Hepatitis Monthly
Year:2015 | Volume:15 | Issue:3|Papers Count:8

Background: Human telomerase reverse transcriptase (hTERT) has become an ideal target for development of anticancer therapy. Small interfering RNAs (siRNAs) are very powerful reagents for gene silencing and show promise for cancer gene therapy. However, only a small number of siRNAs have been demonstrated to be effective. For gene therapy targeting hTERT, it is essential to develop a robust system to fully explore the power of siRNAs.Objectives: We explored a siRNA expression cassette (SEC) to screen highly effective RNAi-targeted sequences for gene therapy of hepatocellular carcinoma (HCC).Materials and Methods: An SEC was developed by flanking H1 and U6 promoters in opposite directions at the siRNA-encoding sequence. Eight SECs specific to hTERT were designed by overlap extension polymerase chain reaction (PCR) and transfected into HepG2 cells with calcium phosphate. The telomerase activity was determined by telomeric repeat amplification protocol (TRAP) silver staining and TRAP real-time PCR analysis. The mRNA and protein expression levels of hTERT were determined by reverse transcription (RT)-PCR and western blot, respectively. Cell viability was determined by the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay and cell apoptosis was measured by the annexin-V/propidium iodide (PI) assay coupled with flow cytometry.Results: Eight hTERT-specific SECs (SEC-1-8) were successfully constructed. In comparison to that of the negative control SEC, the hTERT-specific SECs, especially, SEC-4, SEC-5, SEC-7 and SEC-8 significantly reduced the activity of hTERT in HepG2 cells at 48 hours after transfection. Moreover, the mRNA and protein expression levels of hTERT as well as the cell viability were significantly reduced by SECs. Knockdown of hTERT by SECs in HepG2 cells led to cell apoptosis.Conclusions: Our developed simple SEC was a powerful strategy for screening highly effective RNAi-targeted sequences and showed promise for gene therapy of HCC.

Introduction: Various types of dermatological manifestations have been reported due to hepatitis C virus (HCV) infection and antiHCV therapy. Some of them have been described during IFN-based therapies. PEG-IFN-a-2a/RBV combination is used as the international standard of treatment for HCV infection for a long time. The combination therapy yields an adverse-event profile similar to standard interferon (IFN) therapy. Some of these adverse effects are rheumatologic, neuropsychiatric and dermatological manifestations including alopecia.Case Presentation: We reported a 43-year-old woman with dystrophic anagen effluvium (DAE), rheumatoid arthritis and Hashimoto thyroiditis, which were developed under the combination therapy for chronic HCV infection.Conclusions: Although some cases of alopecia areata (AA) and telogen effluvium (TE) were reported in literature, no case of DAE associated with PEG-INF-a-2a /RBV combination therapy was reported previously.

Background: The only established therapy for chronic viral delta hepatitis, the most severe form of viral hepatitis is treatment with pegylated-interferon a (Peg IFN a).Objectives: In this study, we aimed to determine the efficacy of pegylated-interferon a 2a (Peg-IFN a 2a) and 2b (Peg IFN a 2b) in the treatment of patients infected with chronic delta hepatitis virus.Patients and Methods: The sample size was based on available patients potentially to be recruited. Data of 63 patients receiving either Peg IFN alpha 2a or Peg IFN alpha 2b were retrospectively assessed in the present cohort study performed in Turkey. Of 56 patients completed the study, 41 received Peg IFN α 2a and 15 received Peg IFN α 2b for 12 months. Patients were evaluated for biochemical and virological responses at the end of given treatment and six months after the treatment.Results: Stage of fibrosis was found high in both groups (85.4% vs. 86.7%), while cirrhosis was higher in the group of Peg IFN a 2b (53.3% vs. 34.1%). At the end of treatment, either hepatitis delta virus RNA (HDV RNA) alone or both HDV RNA and hepatitis b virus DNA (HBV DNA) had negative results in 32% of patients. Although HDV RNA negativity was sustained in 30.3% of patients, negativity of both HDV RNA and HBV DNA was decreased to 19.6% six months after completion of the treatment. HBV DNA became positive in one third of patients with response at six months after completion of the treatment (10.7 % of all patients). HDV RNA negativity at month six was found as a predictor of positive response. No significant difference was found between Peg IFN a 2a and Peg IFN a 2b for virological response rate.Conclusions: Treatment with Peg IFN α achieved a sustained negativity of HDV RNA in about one third of patients. Duration of Peg IFN α therapy might be prolonged to at least 24 months or more to prevent the occurrence of Hepatitis B virus (HBV) relapse encountered six months after completion of the treatment.

