Identification, cloning and expression of core protein gene of hepatitis C genotype 4a.

Although Egypt has very high rates of HCV, not much is known about genotype 4a which is the most predominant genotype in Egypt. In the present study, core (C_ED43) gene of the Egyptian strain ED43 of HCV genotype 4a was first analyzed using PC/GENE program. Computer analysis of Core region of the isolate ED43 revealed that the Egyptian genotype 4a is different from those isolated from Europe and Central Africa and that it is closely related to genotype 1b. The DNA region coding for the Core was amplified from HCV_ED43/PUC19 plasmid. The PCR product was then cloned and expressed in E. coli M15 using pQE-30 vector. The expression and antigenicity of the core (Core_4a) protein in E. coli was confirmed by SDS-PAGE and western blotting, which will make it useful for developing assay systems for detecting anti-HCV antibodies and HCV antigen, respectively and which might help in the design of a vaccine against the Egyptian genotype 4a.

Characterization, cloning and expression of NS3 protein gene of hepatitis C genotype 4a.

Clone and express NS3 gene of the Egyptian strain ED43 of HCV genotype 4a in E. coli was studied. Gene and protein sequences of NS3 gene of the ED43 strain were first analyzed using PC/GENE program. DNA homology was 89% the homologies and that of the protein was 78.8% indicating that NS3 gene of the genotype 4a is different from those isolated from other strains. DNA of NS3 region of genotype 4a was amplified from HCV_ED43/PUC19 plasmid. The PCR product was cloned and expressed in E. coli M15 using pQE-30 vector. Fusion protein containing the peptides coded by HCV NS3 (NS3_4a) was expressed by Escherichia coli. The specific HCV antigenicity of the NS3_4a fusion protein was identified by western blotting.

Cytokine secretion profile associated with periportal fibrosis in S. mansoni-infected Egyptian patients.

Periportal fibrosis (PPF) is a major pathological consequence of S. mansoni infection. Ultrasonography is a well-established tool for diagnosis and grading of schistosomiasis-related pathology. This work is performed to study the effect of schistosomiasis mansoni infection on the cytokine secretion profile in S. mansoni-infected patients at various grades of fibrosis, as determined by ultrasonography using Cairo Working Group classification. The levels of IL-2, IL-4, IL5, IL-10, IFN-gamma, and TNF-alpha were measured in the absence of in vitro antigen stimulation and after stimulation with worm and egg antigens. Simple intestinal (INT) patients are characterized by strong proliferation to worm antigen and high levels of IL-10 and TNF-alpha compared to patients at various grades of infection. GradeII (GdII)-infected patients are characterized by higher IL-5 production than are patients with other clinical forms of the disease. Sharp reduction of almost all cytokines in response to both worm and egg antigens was detected in GdIII-infected patients. These results stressed the role of both IL-10 and TNF-alpha in the early stages of hepatic fibrosis, while IL-5 could be employed as a potential predictive marker for advanced stages. In conclusion, PPF is associated with cytokine production profiles that vary with the magnitude of the fibrosis.

T cells are depleted in HCV-Induced hepatocellular carcinoma patients: possible role of apoptosis and p53.

Egypt has possibly the highest Hepatitis C Virus (HCV) prevalence worldwide. A high proportion of HCV infections become chronic and lead to liver cirrhosis and hepatocellular carcinoma (HCC). The cellular and molecular mechanisms behind HCV infection complication are not completely understood although apoptosis has been implicated in this process. Using flowcytometry, we examined whether T lymphocyte; isolated from patients with HCV and HCV-associated HCC (HCV-HCC); are predestined in vivo to undergo spontaneous apoptosis. Also, the role of p53; a key protein in apoptotic process; in the development of HCC was examined. Our data showed that T cells were severely depleted in HCV-HCC patients and its spontaneous apoptosis was higher in patient groups as compared to normal controls. In addition, p53 expression in liver tissue (determined by ELISA) was higher in the HCC patient groups as compared to normal controls and correlated well with the HCC grade. In conclusion, HCV infection induces peripheral T cell apoptosis, depletion and subsequently immune-suppression and this may lead to persistence of infection. Also, p53 is implicated in the poor prognosis of HCV-HCC and could be used as a predictive marker to assess the prognosis of HCC patients.

Immunological identification of Fasciola hepatica antigens containing major human T-cell and B-cell epitopes.

