Association of Cholesterol Ester Transfer Protein Taq IB Polymorphism With Acute Coronary Syndrome in Egyptian National Patients.

BACKGROUND: The association between cholesterol ester transfer protein (CETP) Taq IB polymorphism and coronary artery disease (CAD) has been studied in different populations. Acute coronary syndrome (ACS) is a group of clinical symptoms within acute myocardial ischemia, including unstable angina (UA) and myocardial infarction (MI). Because there are no data reported in the literature concerning the cholesteryl ester transfer protein (CETP) Taq IB polymorphism in Egyptians, our study aimed to investigate the frequency of different CETP Taq IB genotypes in Egyptian patients with ACS and in healthy control individuals. METHODS: The current study was conducted with 70 hospitalized patients who had been diagnosed with ACS and 30 controls. We used real-time polymerase chain reaction (RT-PCR) to determine CETP Taq IB in individuals with different genotypes. RESULTS: The frequency of the GA genotype was significantly lower in UA patients, compared with the control group ( P <.05). CONCLUSIONS: The frequency of the CETP Taq IB genotypes and alleles in all groups was similar to that in other ethnic groups. Individuals with the Taq IB GA genotype may have a lower risk of UA.

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.

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.

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.

Antibody to E1 peptide of hepatitis C virus genotype 4 inhibits virus binding and entry to HepG2 cells in vitro.

AIM: To analyze the neutralizing activity of antibodies against E1 region of hepatitis C virus (HCV). Specific polyclonal antibody was raised via immunization of New Zealand rabbits with a synthetic peptide that had been derived from the E1 region of HCV and was shown to be highly conserved among HCV published genotypes. METHODS: Hyper-immune HCV E1 antibodies were incubated over night at 4 degree Celsius with serum samples positive for HCV RNA, with viral loads ranging from 615 to 3.2 million IU/ mL. Treated sera were incubated with HepG2 cells for 90 min. Blocking of viral binding and entry into cells by anti E1 antibody were tested by means of RT-PCR and flow cytometry. RESULTS: Direct immunostaining using FITC conjugated E1 antibody followed by Flow cytometric analysis showed reduced mean fluorescence intensity in samples pre-incubated with E1 antibody compared with untreated samples. Furthermore, 13 out of 18 positive sera (72%) showed complete inhibition of infectivity as detected by RT-PCR. CONCLUSION: In house produced E1 antibody, blocks binding and entry of HCV virion infection to target cells suggesting the involvement of this epitope in virus binding and entry. Isolation of these antibodies that block virus attachment to human cells are useful as therapeutic reagents.