Background: Hepatitis viruses are one of the most important concerns in the patients on hemodialysis who are at high risk for bloodborne infections. GB virus C (GBV-C)/hepatitis G virus (HGV) is positive-stranded RNA virus with global distribution, which codes structural and nonstructural proteins (such as NS3).Objectives: Review on literature about Iranian HGV isolates showed that there was no report on sequencing and phylogenetic characteristics of NS3 gene and therefore, this study attempted to answers these challenges.Materials and Methods: Briefly, steps followed were as follows: preparation of nucleic acids from three Iranian hemodialysis HGVpositive samples and RNA extraction; reverse transcriptase-PCR (RT-PCR) for amplification of NS3 gene and gel electrophoresis; sequencing and analyzing sequencing data; NCBI Registering; blasting and alignment of sequences; and finally constructing the phylogenetic tree.Results: Nucleotide BLAST results emphasized that most similar sequences to Ir NS3 sequences were those from Europe (Spain, Germany, and the United Kingdom) and the United states and IR sequences were located with five leaves in a branch. In addition, homology of Ir sequences showed96.4% (between IR1 and IR2), 94.9% identity (between IR1 and IR3), and 96.5% identity (between IR2 and IR3).Furthermore, highly variation and significance differences in NS3 between GBC isolates from geographical regions and some little changes in IR sequences were seen. Finally, phylogenetic tree revealed that NS3 genotype of Iranian isolates was probably similar to European countries and USA.Conclusions: Overall, results of present study were consistent with the data reported earlier base on 5’ NTR in Iranian isolates and revealed genotype IR2 is major genotype in Iranian HGV-positive patient. It means there are higher similarities between Iranian and Europe-USA in HGV NS3 gene.

Background: Cardiovascular events account for the main cause of death in patients with non-alcoholic fatty liver disease (NAFLD), and are largely influenced by genetic factors. Although multiple studies showed that tumor necrosis factor-alpha (TNF-a) polymorphisms are risk factors in the progression of NAFLD, few papers on the association of the polymorphisms and the developing coronary artery disease (CAD) in NAFLD patients have been reported.Objectives: The present study was designed to evaluate the association of TNF-a polymorphisms at residues -238 and -308, with the risk of developing CAD in Chinese patients with NAFLD.Patients and Methods: The TNF-a polymorphisms at residues 238 and 308 were genotyped in B-type ultrasonography proven NAFLD patients with (n=246), without (n=247) CAD and healthy controls (n=304), using polymerase chain reaction (PCR). Serum lipid profiles were determined using biochemical methods. Statistical analyses were performed using SPSS statistical software, version 20.0 for Mac.Results: We found a significant association between TNF-a-238 guanine to alanine (GA) polymorphism and carriers of variant allele A between NAFLD patients with and without CAD (P<0.05). Carriers of the A allele of TNF-a-238 had higher serum triglycerides (TG) and low density lipoprotein (LDL) levels in NAFLD patients with CAD (P=0.025 and 0.001, respectively) and a higher TG level in NAFLD patients without CAD (P=0.017), than their non-carrier counterparts.Conclusions: In the Chinese Han population that we studied, NAFLD patients who carry the TNF-a-238 GA polymorphism have an increased risk of developing CAD. Mechanisms underlying this potentially important association require further investigation.

