Mostafa K. El-Awady

Association of IL28B SNP With Progression of Egyptian HCV Genotype 4 Patients to End Stage Liver Disease

Background IL28B single nucleotide polymorphisms (SNPs) play important roles in the management of hepatitis C virus (HCV) infections and are strongly associated with spontaneous and treatment-induced HCV clearance. Objectives In the present study, the association between IL28B variants and the progression of HCV infection in Egyptian patients infected with type 4a virus will be examined. Patients and Methods Frequencies of the protective genotype C/C of SNP, rs12979860 were determined in healthy subjects, spontaneous resolvers, and chronic HCV type 4 patients with low F scores and in patients with end stage liver disease (ESLD). This study included a total of 404 subjects. Patients infected with HCV type 4a (n = 304) were divided into; chronic hepatitis C (CHC) with low F scores (CHC, n = 110), end stage liver disease (n = 110), liver cirrhosis (LC) (n = 35) and hepatocellular carcinoma (HCC) patients (n = 75), spontaneous resolvers of HCV infection (n = 84) were also included. A healthy group representing the Egyptian population (n = 100) was also included in the genotyping of IL28B. The later was typed via a polymerase chain reaction based restriction fragment length polymorphism (PCR-RFLP) assay analysis on purified genomic DNA extracted from all individuals. Results A significant increase (P < 0.0005) was observed in frequencies of IL-28B rs12979860 C/C genotypes in the healthy population, than in the CHC, LC and HCC groups (C/C = 48%, 13%, 0%.and 0% respectively). On the other hand the C/C genotype was significantly higher (P < 0.0005) in spontaneous resolvers than in healthy subjects. A comparable significant increase in the frequency of C/T allele accompanied by mild elevation of T/T allele frequency, were detected along the progression towards ESLD. Conclusions Genotype C/C is associated with viral clearance during acute infection. The sharp decline in the C/C genotype from healthy to CHC subjects and the total absence of the C/C genotype in ESLD suggests a central role of this genotype against HCV disease progression.

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

BackgroundThis 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.MethodsPBMC from 26 patients with chronic HCV infection were cultured for 72 hours in presence and absence of 50 μg 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).ResultsQuantification 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.Conclusionthe 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.

Assay for hepatitis C virus in peripheral blood mononuclear cells enhances sensitivity of diagnosis and monitoring of HCV-associated hepatitis.

Hepatitis C virus (HCV) is a major etiological factor in chronic hepatitis affecting up to 24% of blood donors in Egypt. Since fluctuating levels of HCV RNA loads, including undetectable values, have been frequently observed in sera of chronic hepatitis patients, this study was designed to assess the sensitivity of PCR amplification for the plus- and minus-RNA strands in peripheral blood mononuclear cells (PBMC) compared to single serum PCR assay. Since the latter test detects viremia in only 79.5% of seropositive cases, the highest sensitivity for HCV diagnosis was achieved (93.20% when applying the combined triple test including PCR amplification of plus-strand in serum, together with plus-strand in PBMC and minus-strand in PBMC. The results of this study indicate that the triple test provides significant information on extrahepatic replication of HCV in a sizable sample of seropositive subjects (429 cases) and improves the assessment of HCV viremia. The cost/effectiveness and speed were upgraded by using capillary/air rapid thermal cycler. The use of the triple assay in HCV diagnosis and post-therapy monitoring is recommended.

