Establishment of a platform for molecular and immunological characterization of the RNA-dependent-RNA-polymerase NS5B of an Egyptian HCV isolate.

The present work aimed at establishing a platform to enable frequent characterization of the HCV RNA-dependent-RNA-polymerase from Egyptian clinical isolates. Subjecting amplified HCV-NS5B coding gene from Egyptian patient's serum to sequencing, multiple alignment, and phylogenetic analysis confirmed its subtype 4a origin. Nucleotide sequence analysis revealed presence of an additional start codon at the beginning of the NS5B gene. Peptide sequence alignment demonstrated presence of unique amino acid residues in our 4a-NS5B sequence distinct from the JFH-1-NS5B sequence as well as unique amino acids compared to other genotypes. The distinct molecular structure of the herein characterized 4a-NS5B from the 2a-JFH-1-NS5B was further demonstrated both in the built 3D models and the Ramachandran plots corresponding to each structure. Both the unique amino acid residues and 3D structure of the 4a-NS5B may influence both genotype 4a replication rate and response to therapy in comparison to other genotypes. Many resistance mutations to polymerase inhibitors were found both in ours and other genotypes' sequences. The presence of the required amino acid motifs for the RNA dependent RNA polymerase activity encouraged to clone the NS5B570-encoding sequence downstream CMV promotor in a mammalian expression vector. Such construct was used for both prokaryotic expression in bacteria and for DNA immunization. Successful mammalian expression and induction of specific immune response were demonstrated by ELISA and Western blotting. The potential of both the raised antibodies and the expressed NS5B to differentiate between HCV-infected and control human sera were demonstrated which reflect their diagnostic value.

Anti-cyclic citrullinated peptide antibodies as a discriminating marker between rheumatoid arthritis and chronic hepatitis C-related polyarthropathy.

Articular involvement is a frequent extrahepatic manifestation of hepatitis C virus (HCV) infection. The distinction between HCV-related polyarthropathy and true RA may be very difficult, especially with recent onset RA before articular damage and erosions develop. The objective of the study is to assess the diagnostic utility of anti-CCP antibodies and compare it with that of rheumatoid factor (RF) in distinguishing between rheumatoid arthritis (RA) and HCV-related polyarthropathy. Anti-cyclic citrullinated peptide (CCP) antibodies and RF were determined in the sera of 30 patients with RA and 22 patients with HCV-related polyarthropathy. Anti-CCP antibodies were positive in 83.3% of patients with RA and in 4.5% in patients with HCV and polyarthropathy. RF was positive in 90% of RA patients and in 81.1% of HCV patients with polyarthropathy. The anti-CCP antibodies showed higher specificity for RA compared with RF (95.4 vs. 18.2%). However, the sensitivity of anti-CCP was comparable to that of RF (83.3 vs. 90%). In conclusions, anti-CCP antibodies are reliable laboratory markers to differentiate between RA and HCV-related polyarthropathy.

Adiponectin: an adipocyte-derived hormone, and its gene encoding in children with chronic kidney disease.

BACKGROUND: The prevalence of cardiovascular disease (CVD) and inflammation is high in patients with chronic kidney disease (CKD). Adiponectin (ADPN) is an adipocytokine that may have significant anti-inflammatory and anti-atherosclerotic effects. Low adiponectin levels have previously been found in patients with high risk for CVD. METHODS: On seventy eight advanced CKD (stages 4 and 5) pediatric patients undergoing maintenance hemodialysis( MHD) or conservative treatment (CT) the following parameters were studied: body mass index, left ventricular mass index(LVMI), serum adiponectin , cholesterol, HDL-cholesterol, high sensitivity C-reactive protein (hs CRP),interleukin 6(IL6) and single-nucleotide polymorphisms (SNPs) in the ADIPOQ gene at positions 45, and 276. Seventy age-and gender-matched healthy subjects served as control subjects. RESULTS: Markedly (P = 0.01) elevated plasma adiponectin levels were observed in CKD patients, especially CT patients, compared to control subjects. The wild type of ADIPOQ 45T > G (T) allele is the main gene for patients and controls. MHD and CT patients had significantly higher frequency of the TT genotypes of +276G > T gene (P = 0.04) compared with control subjects. A significant positive correlation was observed between plasma adiponectin and IL6 level, whereas negative correlations were found between adiponectin level, cholesterol, HDL cholesterol and hs CRP. In a stepwise backward multiple regression model only IL6 (P = 0.001) was independently associated with plasma adiponectin levels. The adiponectin gene the 276 GT+TT genotypes were associated with a higher level of adiponectin . CONCLUSIONS: The present study demonstrated that ADPN is related to several metabolic and inflammatory CV risk factors in a manner consistent with the hypothesis that this protein might have a protective role against these factors. We observed an association between the +276G>T SNP in the adiponectin gene and CKD in children. Genetic variation of +276 gene seemed to have a positive impact on circulating adiponectin levels in CKD patients.

Bone mineral density, bone turnover markers, lean mass, and fat mass in Egyptian children with congenital adrenal hyperplasia.