Background: Hepatitis A, a fecal-oral transmitted disease, which has been considered endemic in developing countries, seems to change its pattern in developing countries because of their improved socioeconomic status.Objectives: In the present study, we aimed to determine the need of vaccination in 270 students at AJA University of Medical Sciences.Materials and Methods: The serum level of anti-HAV antibody was checked in 270 students of AJA University of medical students, and effect of different factors, including age, gender, pre-university entrance exam region, familial education, familial income, clean water availability, and previous history of jaundice were tested.Results: Of total 270 students, 30 were female. Their age ranged between 18 and 30 years old with the mean age of 20.58 years and just 34% of students had positive level of anti-HAV antibody. Age and sex had no role in positive serum level of anti-HAV antibody. According to analyzed data, lack of clean water availability, pre-university entrance exam region, lower family education, and poor health status estimation increased statistically the risk of HAV infection.Conclusions: Because 66% of students were anti-HAV antibody negative and they will work as health care workers in future, our study suggest vaccinating all students accepted at AJA University of Medical Sciences.

Background: Single-nucleotide polymorphisms (SNP) in the inosine triphosphate pyrophosphatase (ITPA) gene correlate with ribavirin (RBV)-induced anemia in patients with chronic hepatitis C (CHC) receiving combination therapy. Managing anemia is an early priority in the treatment process.Objectives: The aim was to develop a predictive index based on ITPA SNP status to identify CHC patients at risk of anemia.Patients and Methods: A total of 418 eligible East Asian patients diagnosed with CHC genotype 1 (G1) received combination therapy in this study. Participant DNA was genotyped for a functional ITPA SNP (C/C, A/A or C/A) on chromosome 20 at rs1127354. A predictive index was constructed by incorporating independent factors identified for severe anemia events (hemoglobin<10 g/dL). Areas under the receiveroperating characteristic curves (AUCs) represented the diagnostic accuracies of the predictive index in randomly assigned development and validation cohorts.Results: Multiple logistic regressions identified age (≥ 50 y: OR=9.7, 95% CI=5.0- 18.6), ITPA rs1127354 (C/C: OR=3.3, 95% CI=1.8- 5.8) and baseline hemoglobin (<14.0 g/dL: OR 6.4, 95% CI=3.3-12.1; 14.0 - 14.9: OR = 2.4, 95% CI=1.2-4.6) as predictors of severe anemia throughout the treatment. For severe anemia, the predictive index incorporating age, ITPA SNP status and baseline hemoglobin yielded diagnostic accuracies (AUCs) of 0.830 (95% CI=0.783-0.871) in the development (n=324) and 0.902 (0.826-0.925) in the validation (n=81) cohorts.Conclusions: In patients with CHC G1 and receiving combination therapy, ITPA SNP-based index was an accurate and practical solution for prediction of severe anemia.

Background: A potential treatment for healing hepatic tissue is delivering isolated hepatic cells to the site of injury to promote hepatic cells formation. In this technology, providing an appropriate injectable system for delivery of hepatic cells is an important issue. In this regard, fibrin scaffolds were designed with many advantages over other scaffolds like cell delivery vehicles for biodegradation, biocompatibility and hemostasis.Objectives: The aim of this study was to determine suitable cell culture circumstances for HepG2 cell proliferation and differentiation in 3D fibrin scaffolds by evaluating Ca2+ concentrations, cell numbers, various ratios of plasma/RPMI 1640 and thickness of fibrin scaffold.Materials and Methods: In a one-stage experimental design, Box-Behnken design strategy was performed by Minitab 15 software (version 15, Minitab. State College, PA) with three factors at three levels (low, medium and high) and 27 runs for identification of the effects of ratio of plasma/RPMI 1640, Ca2+ concentration and thickness on the formation of fibrin gel scaffold and 3D HepG2 culture.Results: The optimal concentrations for fibrin scaffold fabrication were achieved by adding 0.15 mol CaCl2 (50 mL) and 1×105 cells to 1:4 of plasma/RPMI 1640 ratio (500 mL with 2.3 mm thickness per well).Conclusions: Our approach provided easy handle method using inexpensive materials like human plasma instead of purified fibrinogen to fabricate fibrin scaffold.