Conserved peptides within the E2 region of Hepatitis C virus induce humoral and cellular responses in goats

The reason(s) why human antibodies raised against hepatitis C virus (HCV) E2 epitopes do not offer protection against multiple viral infections may be related to either genetic variations among viral strains particularly within the hypervariable region-1 (HVR-1), low titers of anti E2 antibodies or interference of non neutralizing antibodies with the function of neutralizing antibodies. This study was designed to assess the immunogenic properties of genetically conserved peptides derived from the C-terminal region of HVR-1 as potential therapeutic and/or prophylactic vaccines against HCV infection. Goats immunized with E2-conserved synthetic peptides termed p36 (a.a 430–446), p37(a.a 517–531) and p38 (a.a 412–419) generated high titers of anti-p36, anti-p37 and anti-P38 antibody responses of which only anti- p37 and anti- p38 were neutralizing to HCV particles in sera from patients infected predominantly with genotype 4a. On the other hand anti-p36 exhibited weak viral neutralization capacity on the same samples. Animals super-immunized with single epitopes generated 2 to 4.5 fold higher titers than similar antibodies produced in chronic HCV patients. Also the studied peptides elicited approximately 3 fold increase in cell proliferation of specific antibody-secreting peripheral blood mononuclear cells (PBMC) from immunized goats. These results indicate that, besides E1 derived peptide p35 (a.a 315–323) described previously by this laboratory, E2 conserved peptides p37 and p38 represent essential components of a candidate peptide vaccine against HCV infection.

Schistosoma hematobium soluble egg antigens induce proliferation of urothelial and endothelial cells

Abstract Bladder carcinoma accounts for 26% of reported human malignancies in Egypt, and has been strongly associated with urinary schistosomiasis. Nevertheless, the immediate role of schistosomal egg proteins in bladder carcinogenesis is unexplored. We investigated the effects of crude soluble egg antigens (SEA) of Schistosoma hematobium on urothelial cell proliferation. The proliferation of bovine endothelial Endo, human urothelial J82 and smooth muscle SMC cell lines was assessed by low-density growth assays. SEA induced proliferation of both J82 and Endo cells in a dose-dependent fashion, but not SMC. Preboiling or proteinase K treatment of SEA abolished its effect. In addition, SEA enhanced urothelial expression of B-cell translocation protein (BTG1) and human proliferating cell nuclear antigen (PCNA) mRNAs. Given the strong correlation between cell proliferation and carcinogenesis, the findings suggest that crude SEA may play some role in schistosomal bladder carcinogenesis.

Screening of dystrophin gene deletions in Egyptian patients with DMD/BMD muscular dystrophies

Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are allelic disorders caused by mutations within the dystrophin gene. Our study has identified 100 Egyptian families collected from the Human Genetics Clinic, National Research Center, Cairo. All cases were subjected to complete clinical evaluation pedigree analysis, electromyography studies, estimation of serum creatine phosphokinase enzyme (CPK) levels and DNA analysis. Multiplex PCR using 18 pairs of specific primers were used for screening of deletion mutations within the dystrophin gene. A frequency of 55% among the families. Sixty per cent of detected deletions involved multiple exons spanning the major or the minor hot spot of the dystrophin gene. The remainder 40% which mainly involved exon 45. Comparing these findings with frequencies of other countries it was found that our figures fall within the reported range of 40%– for deletions. The distribution of deletions in our study and other different studies was variable and specific ethnic differences do not apparently account for specific deletions. In addition this study concluded that employment of the 18 exon analysis is a cost effective and a highly accurate (97% to launch a nationwide program.

Status of diphtheria immunity in the Egyptian population.

Abstract In an attempt to evaluate the levels of immunity against diphtheria in the Egyptian population, 709 healthy subjects aged 2 months–105 years and from six regions of Egypt were investigated. An ELISA was used to determine the serum concentration of anti-diphtheria IgG in each subject. Following widely-used criteria for defining levels of protection, 34.1% of the subjects were categorized as susceptible to diphtheria (with < 0.01 IU antitoxin/ml serum), 43.7% were considered to have basic protection (0.01– < 0.1 IU/ml) and only the other 22.1% appeared fully protected (≥ 0.1 IU/ml). The results revealed that most of the subjects aged 2 months–50 years had a basic or fully protective level of IgG against diphtheria, although males were slightly more likely to be unprotected than females (36.2% v. 31.6%) and certain age-groups appeared to be much more likely to be susceptible than others. If outbreaks of diphtheria like those seen in recent years in the former Soviet Union are to be avoided in Egypt, the most susceptible groups of the population need to be given booster immunizations.

