SOCS1 and pattern recognition receptors: TLR9 and RIG-I; novel haplotype associations in Egyptian fibrotic/cirrhotic patients with HCV genotype 4.

In this paper we explore the role of suppressor of cytokine signaling 1 (SOCS1) (rs243327), the regulator of toll-like receptor-9 (TLR9) (rs352140), retinoic acid inducible gene-I (RIG-I) (rs669260), and cluster of differentiation 152 (CD152) (rs231776) in fibrotic/cirrhotic patients. Single nucleotide polymorphisms (SNPs) within these genes as well as haplotype analyses were performed on a cohort of 120 Egyptian fibrotic patients. Fibrosis had progressed from HCV genotype 4 infections. Using RT-PCR, SNPs were evaluated in the DNA collected from each patient using TaqMan® genotyping assays. A regression model was used to evaluate allelic and haplotypic associations with a fibrosis/cirrhotic scale. The necroinflammatory A score was adjusted for non-genetic covariates. The genotype distributions for SOCS1 (rs243327) and TLR-9 (rs352140) differed significantly between the F1-F3 and F3-F4 groups. On the other hand, the genotype distributions for RIG-I (rs669260) and CD152 (rs231776) genes did not significantly differ. The allele frequency was calculated using Hardy-Weinberg Equilibrium (HWE) for the SOCS1 (rs243327), RIG-I (rs669260), and CD152 (rs231776) genes. These calculated frequency values indicated the need to compare them to another population for that locus. However, TLR9 (rs352140) did not show similar results. The A allele in SOCS1, TLR9, and RIG-I SNPs was an adverse prognostic factor for liver fibrosis and liver activity. Haplotype analysis revealed a significant association between SOCS1 and TLR9 in fibrotic/cirrhotic patients. This indicated the presence of the A allele in either gene, which is considered a risk factor for the progression of liver disease to cirrhosis. SOCS1 rs243327, TLR9 rs352140, and RIG-I rs669260 polymorphisms might affect liver pathophysiology and the cirrhotic outcome following genotype 4 HCV infection. Therefore, performing this specific SNP testing may be of value for the stratification of the population at risk.

Relation of interleukin-1ß gene to treatment response in chronic patients infected with HCV genotype 4.

INTRODUCTION: Hepatitis C virus (HCV) infection results in chronic hepatitis in more than 70% of infected patients, while 20-30% of patients recover spontaneously. This strengthens the role of the host genetic factors in either spontaneous or drug-induced viral clearance. The aim of this study was to investigate the relationship between interleukin-1ß +3953 gene polymorphism and the response to interferon therapy in chronic HCV patients infected with genotype 4. METHODOLOGY: The interleukin-1ß (+3953 C/T) (rs1143634) gene was amplified in 115 chronic HCV patients. Interleukin-1ß single nucleotide polymorphism (SNP) plus several clinical and pathological factors were statistically analyzed in correlation with response to therapy. RESULTS: Genotypes C/T and T/T had a significant association with non-response to treatment compared to genotype C/C, which had a strong association with response to treatment (95% confidence; 6.4884-48.5818, p = 0.0001). Furthermore, analysis of allele frequency in this cohort revealed that the T allele is associated with non-response, higher fibrosis, and higher hepatic activity, while the C allele had a significant association with sustained virologic response lower fibrosis, and lower hepatic activity (p value = 0.0001). CONCLUSION: This is the first study to examine the correlation between interleukin-1ß (+3953 C/T) (rs1143634) gene polymorphism and the response of interferon therapy in genotype 4 HCV-infected patients. The results encourage further assessment of this SNP as a marker to predict response to therapy and disease progression, which can have major implications in saving money, time, and in avoiding unnecessary adverse effects.

Heterogeneity and new epitopes of hepatitis C virus genotype 4.

