Cytokine Profiles and Cell Proliferation Responses to Truncated ORF2 Protein in Iranian Patients Recovered from Hepatitis E Infection.

Background. The aim of this study was to evaluate hepatitis E virus (HEV) specific cellular immune responses to truncated ORF2 protein in Iranian patients recovered from HEV infection. Information about HEV-specific immune responses could be useful in finding an effective way for development of HEV vaccine. Methods. A truncated form of HEV ORF2 protein containing amino acids 112-608 was used to stimulate peripheral blood mononuclear cells (PBMCs) separated from HEV-recovered and control groups. Finally, the levels of four cytokines, IFN-γ ELISPOT, and cell proliferative responses following stimulation with the truncated ORF2 protein were assessed in the both groups. Results. The truncated ORF2 protein was able to induce IFN-γ ELISPOT and cell proliferation responses and to produce significant amounts of IFN-γ and IL-12 cytokines, but low amounts of IL-10 and IL-4 cytokines in vitro. These responses were significantly higher in the recovered group compared to the control group. These results indicate the antigenic nature of the truncated ORF2 protein and production of T helper type 1 cytokines. Conclusion. The truncated ORF2 protein can effectively induce significant cellular immune responsesand can be introduced as a potential vaccine candidate. However, further studies are required to evaluate this protein in vivo.

Determining the Role of Helicobacter pylori in Chronic Sinus Infections Using the Polymerase Chain Reaction.

BACKGROUND: Helicobacter pylori are becoming increasingly recognized as a possible pathological cause of chronic rhinosinusitis (CRS). OBJECTIVES: Considering the prevalence of CRS and its impact on quality of life, we decided to determine the role of H. pylori in chronic sinus infections by using the PCR technique. PATIENTS AND METHODS: In a case-control analytical epidemiologic survey, the study population was selected by consecutive sampling from patients with CRS undergoing endoscopic sinus surgery during years 2010 - 2012. Patients were divided into two groups. The study group consisted of patients with CRS and the control group consisted of patients with nasal obstruction caused by concha bullosa, without inflammation or infection of the sinuses. Sampling was performed during surgery from the infected tissue and from the middle turbinate mucosa. Eventually, bacterial DNA was extracted and used for the PCR test, in order to isolate H. pylori. RESULTS: Nine patients (18%) with CRS had H. pylori isolated from their samples whereas in the control group, H. pylori were only found in two patients (4%); this difference was statistically significant (P = 0.025). The indicator wasn't statistically significant between males and females. There was no statistical correlation in relative frequency of H. pylori for different age groups (P > 0.05). CONCLUSIONS: There was a significant correlation between CRS and presence of H. pylori in sinonasal mucosa. This relationship may reflect the role of H. pylori as one of the pathogenic factors in the development of CRS. However, further studies are required to confirm this role.

Codon-Optimized Expression and Purification of Truncated ORF2 Protein of Hepatitis E Virus in Escherichia coli.

BACKGROUND: Hepatitis E virus (HEV) is a causative agent of acute hepatitis among people of different age groups and has high mortality rate of up to 30% among pregnant women. Therefore, primary prevention of HEV infection is essential. OBJECTIVES: The aim of this study was to obtain the highly purified truncated open reading frames 2 (ORF2) protein, which might be a future HEV vaccine candidate. MATERIALS AND METHODS: The truncated orf2 gene (orf2.1), encoding the 112-660 amino acid of HEV capsid protein sequence, was optimized, synthesized, and cloned into pBluescript II SK(+) vector. After subcloning into expression vector pET-30a (+), a 193-nucleotide fragment was deleted from the construct and the recombinant plasmid pET-30a-ORF2.2 (orf2.2 encodes 112-608 amino acid sequence of HEV capsid protein) was constructed and used for transformation of Escherichia coli BL21 cells. After induction with isopropyl-ß-D-thiogalactopyranoside (IPTG) and optimizing the conditions of expression, the target protein was highly expressed and purified by Ni(2+)-chelate affinity chromatography. The expressed and purified protein was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. RESULTS: The subcloning was confirmed by PCR, restriction enzyme digestion, and DNA sequencing of recombinant plasmid pET30a-ORF2.2. The results obtained from optimizing the expression conditions showed that the highest expression of the protein was obtained by adding IPTG at a final concentration of 1 mM at 37℃ for four hours. The expression and purification of truncated ORF2 protein was confirmed by SDS-PAGE and western blotting. SDS-PAGE analysis showed a protein band of about 55 kDa. SDS-PAGE of the purified protein revealed that the highest amount of target protein in elution buffer at the pH of 4.5 was obtained. The yield of the purified protein was about 1 mg/L of culture media. CONCLUSIONS: In this study, the optimized truncated ORF2 protein was expressed in E. coli successfully and the highly purified protein was obtained, which can be a potential vaccine candidate and as an antigen in ELISA to diagnose HEV infections.

