Characterization of NS3 protease from an Egyptian HCV genotype 4a isolate.

The role of the NS3 protease in HCV replication was demonstrated by the ability of a protease inhibitor cocktail (10 microg/ml) to abolish the induced cytopathic effect in RAW macrophages upon infection with Egyptian sera. The HCV protease gene was amplified from Egyptian sera by nested PCR and cloned downstream of the CMV promotor in a mammalian expression plasmid, which was then used to transform bacteria. Colonies carrying the gene in the correct orientation were subjected to large-scale plasmid purification followed by sequencing. Phylogenetic comparison of the sequence obtained with published sequences from different genotypes confirmed that our sequence belongs to genotype 4a. Of the other genotypes, the most closely related ones were from genotype 1. Multiple alignments of protease peptides showed that the catalytic triads and binding residues for substrate, Zn2+ and the NS4 cofactor are conserved among different isolates, including ours, and confirmed the closer homology between NS3 of genotypes 4 and 1. The HCV-protease-encoding construct was successfully transcribed in both mammalian cells and mice. Mouse antibodies produced against the protease-encoding-construct detected the 18-kDa enzyme in lysates of cells transfected with the construct by Western blotting, and in the media of infected cells by ELISA.

Prevalence of Xmnl Gγ polymorphism in Egyptian patients with ß-thalassemia major.

BACKGROUND AND OBJECTIVES: ß-thalassemia results from a deficiency of ß-globin chains leading to an excess in a globin chains resulting in hypochromic microcytic red cells, ineffective erythropoiesis and hemolytic anemia. It is a result of a decline of HbF synthesis during the first year of life. F-cell levels are influenced by a sequence variant (C->T) at position -158 upstream of the -globin gene, so the frequency of the Xmnl Gγ polymorphism in Egyptian patients with b-thalassemia major needed evaluation to decide on the value of HbF augmentation drugs in treating Egyptian b-thalessemia. DESIGN AND SETTING: A cross-sectional study including 30 ß-thalassemia major patients diagnosed and attending the Pediatric Hematology Unit, Children's University Hospital, Ain Shams University, Cairo, Egypt, in the period from October 2008 to October 2009. PATIENTS AND METHODS: The 17 males and 13 females underwent a medical history and physical examination. Tests included a complete blood count, hemoglobin electrophoresis, serum ferritin, and detection of Xmnl Gγ polymorphism by PCR. RESULTS: The mean (SD) age was [2]10.2 (6.9) years. The most frequent genotype observed was homozygosity for the absence of the site Xmnl (-/-) in 96% of cases. Heterozygosity (+/-) genotype was detected in 4% of cases, while homozygosity for the site XmnI (+/+) genotype was absent. Genotype was not related to age at first transfusion, fetal hemoglobin level or transfusion frequency. CONCLUSION: Despite the small sample size, the study demonstrated that Egyptian ß-thalessemia patients have low frequency of positivity for the Xmnl polymorphism whether in heterozygous (+/-) or homozygous (+/+) state.

Effect of different monotherapies on serum nitric oxide and pulmonary functions in children with mild persistent asthma.

INTRODUCTION: Common medications used to treat mild persistent asthma are glucocorticoids, leukotriene receptor antagonists and theophylline. The aim of the study was to evaluate monotherapy with either inhaled steroids, oral leukotriene receptor antagonist or theophylline in Egyptian children with mild persistent asthma by determining their clinical, laboratory and spirometric responses to treatment. MATERIAL AND METHODS: Thirty-nine mild asthmatic children between 8 and 13 years of age were included in the study. Patients were classified according to therapy received into four groups: oral leukotriene receptor antagonist (montelukast), inhaled corticosteroid (fluticasone propionate), sustained-release (SR) theophylline, and no treatment. Pulmonary function testing was performed at the start of therapy and 8 weeks later using spirometry. Eosinophil count and serum nitric oxide were estimated in the blood. Minitab statistical package was used for analysis of data. RESULTS: Follow-up after 8 weeks revealed significant improvement in FEV1% in groups 1 (p < 0.01) and 3 (p < 0.05), significant improvement in PEFR in groups 1 (p < 0.05) and 2 (p < 0.01), significant decline in serum NO levels in groups 1 (p < 0.05) and 2 (p < 0.05), as well as significant improvement in eosinophil count in groups 1, 2 and 3 (p < 0.01, < 0.001, < 0.01 respectively). There was a statistically significant positive correlation between the decline in serum NO and the decline in blood eosinophil % in group 2 (p < 0.05). CONCLUSIONS: Inhaled corticosteroids and montelukast have a significant role in controlling the pulmonary functions and the inflammatory process in children with mild persistent asthma, although inhaled corticosteroids seem to yield a better response. Children with mild persistent asthma should receive a controller medication, and SR theophylline may be a good cost-benefit alternative for low socio-economic groups of patients.

Schistosoma mansoni soluble egg antigens enhance HCV replication in mammalian cells.

BACKGROUND: This work demonstrates successful propagation of HCV in HepG2 and human blood cells as well as viral shedding into their culture media. The influence of Schistosoma mansoni crude soluble egg antigens (SEA) on the rate of viral propagation in both mammalian cells was also monitored. METHODOLOGY: HepG2 cells were inoculated with HCV viremic human sera and some wells were exposed to HCV infection in presence of SEA. Cells were harvested for RT-PCR and Western blotting analysis. HepG2 media was collected for HCV ELISA. Blood samples from HCV-infected humans were cultured in the presence and absence of SEA. Media were collected at different time points post culturing and subjected to HCV ELISA. RESULTS: The ELISA concentration of HCV antigens were generally higher in media of infected HepG2 cells compared to media of control cells at all time intervals post infection. Western blots showed reactivity to immunogenic peptides of different molecular weights in lysate of infected HepG2 cells that were not evidenced in uninfected cells. In presence of SEA, RT-PCR results revealed earlier detection of viral RNA in infected HepG2 cells compared to in absence of such bilharzial antigen. Also, ELISA results revealed higher levels of detected HCV antigens in media of both infected HepG2 and blood cells cocultured with S. mansoni SEA compared to that of cultured infected cells in absence of the parasite antigens. CONCLUSION: HepG2 cells as well as whole blood cultures maintain HCV replication. Furthermore, SEA has the potential to enhance HCV propagation.