BACKGROUND: Hepatitis C virus (HCV) was found to have a major role in human liver disease by its ability to face the host-cell defenses and the immune system. Heterogeneity of HCV was the key for its adaptation to its host and represented a significant hurdle for the development of both effective vaccines as well as for novel therapeutic interventions. OBJECTIVES: Due to the heterogeneity of HCV virus because of both high replication and high mutation rate in vivo, this study was conducted to analyze different isolates of Egyptian patients of genotype 4, of the most mutant regions of the virus (E1 and E2) as they played an important role in viral persistence by escaping from the immune system of the host body. PATIENTS AND METHODS: This study was conducted through PCR amplification of E1 and E2 regions, sequencing and phylogenetic analysis, calculating synonyms and non-synonyms substitutions, finding the possible glycosylation sites and different epitope domains. RESULTS: The present work figured out that the heterogeneity of the quasispecies of our local strains 4a was high showing up 15% diversity. This study also showed four glycosylation sites that play an important role in the entry of the virus and protein folding. Besides, different epitpoes were identified in different regions of the E1 and E2 domains; a finding which would help in determining the neutralizing and non- neutralizing antibodies. CONCLUSIONS: This study would help in understanding the driving forces of genetic diversity and would be fundamental for representing potential candidate targets for antibodies and the development of vaccine trials.

Association between low molecular polypeptide 7 single nucleotide polymorphism and response to therapy in hepatitis C virus infection.

AIM: To investigate the relationship between low molecular polypeptide-7 (LMP-7) gene polymorphism and response to interferon (IFN) therapy in chronic hepatitis C virus (HCV) patients. METHODS: LMP-7 polymorphism at codon 49 with nucleotide substitution from A to C was amplified in 104 chronic HCV patients of genotype 4. The amplicons were digested with restriction endonuclease BsmI and the produced restriction fragment length polymorphism was analyzed. Patients received IFN + regional blood volume therapy for 48 wk and the frequency of this single nucleotide polymorphism (SNP) was statistically correlated with treatment response. The exclusion criteria for these patients were stated by the national health program for treating viral hepatitis. Main exclusion criteria included co-infection with hepatitis B virus or schistosomiasis, thyroid dysfunction, uncontrolled diabetes mellitus, history of long term drug or alcohol intake and autoimmune hepatitis. Multivariate analyses were done to correlate LMP-7 SNP plus several factors such as age, gender, weight, serum alpha-fetoprotein (AFP) and alanine aminotransferase levels, liver activity, fibrosis score and viral load with response to therapy. RESULTS: The data presented in this study clearly demonstrated statistically significant differences between sustained virological response (SVR) (defined as the absence of HCV RNA levels in the patient's sera at least 6 mo after discontinuation of treatment) and non-response (NR) (where HCV RNA levels in the patient's sera never become undetectable for 6 mo during or after treatment). Variables were described as odds ratio with 95%CI. The data were considered significant if P values were ≤ 0.05; highly significant if P < 0.01 and very highly significant if P < 0.001. Current data showed that 91.7% of patients carrying LMP-7 C/C allele were associated with SVR, while the other two genotypes C/A and A/A were associated with NR patients, 83.3% and 64.3% respectively, showing that genotype CC was strongly associated with response to interferon (95%CI: 12.0719-134.6572, P = 0.0001). The majority of parameters recorded in SVR and NR patients included higher values of mean age (P = 0.004), alanine aminotransferase (P = 0.001), AFP (P = 0.001), body weight (P = 0.025), viral load (P = 0.025), higher fibrosis and histological activity index indices among NR vs SVR patients. Also, the multivariate statistical analysis of the different factors of fibrosis score, liver activity grade, genotypes and alleles of LMP-7 gene polymorphism in responders and NRs of HCV patients in this study showed that HCV patients with A allele had a very highly significant association with the NRs, high fibrosis and higher liver activity, while the C allele had a very highly significant association with the responders, low fibrosis and lower liver activity (95%CI: 3.5800-13.2519, P = 0.0001). CONCLUSION: LMP-7 SNP is a candidate gene that should be considered when designing a mathematical model for predicting response to therapy and disease progression in HCV patients.