Prediction of relapse after interferon therapy in hepatitis C virus‐infected patients by the use of triple assay

Background and Methods: In recent years, several tests have been used as predictive factors for relapse after the end of interferon therapy of chronic hepatitis C carriers. Serum hepatitis C virus (HCV)‐RNA has proved insensitive for predicting relapse because more than 50% of patients with undetectable reverse transcription–polymerase chain reaction (RT‐PCR) levels of HCV at the end of therapy become positive for viral RNA after interferon withdrawal. The detection of a minus RNA strand of HCV in liver also failed to efficiently predict relapses in chronic hepatitis patients. Furthermore, the use of a whole blood assay of HCV‐RNA has been controversial. We used a triple assay comprised of RT‐PCR tests for the detection of HCV‐RNA plus strand in serum and peripheral blood mononuclear cells (PBMC), together with testing for the minus strand in PBMC for prediction of relapse after interferon + ribavirin combination therapy in 45 patients with chronic hepatitis C.

Establishment of Hybrid Cell Lines Producing Monoclonal Antibodies to a Synthetic Peptide from the E1 Region of the Hepatitis C Virus

Abstract We aimed at establishing hybridoma cells secreting monoclonal antibodies (mAbs) against E1 synthetic peptide of HCV. BALB/c mice were immunized with HCV E1‐synthetic peptide (GHRMAWDMM) and its spleenocytes were fused with the P3NS1 myeloma cell line. Two highly reactive and specific mAbs (10C7 IgG2b mAb, and 10B2 IgG1 mAb) were generated. The target HCV E1 antigen was identified at ∼38 kDa in serum of infected individuals. A newly developed ELISA detected the target antigen in 90% of sera from HCV RNA infected individuals with a specificity of 84%. So, the generated mAbs may provide promising probes for serodiagnosis of HCV infection.

Murine neutralizing antibody response and toxicity to synthetic peptides derived from E1 and E2 proteins of hepatitis C virus.

INTRODUCTION The highest estimated prevalence of HCV infection has been reported in Egypt, nearly 12% mostly type 4. Currently, a commercial vaccine to protect this high risk population as well as global HCV infected patients is not available. OBJECTIVES In the present study, we aim at: (1) examining the viral binding capacities of purified monospecific polyclonal murine antibodies raised against genetically conserved viral protein sequences, i.e. synthetic peptides derived from those sequences located within envelope proteins and (2) assessment of immunogenic properties and safety parameters of those peptides individually and in a vaccine format in mice. METHODS Purified IgG Abs from immunized mice were used in immunocapture RT-PCR experiments to test viral neutralization by Abs raised against each of 4 peptides termed p35 (E1), p36 (E2), p37 (E2) and p38 (E2). Swiss mice were immunized with each of the 3 peptides (p35, p37 and p38) which generated neutralizing antibodies in immunocapture experiments. Antibody responses to corresponding peptides were determined using different routes of administration, different adjuvants, different doses and at different time points post-injection. To explore the dose range for future pharmacological studies, three doses namely 50 ng, 10 μg and 50 μg/25 gm mouse body weight were tested for biochemical and histopathological changes in several organs. RESULTS Murine Abs against p35, p37 and p38 but not p36 showed HCV neutralization in immunocapture experiments. Subcutaneous injection of peptides elicited higher responses than i.m. and i.p. Immunization with Multiple Antigenic Peptide (MAP) form or coupled to Al PO4 elicited the highest Ab responses. Peptide doses of 50 ng/25 gm body weight or less were effective and safe, however dose assessment still requires further study. Histopathological changes were observed in animals that received doses ∼1000 times higher than the potential therapeutic dose. CONCLUSION Exploration of humoral immunogenicity, neutralization capacity and safety suggested that the peptides presented herein are candidate vaccine components for further preclinical assessment.