Impaired Akt phosphorylation in B-cells of patients with common variable immunodeficiency.

Common variable immunodeficiency (CVID) is a heterogeneous group of primary immunodeficiency characterized by recurrent infections. We evaluated whether defective PI3K/Akt/FoxO pathway could influence B-cell fate. Determination of B-cell subsets in CVD patients and healthy donors (HDs) were performed using flow cytometry. We evaluated mRNA and protein expression of PI3K, Akt and FoxO using real-time PCR and flow cytometry, respectively. Moreover, phosphorylated Akt (pAkt) expression in B-cells has been measured by flowcytometry. We identified a significant reduction in the percentage of marginal zone like B-cells, memory B-cells (total, switched and unswitched) and plasmablasts in patients, as these decreased B-cell subsets had a significant negative correlation with increased apoptosis in patients. Surprisingly, we identified decreased pAkt expression in B-cells of patients than HDs. We described for the first time impaired pAkt expression in B-cells of CVID patients that had a significant correlation with antibody response to the vaccine and worse clinical complications.

Molecular characterization of hepatitis B virus (HBV) strains circulating in the northern coast of the Persian Gulf and its comparison with worldwide distribution of HBV subgenotype D1.

Iran is a large country that covers the northern coast of the Persian Gulf. Iranian residents of this coastal region interact closely with people from neighboring countries because of historical and cultural relationships, as well as economic activities. In addition, the inhabitants of this border region have experienced several wars, which have affected public health infrastructures. This study characterized for the first time, the evolution of the full-length genome of HBV strains in asymptomatic carrier patients living in this particular region. In addition, this study was compared and complemented by a comprehensive evolutionary analysis of the worldwide geographical distribution of HBV subgenotype D1. Evolutionary analysis demonstrates that patients living in the northern coast of the Persian Gulf are mainly infected with HBV subgenotype D1, subtype ayw2. Specific mutations related to advanced liver disease were found more frequently in these strains compared to other strains isolated from asymptomatic carriers from other regions of Iran. This global comprehensive analysis showed that HBV subgenotype D1 strains have a worldwide distribution and that human mobility and immigration had a large impact on dispersal of HBV subgenotype D1, subtype ayw2 in Middle Eastern countries such as Iran, Syria, and Turkey. In addition to association of subtype ayw2 with subgenotype D1, it was demonstrated that other HBV subtypes like adw2, ayw1, and ayw3 are associated with HBV subgenotype D1 in different regions of the world. This study also revealed a remarkable distribution of subgenotype D1, subtype ayw4 although this particular subtype is associated with subgenotype D4 of HBV in European countries.

Evolutionary analysis of HBV "S" antigen genetic diversity in Iranian blood donors: a nationwide study.

The genetic diversity of the HBV S gene has a significant impact on the prophylaxis and treatment of hepatitis B infection. The effect of selective pressure on this genetic alteration has not yet been studied in Iranian blood donors. To explore HBV evolution and to analyze the effects and patterns of hepatitis B surface antigen (HBsAg) mutations on blood screening assays, 358 Iranian blood donors diagnosed as asymptomatic HBV carriers were enrolled in this nationwide study. Large S and partial S genes were amplified and sequenced. HBV (sub) genotypes and synonymous and nonsynonymous mutations were investigated. The impact of naturally occurring mutations on HBsAg ELISA results was explored. Phylogenetic analyses revealed that isolated strains were of genotype D. The dominant subgenotype/subtype was D1/ayw2. Deletions and naturally occurring stop codons in the pre-S1 and major hydrophilic region (MHR) were identified. In total, 32.8% of the studied strains harbored 195 single or multiple mutations in the MHR, the majority of which were located at the first loop of the "a determinant" domain. The ayw2 subtype showed a significant effect on the ELISA signal/cut-off value and carried fewer mutations in the MHR. Nonsynonymous/synonymous substitution value indicated that negative selection was the dominant evolutionary force in the HBV S gene. This nationwide study revealed that mutation frequency of HBsAg among Iranian blood donors was much higher than previous reports from the different local regions. These findings regarding the significant differences in reactivity of ELISA among different subtypes of HBV and its correlation with the number of mutations at the MHR will be valuable to public health authorities.