INTRODUCTION: Aim of this paper is to assess bone mineral density (BMD) and body composition, by dual energy X-ray absorptiometry (DXA), and various markers of bone growth, in a group of children with congenital adrenal hyperplasia (CAH) on long-term glucocorticoid therapy. MATERIAL AND METHODS: A case-control study included thirty patients with CAH with different states of metabolic control. Their mean age was 7.5 ±4.2 years. All patients are subjected to BMD using DXA at the neck of the femur and lumbar spine. A blood sample was taken for assessment of osteocalcin, osteoprotegerin, and procollagen type 1, as markers of bone formation, as well as RANKL and urinary deoxypyridinoline (DPD), as markers of bone resorption. RESULTS: We found no difference in BMD in patients and control subjects; however, patients showed significantly lower serum osteocalcin (p = 0.008) and osteoprotegerin (p = 0.0001) and significantly higher serum RANKL levels (p = 0.0001). Our results show that patients had significantly lower lean body mass (p = 0.005) and fat/lean ratio (p = 0.008) compared to matched controls. The duration of treatment showed a significant negative correlation with procollagen type 1 (r = -0.49, p = 0.02) and lean mass % (r = -0.43, p = 0.04); however, it showed a significant positive correlation with total fat mass % (r = 0.6, p = 0.0006), and fat/lean ratio (r = 0.43, p = 0.04). Dose of steroid had a significant positive correlation with BMI SDS (r = 0.4, p = 0.02). CONCLUSIONS: Bone mineral density is normal but bone turnover is low in patients with CAH. There is an increase in fat/lean mass in patients with CAH.

Red cell alloimmunization and autoantibodies in Egyptian transfusion-dependent thalassaemia patients.

INTRODUCTION: The objective of this study was to explore the frequency of red cell alloantibodies and autoantibodies among ß-thalassaemia patients who received regular transfusions. MATERIAL AND METHODS: This study included 501 patients with ß-thalassaemia. This work planned to study the presence of alloantibodies and autoantibodies to different red cell antigens in multitransfused thalassaemia patients using the ID. Card micro typing system. RESULTS: Of a total of 501 ß-thalassaemia patients included in the study, 11.3% of patients developed alloantibodies; 9.7% of these alloantibodies were clinically significant. The most common alloantibodies were anti-K, anti-E and anti-C. The rate of incidence of these alloantibodies was 3.9%, 3.3% and 1.7% respectively. Autoantibodies occurred in 28.8% of the patients and 22.1% of these antibodies were typed IgG. There was a significant association between splenectomy with alloimmunization and autoantibody formation (p = 0.03, p = 0.001 respectively). There was no significant association between alloantibody, autoantibody formation and number of transfused packed red cells. CONCLUSIONS: Alloimmunization to minor erythrocyte antigens and erythrocyte autoantibodies of variable clinical significance are frequent findings in transfused ß-thalassaemia patients. There is an association between absence of the spleen and the presence of alloimmunization and autoantibody formation.

Prevalence of celiac disease, Helicobacter pylori and gastroesophageal reflux in patients with refractory iron deficiency anemia.

OBJECTIVE: The aim of this article is to determine the prevalence of celiac disease (CD), Helicobacter pylori (H. pylori) and gastroesophageal reflux (GER) in patients with resistant iron deficiency anemia (IDA). PATIENTS: The study included 25 patients <18 years of age with refractory IDA (not responding to iron therapy for 3 months in a dose of 6 mg elemental iron/kg/day). METHODS: All patients included in the study were subjected to careful history taking and thorough clinical examination. Blood sample was taken for analysis of antibodies for CD including: antigliadin antibody (AGA), antiendomysial antibody (EMA), antireticulin antibody (ARA) and antitissue Transglutaminase (tTg) IgG antibody. Anti-H. pylori IgG antibodies and a (13)C-urea breath test (UBT) was done to all patients to diagnose H. pylori. Upper gastrointestinal tract endoscopy was done for all patients to evaluate for the presence of some etiologies of intractable anemia as chronic blood loss. These included: CD, H. pylori infection and GER. The upper gastrointestinal tract endoscopy was also done to evaluate the presence of bleeding spots, ulcers or angiomatous malformations. In addition, gastric antral biopsies were taken for diagnosis of H. pylori infection by the following tests: rapid urease test, histopathological examination and culture. RESULTS: CD was positive in 11 out of 25 patients (44%), H. pylori infection in 12 out of 25 patients (48%), while GER was diagnosed in 11 out of 25 patients (44%). Patients with CD had age of presentation < or =2 years in two patients (18.2%) while the remaining nine patients (81.8%) had age of presentation >2 years and it was statistically significant (p = 0.05*). Also patients with H. pylori had age of presentation < or =4 years in five patients (41.7%) and the remaining seven patients (81.8%) had age of presentation >4 years and it was statistically significant (p = 0.03*). Logistic regression analysis demonstrated that the risk factors for severity of anemia were age of patients and duration of anemia. On the other hand, other parameters have no significant influence on the severity of anemia. Also risk factors of short stature were age of presentation of anemia, degree of anemia and H. pylori infection. AGA had the highest sensitivity (100%) followed by antiendomysium antibody (81.8%) while the tTG antibody had the highest specificity (85.7%) for diagnosis of CD. UBT and histopathology had the highest sensitivity (100%) for diagnosis of H. pylori while rapid urease test, culture, H. pylori stool antigen and anti-H. pylori IgG antibody had the highest specificity (100%). In conclusion, refractory IDA may be due to clinically unapparent H. pylori gastritis and CD. CD is one of the most common causes of intestinal malabsorption during childhood which leads to impairment of iron absorption. Apart from offering them gluten-free diet rich in iron, early detection and treatment of IDA and prophylactic iron and folic acid supplementation will go a long way to optimize their mental and psychological functions. Eradication of H. pylori infection with concomitant iron therapy should correct the anemia.