A Study of CC-Chemokine Receptor 5 (CCR5) Polymorphism on the Outcome of HCV Therapy in Egyptian Patients.

BACKGROUND: Chronic hepatitis C virus (HCV) infection is a globally serious public health issue. OBJECTIVES: In this study, we investigated CC chemokine receptor 5 (CCR5-59029) polymorphism which is considered an important component of the immune system in determining the outcome of HCV infection. Its critical role as a marker in response to interferon therapy of HCV infection is also investigated besides its effect on other clinical patient factors. PATIENTS AND METHODS: This study was conducted on 82 Egyptian patients with chronic Hepatitis C Virus (HCV) infection who received PEG-INF + Ribavirin treatment for 48 weeks. The study was also conducted on 50 healthy controls (with negative results for HCV antibody and RNA PCR). Full history of patients in this study was recorded. Clinical and histological examinations, qualitative HCV nested RT-PCR, quantitative real -time PCR, and genotyping of HCV RNA genome were performed. CCR5-59029 polymorphism with nucleotide substitution from G to A was amplified. The amplicons were digested with restriction endonuclease Bsp 1286I, and produced RFLPs of the CCR5 genotypes were determined. RESULTS: The present study showed a significant association between the functional SNP of CCR5 gene and the viral response to interferon in chronic HCV Egyptian patients. It was shown that the higher fibrosis stages (F2-F4) had significant association with nonresponse to treatment compared to the lower fibrosis stages (F0-F1) (95% confidence: 5.497 - 55.074, P = 0.0001). In addition, worse liver activity grade (A2-A3) had a very highly significant association with non-responder HCV patients compared to those with better liver activity grade (A1) (95% confidence: 2.242 - 20.974, P = 0.0007). Most importantly HCV patients with G allele had a high significant association with nonresponse to treatment, higher fibrosis stages and worse liver activity grades, while the A allele had a high significant association with sustained response, low fibrosis stages and relatively better liver activity grade (95% confidence: 3.347 - 15.036, P = 0.0001). CONCLUSIONS: SNPs within the CCR5 gene should be considered as an important factor used in combination with other host gene SNPs when developing a mathematical model for anticipating response to HCV therapy.

Cytotoxic effects of 2-methoxyestradiol in the hepatocellular carcinoma cell line HepG2.

AIM: The study was designed to examine the potential cytotoxicity of 2-methoxyestradiol (2ME2), a natural 17beta-estradiol metabolite, in hepatocellular carcinoma and the possible underlying mechanisms for this cytotoxicity. METHODS: The cell line HepG2 was treated with different concentrations of 2ME2 for 48 and 72 h. RESULTS: Using the sulforhodamine B assay, HepG2 was sensitive to the cytotoxic effect of 2ME2. 2ME2 induced cell arrest at the G(2)/M phase and a significant high percentage of apoptotic cells compared to the control group. Also, 2ME2 induced a significant increase in caspase 9 enzymatic activity after 48 and 72 h of treatment compared with control values. The DNA laddering was observed only in cells treated for 72 h. Furthermore, 2ME2 induced a significant decrease in the expression levels of vascular endothelial growth factor (VEGF) gene compared to the control values. CONCLUSION: 2ME2 exerts cytotoxic activity in the HepG2 cell line by preferential cell blocking at the G(2)/M phase as well as induction of apoptosis as evidenced by increased caspase 9 enzymatic activity and observed DNA laddering in 2ME2-treated HepG2 cells. In addition, a reduction in hypervascularity is an important postulated mechanism as indicated by the significant reduction in the expression of VGEF, one of the most important angiogenic factors.

HepG2 cells support viral replication and gene expression of hepatitis C virus genotype 4 in vitro.