Study of hepatitis B prevalence in parallel with the most frequent HBV genotype in South Iranian blood donors.

BACKGROUND: Hepatitis B virus (HBV) is one of the leading causes of acute and chronic liver diseases and in turn responsible for a million of worldwide annual deaths. HBV has been classified into eight main groups, designated as A-H with different geographical distribution. Some genotypes are associated with different clinical outcomes and particular viral mutations. Current study was aimed to investigate the genotype and prevalence of HBV in blood donors of south of Iran. METHODS: This experimental study investigated the prevalence of HBV positivity in 198,289 Iranian blood donors from south of Iran by both ELISA- and PCR-based methods. Within 198,289 donors, 120 HBsAg(+) cases were selected, HBV-DNA was extracted, and the p gene sequences were amplified by nested-PCR. The HBV genotypes were determined by direct sequencing of the polymerase gene of HBV. Phylogenetic trees also were constructed by the neighbor-joining (NJ) method. RESULTS: Findings of this study indicated that 0.184%, 0.329%, and 0.215% of blood donors were HBsAg(+) among Isfahan, Kerman, and Yazd provinces, respectively. Only 69 (57.5%) cases of 120 HBsAg(+) donors were HBV-DNA(+) . Sequencing analysis revealed that all of HBV-infected donors had the D genotype of HBV. CONCLUSIONS: In conclusion, the prevalence of genotype D was 100% in Iranian HBV blood donors. These findings may have an impact on the immunological and genetic diagnosis of HBV, selection of diagnostic kits, and viral quality control panels to evaluate diagnostic methods.

Comparing the effect of IL-12 genetic adjuvant and alum non-genetic adjuvant on the efficiency of the cocktail DNA vaccine containing plasmids encoding SAG-1 and ROP-2 of Toxoplasma gondii.

Various methods are available for enhancing the potency of DNA vaccines, including employment of different forms of adjuvant. The current study was carried out to evaluate and compare the effects of genetic and non-genetic adjuvants on the immune response stimulated by DNA vaccine. Thus, two adjuvants, IL-12 (genetic adjuvant) and aluminum hydroxide (alum, non-genetic adjuvant), were used with cocktail DNA vaccine containing plasmids encoding complete rhoptry antigen 2 (ROP-2) and surface major antigen 1 (SAG-1) of Toxoplasma gondii. The efficacy of pcROP2+pcSAG1 in stimulation of the immune response against toxoplasmosis with and without adjuvant was evaluated in female BALB/c mice by measuring the level of total IgG antibody and cytokines. The results obtained indicated that after challenging the mice with the fatal RH strain of T. gondii, the survival rates of mice immunized with pcROP2+pcSAG1 (DNA cocktail), pcSAG1+pcROP2+alum, and pcSAG1+pcROP2+IL-12 were significantly greater than that of the control groups (p<0.05). Moreover, measurement of total IgG antibody indicated the significant difference between the control and experimental groups (p<0.05). Finally, the results obtained by measurement of cytokines (IFN-γ and IL-4) showed high levels of IFN-γ and low levels of IL-4 in groups vaccinated with pcROP2+pcSAG1 (DNA cocktail), pcSAG1+pcROP2+alum, and pcSAG1+pcROP2+IL-12 as the experiment groups, in comparison with the controls groups (PBS, pc-DNA3, alum+PBS, and pCAGGS-IL-12+pcDNA3). The results of the study showed that use of adjuvants (IL-12 and alum) coincident with DNA cocktail leads to significant change in the survival rates of the experiment groups in comparison with control groups. Also, there is no significant difference between adjuvants to induce immune responses.

A Study on the Association between CCRΔ32 Mutation and HCV Infection in Iranian Patients.

BACKGROUND: Mutations in the coding region of the Chemokine Receptor 5 (CCR5) genes reduce or eliminate CCR5 expression in immune cells and progression of HCV infection. This study aimed to investigate the role of this mutation in HCV infection in Iranian patients in comparison with healthy individuals. METHODS: 100 HCV infected patients and 100 healthy individuals were randomly selected. The CCR5Δ32 genotypes were determined using specific primers and PCR method. RESULTS: The agarose gel electrophoresis showed a189-bp fragment from wild type for both alleles of CCR5 gene. The CCR5-Δ32 allele was not found in any HCV infected and healthy subjects. CONCLUSION: The mutation in CCR5 gene was not detected in any of the two groups; therefore, the role of CCR5 gene expression in immune cells and progression of HCV infection needs to be studied in larger samples in our country.

