbladder wall and PLT level in peripheral blood. A significantly higher expression of polysaccharides in gallbladder epithelium was demonstrated in young patients admitted in the emergency mode to the hospital. These correlations in young patients may suggest a role of both mucus components in pathogenesis of cholelithiasis in this age group. A quantitative appraisal of mucus component expression in the two parts of gallbladder mucosa (epithelium vs. entire mucosa) using spatial visualization technique permitted to more accurately compare production of glycoproteins and of polysaccharides in patients with cholelithiasis and to demonstrate additional correlations of a potential clinical significance.

Insulin-like growth factor-1 mRNA isoforms and insulin-like growth factor-1 receptor mRNA expression in chronic hepatitis C.

AIM To evaluate the expression of different insulin-like growth factor (IGF)-1 mRNA isoforms and IGF-1 receptor (IGF-1R) mRNA in hepatitis C virus (HCV)-infected livers. METHODS Thirty-four liver biopsy specimens from chronic hepatitis C (CH-C) patients were obtained before anti-viral therapy. Inflammatory activity (grading) and advancement of fibrosis (staging) were evaluated using a modified point scale of METAVIR. The samples were analyzed using quantitative real-time PCR technique. From fragments of liver biopsies and control liver that were divided and ground in liquid nitrogen, RNA was isolated using RNeasy Fibrous Tissue Mini Kit according to the manufacturers instruction. Expression levels of IGF-1 mRNA isoforms (IGF-1A, IGF-1B, IGF-1C, P1, and P2) and IGF-1R mRNA were determined through normalization of copy numbers in samples as related to reference genes: glyceraldehyde-3-phosphate dehydrogenase and hydroxymethylbilane synthase. Results on liver expression of the IGF-1 mRNA isoforms and IGF-1R transcript were compared to histological alterations in liver biopsies and with selected clinical data in the patients. Statistical analysis was performed using Statistica PL v. 9 software. RESULTS The study showed differences in quantitative expression of IGF-1 mRNA variants in HCV-infected livers, as compared to the control. Higher relative expression of total IGF-1 mRNA and of IGF-1 mRNAs isoforms (P1, A, and C) in HCV-infected livers as compared to the control were detected. Within both groups, expression of the IGF-1A mRNA isoform significantly prevailed over expressions of B and C isoforms. Expression of P1 mRNA was higher than that of P2 only in CH-C. Very high positive correlations were detected between reciprocal expressions of IGF-1 mRNA isoforms P1 and P2 (r = 0.876). Expression of P1 and P2 mRNA correlated with IGF-1A mRNA (r = 0.891; r = 0.821, respectively), with IGF-1B mRNA (r = 0.854; r = 0.813, respectively), and with IGF-1C mRNA (r = 0.839; r = 0.741, respectively). Expression of IGF-1A mRNA significantly correlated with isoform B and C mRNA (r = 0.956; r = 0.869, respectively), and B with C isoforms (r = 0.868) (P < 0.05 in all cases). Lower expression of IGF-1A and B transcripts was noted in the more advanced liver grading (G2) as compared to G1. Multiple negative correlations were detected between expression of various IGF-1 transcripts and clinical data (e.g., alpha fetoprotein, HCV RNA, steatosis, grading, and staging). Expression of IGF-1R mRNA manifested positive correlation with grading and HCV-RNA. CONCLUSION Differences in quantitative expression of IGF-1 mRNA isoforms in HCV-infected livers, as compared to the control, suggest that HCV may induce alteration of IGF-1 splicing profile.

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).

The prevalence of mixed genotype infections in Polish patients with hepatitis C

OBJECTIVES Hepatitis C virus (HCV) genotype has been described as an independent predictor of the response to therapy. A mixed infection with two types of HCV is probably an uncommon event. The aim of this study was to determine the occurrence of mixed infection with two different HCV genotypes in adult patients with chronic hepatitis C eligible for treatment. METHODS Plasma samples and clinical and demographic data were collected from 1159 patients with hepatitis C. The INNO-LiPA HCV assay was used to identify the HCV genotypes. RESULTS The dominant genotype was genotype 1, which was found to be responsible for 83.9% of infections, with subtype 1b being the most common. A mixed genotype infection was detected in 26 patients (2.2%). The most common mixed genotype was 1a+1b detected in 17/26 patients (65%). Antiviral therapy led to complete elimination of both genotypes in 50% of patients with 1b+3a infection and in 33% of patients with 1b+4a infection. CONCLUSIONS The results obtained showed that infection with mixed HCV genotypes in Polish patients with hepatitis C is uncommon. The selective elimination of genotypes 3a and 4a after therapy confirms the greater resistance to treatment of genotype 1b. In the context of new anti-HCV drug development, further investigations are needed to determine the clinical importance of mixed HCV infection.