AIM: To establish a cell culture system with long-term replication of hepatitis C virus (HCV) genome and expression of viral antigens in vitro. METHODS: HepG2 cell line was tested for its susceptibility to HCV by incubation with a serum from a patient with chronic hepatitis C. Cells and supernatant were harvested at various time points during the culture. Culture supernatant was tested for its ability to infect naive cells. The presence of minus (antisense) RNA strand, and the detection of core and E1 antigens in cells were examined by RT-PCR and immunological techniques (flow cytometry and Western blot) respectively. RESULTS: The intracellular HCV RNA was first detected on d 3 after infection and then could be consistently detected in both cells and supernatant over a period of at least three months. The fresh cells could be infected with supernatant from cultured infected cells. Flow cytometric analysis showed surface and intracellular HCV antigen expression using in house made polyclonal antibodies (anti-core, and anti-E1). Western blot analysis showed the expression of a cluster of immunogenic peptides at molecular weights extended between 31 and 45 kDa in an one month old culture of infected cells whereas this cluster was undetectable in uninfected HepG2 cells. CONCLUSION: HepG2 cell line is not only susceptible to HCV infection but also supports its replication in vitro. Expression of HCV structural proteins can be detected in infected HepG2 cells. These cells are also capable of shedding viral particles into culture media which in turn become infectious to uninfected cells.

Soluble egg antigen of Schistosoma Haematobium induces HCV replication in PBMC from patients with chronic HCV infection.

BACKGROUND: This study was conducted to examine, in vitro , the effect of soluble egg antigen (SEA) of S. haematobium on intracellular HCV RNA load in peripheral mononuclear cells (PBMC) as well as on cell proliferation in patients with chronic HCV infection. METHODS: PBMC from 26 patients with chronic HCV infection were cultured for 72 hours in presence and absence of 50 mug SEA/ml medium. Intracellular HCV RNA quantification of plus and minus strands was assessed before and after stimulation. PBMC from five healthy subjects were cultured for 7 days, flow cytometric analysis of DNA content was used to assess the mitogenic effect of SEA on PBMC proliferation compared to phytoheamaglutinine (PHA). RESULTS: Quantification of the intracellular viral load showed increased copy number/cell of both or either viral strands after induction with SEA in 18 of 26 patients (69.2%) thus indicating stimulation of viral replication. Flow cytometric analysis showed that mean +/- S.D. of percent values of cell proliferation was induced from 3.2 +/- 1.5% in un-stimulated cells to 16.7 +/- 2.5 % and 16.84 +/- 1.7 % in cells stimulated with PHA and SEA respectively. CONCLUSION: the present study supports earlier reports on SEA proliferative activity on PBMC and provides a strong evidence that the higher morbidity observed in patients co-infected with schistosomiasis and HCV is related, at least in part, to direct stimulation of viral replication by SEA.

Antisense oligonucleotide inhibition of hepatitis C virus genotype 4 replication in HepG2 cells.

BACKGROUND: Hepatitis C (HCV) viral infection is a serious medical problem in Egypt and it has a devastating impact on the Egyptian economy. It is estimated that over 15% of Egyptians are infected by the virus and thus finding a cure for this disease is of utmost importance. Current therapies for hepatitis C virus (HCV) genotype 4 with interferon/ribavirin have not been successful and thus the development of alternative therapy for this genotype is desperately needed. RESULTS: Although previous studies utilizing viral subgenomic or full cDNA fragments linked to reporter genes transfected into adhered cells or in a cell free system showed promise, demonstration of efficient viral replication was lacking. Thus, we utilized HepG2 cells infected with native HCV RNA genomes in a replication competent system and used antisense phosphorothioate Oligonucleotides (S-ODN) against stem loop IIId and the AUG translation start site of the viral polyprotein precursor to monitor viral replication. We were able to show complete arrest of intracellular replication of HCV-4 at 1 uM S-ODN, thus providing a proof of concept for the potential antiviral activity of S-ODN on native genomic replication of HCV genotype 4. CONCLUSION: We have successfully demonstrated that by using two S-ODNs [(S-ODN1 (nt 326-348) and S-ODN-2 (nt 264-282)], we were able to completely inhibit viral replication in culture, thus confirming earlier reports on subgenomic constructs and suggesting a potential therapeutic value in HCV type 4.