Expression of Plasmid Encoded GRA4 Gene of Toxoplasma gondii RH Strain in CHO Eukaryotic Cells.

BACKGROUND: Toxoplasmosis is a common infection all around the world. During pregnancy; it may lead to congenital disorders or abortion in human and animals. Severe damage of toxoplasmosis indicates to require effective vaccine. One of dense granules antigen is GRA4 that secrete from tachyzoite and bradyzoite. GRA4 genome is unique without intron and is one of the major immunogenic proteins from Toxoplasma gondii. METHODS: We confirmed the cloning of GRA4 gene into pcDNA3 by restriction enzyme and PCR of GRA4 gene with pcGRA4 plasmids as template. Then with using calcium-phosphate method we transfected the pcGRA4 into CHO (Chinesehamster ovary) cells. The yielded protein was separated by SDS-PAGE and moved by electroblotting to nitrocellulose paper. RESULTS: Result of SDS-PAGE analysis showed the appearance of band approximately 42 kDa which was absent in the negative control, that was able to identify toxoplasmosis antibody IgM+ serum in western blot analysis. CONCLUSION: pcGRA4 plasmid is able to synthesis of antigenic protein in CHO cells. The ability of pcGRA4 for induction of protective immune response against toxoplasmosis will be evaluated in mouse model.

Identification of Candida Species Using MP65 Gene and Evaluation of the Candida albicans MP65 Gene Expression in BALB/C Mice.

BACKGROUND: Systemic candidiasis is a major public health concern. In particular, in immunocompromised people, such as patients with neutropenia, patients with Acquired Immune Deficiency Syndrome (AIDS) and cancer who are undergoing antiballistic chemotherapy or bone marrow transplants, and people with diabetes. Since the clinical signs and symptoms are nonspecific, early diagnosis is often difficult. The 65-kDa mannoprotein (MP65) gene of Candida albicans is appropriate for detection and identification of systemic candidiasis. This gene encodes a putative b-glucanase mannoprotein of 65 kDa, which plays a major role in the host-fungus relationship, morphogenesis and pathogenicity. OBJECTIVES: The current study aimed to identify different species of Candida (C. albicans, C. glabrata and C. parapsilosis) using the Polymerase Chain Reaction (PCR) technique and also to evaluate C. albicans MP65 gene expression in BALB/C mice. MATERIALS AND METHODS: All yeast isolates were identified on cornmeal agar supplemented with tween-80, germ tube formation in serum, and assimilation of carbon sources in the API 20 C AUX yeast identification system. Polymerase Chain Reaction was performed on all samples using species-specific primers for the MP65 65 kDa gene. After RNA extraction, cDNA synthesis was performed by the Maxime RT Pre Mix kit. Candida albicans MP65 gene expression was evaluated by quantitative Real-Time (q Real-Time) and Real-Time (RT) PCR techniques. The 2-ΔΔCT method was used to analyze relative changes in gene expression of MP65. For statistical analysis, nonparametric Wilcoxon test was applied using the SPSS version 16 software. RESULTS: Using biochemical methods, one hundred, six and one isolates of clinical samples were determined as C. albicans, C. glabrata and C. parapsilosis, respectively. Species-specific primers for PCR experiments were applied to clinical specimens, and in all cases a single expected band for C. albicans, C. glabrata and C. parapsilosis was obtained (475, 361 and 124 base pairs, respectively). All species isolated by culture methods (100% positivity) were evaluated with PCR using species-specific primers to identify Candida species. Relative expression of Mp65 genes increased significantly after C. albicans injection into the mice (P < 0.05). CONCLUSIONS: The results of the current study showed that the PCR method is reproducible for rapid identification of Candida species with specific primers. Mp65 gene expression of C. albicans after injection into the mice was 2.3 folds higher than before injection, with this difference being significant. These results indicated that increase of Mp65 gene expression might be an early stage of infection; however definitive conclusions require further studies.