Fasciola hepatica whole worm homogenate (Fhwwh) separated fractions were used in enzyme linked immunoelectrotransfer blot (EITB) to identify the antigen(s) which induces antibody formation in human fascioliasis. The immuno-reactive antigens recognized by the infected patients were 25-29 kDa and 12 kDa. Antigens were biochemically purified by model 491-prep cell fraction (BIO-RAD). The capability of the purified target antigens to induce humoral and cellular responses with cells and sera of infected patients was investigated using enzyme linked immunosorbent assay (ELISA) and lymphoproliferative responses techniques. The 25-29 kDa cluster of antigen(s) were found to be more efficient in inducing lymphoproliferative response than 12 KDa, thus, it was considered as the target antigens used in the generation of human monospecific polyclonal immunopurified antibody probes (IPAb). The specificity and immunoreactivity of the IPAb with Fhwwh, F. hepatica excretory secretory products (FhESP) and S. mansoni adult worm antigen (SAWA) were evaluated by using EITB. Results showed that IPAb was immunoreactive with 25-29 and 12 kDa antigens of both Fhwwh and FhESP. It was concluded that the most immunogenic F. hepatica target antigen(s) were 25-29 and12 KDa antigens and there is a cross-reactivity between 25-29 and 12 KDa antigens in both Fhwwh and FhESP. The involvement of IPAb in antibody dependent cell mediated cytotoxicity in-vitro technique was studied. Results indicated that human neturophils were more effective in adhering to the IPAb-coated flukes at early stages of development.

Cloning and sequence analysis of genes encoding Fasciola hepatica immunodominant antigens.

Fasciola hepatica lambdagt11 cDNA expression library was immuno-screened with IPAb, two clones were isolated and identified as Fhlambda400 Fhlambda800. Both clones were sequenced, FhA400 contained 305 translated bases encoding 11.509 kDa and designated as SFh12, while Fhlambda800 contained 311 translated bases encoding protein of 11.058 kDa designated as SFh11. The DNA sequence homology search of Fhlambda400 revealed a relatively high degree of identity with F. hepatica amoebapore-like protein mRNA (GenBank accession No. AF286903). However, Fhlambda800 revealed the highest similarity with F. hepatica tegumental antigen (T1) mRNA (GenBank accession No. AF153056). The protein homology search of SFh12 gave 100% identity with amoebapore-like protein (APLP), while SFh-\11 showed 75% identity with F. hepatica tegumenttal antigen (TA). The biochemical analysis of the deduced proteins was identified; in addition the predicted T- & B-cell epitopes have been also evaluated. However, histological localization of identified antigens was achieved using the IPAb in an indirect immunoflorescent antibody assay (IFA). Results revealed that the IPAb labeled the outer glycocalyx in a characteristic, pattern, which proved that the identified antigens were tegumental in origin and that infected Fasciola subjects induced antibodies directed mainly against tegumental components.

Application and assessment of a dipstick assay in the diagnosis of hydatidosis and trichinosis.

The aim of the present work was to apply and evaluate a dipstick assay for the serodiagnosis of human hydatidosis as well as human and experimental trichinosis using camel hydatid cyst fluid (HCF) and Trichinella spiralis muscle larval (TSML) antigens, respectively, and compare this to enzyme-linked immunoelectrotransfer blot (EITB) and Falcon assay screening test-enzyme-linked immunosorbent assay (FAST-ELISA). Sera samples were collected from patients with confirmed hydatidosis and trichinosis and with other parasitic diseases as well as from normal healthy individuals. Also, sera samples were collected from mice experimentally infected with T. spiralis which were sacrificed at different time points post-infection (PI). HCF and TSML antigens were used in EITB after separation and characterization of their antigenic components using 5-22.5% sodium dodecyl sulphate-polyacrylamide gel electrophoresis under non-reducing condition. For the diagnosis of hydatidosis, the sensitivity, specificity and diagnostic accuracy of the dipstick assay and EITB were 100, 91.4 and 95.1% while those of FAST-ELISA were 96.2, 100 and 98.4%, respectively. For the diagnosis of human trichinosis, the sensitivity, specificity and diagnostic accuracy of the dipstick assay and EITB were 100% while those of FAST-ELISA were 85.7%. FAST-ELISA proved to be more sensitive in the early diagnosis of experimental T. spiralis infection (100% sensitivity from the second week PI) than the dipstick and EITB (100% sensitivity from the third week PI). All tests retained their sensitivity till the 12th week PI. Since the dipstick assay is extremely easy to perform with a visually interpreted result within 15 min, in addition to being both sensitive and specific, the test could be an acceptable alternative for use in clinical laboratories lacking specialized equipment and the technological expertise needed for EITB and FAST-ELISA.

Cellular and humoral immune responses to recombinant Smp17.7 Schistosoma mansoni antigen.

To determine the immunological responses to S. mansoni antigen rSmp17.7, a total of 184 subjects, 174 patients from a schistosomiasis endemic area, and 10 controls were used. Proliferation, cytokine profile in culture supernatants from antigen-stimulated peripheral blood mononuclear cells and specific IgG1, IgG3, IgG4, IgA, IgM & IgE levels were assessed. The highest stimulation index to rSmp17.7 was detected in S. mansoni patients. The evaluation of the cytokine profile [IL-2, IL-4 & IFN-gamma] in response to this antigen showed a significant increase as demonstrated by ELISA. Specifically, IFN-gamma and IL-2 were significantly detected by flow cytometry. IgG1 and IgM were the only Igs which showed a significant increase. These results highlight the importance of rSmp17.7 as a candidate vaccine for schistosomiasis. The results pave the way to understand the mechanism of schistosome-vaccine efficacy.