Serotype determination of adenoviruses in children with respiratory infection.

OBJECTIVE: To determine adenovirus serotypes among children with acute respiratory infection (ARI) in Khoozestan province, Iran during years 2010-2011. METHODS: One hundred sixty three nasopharyngeal swabs were collected from children between 1 and 15 y who were hospitalized for the acute respiratory infection. The viral DNA was extracted from the nasopharyngeal swabs and adenoviruses were detected by Nested PCR. Positive PCR samples were sequenced in order to confirm the adenovirus serotypes. RESULTS: Out of 163 samples, 30 (18.4 %) were positive for adenoviruses by nested PCR. Twenty two PCR products were sequenced and recognized as Ad5 and Ad2 serotypes including 19 (86.3 %) Ad5 and 3 (13.7 %) Ad2. CONCLUSIONS: This study reveals that adenoviruses with Ad5 predominance are important cause of respiratory tract infection in children.

Cloning of fliC Gene From Salmonella typhimurium in the Expression Vector pVAX1 and Evaluation of its Expression in Eukaryotic Cells.

BACKGROUND: Flagellin is the main structural protein of the flagella of many pathogens including Salmonella typhimurium. It is a potent trigger of innate immune responses that enhance adaptive immune responses to a variety of protein antigens. Flagellin has intrinsic adjuvant activity mediated through toll-like receptor (TLR) 5 and is an attractive candidate for highly effective vaccine adjuvant conferring enhanced antibody and cellular immune responses to proteins or peptides. In the present study, we cloned the fliC gene from S. enterica typhimurium in eukaryote vector pVAX1 and evaluated its expression in eukaryotic cells. OBJECTIVES: The main aim of the present study was to construct a DNA vaccine expressing fliC as an adjuvant. MATERIALS AND METHODS: The fliC gene of S. typhimurium (ATCC 14028) was amplified by PCR with specific primers and cloned into the pPrime cloning vector and successfully subcloned into expression vector pVAX1. The recombinant plasmid pVAX-fliC was finally expressed in eukaryotic cells. RESULTS: Cloning and subcloning of the fliC gene were confirmed by colony PCR, restriction enzymes digestion and DNA sequencing of the recombinant plasmids pPrime-fliC and pVAX-fliC. The expression of flagellin protein in eukaryotic cells was approved by immunofluorescence assay (IFA), western blotting analysis and the reverse transcriptase polymerase chain reaction (RT-PCR) method. CONCLUSIONS: The results of this study demonstrated that the fliC gene in recombinant plasmid pVAX-fliC was successfully expressed in eukaryotic cells and produced flagellin protein, which could be used as an effective adjuvant for DNA vaccine research.

Investigation of Possible Correlation between Giardia duodenalis Genotypes and Clinical Symptoms in Southwest of Iran.

BACKGROUND: Giardia duodenalis is one of the most important human enteric parasites throughout the world. Clinical symptoms of this parasite vary from asymptomatic infection to chronic diarrhea. Still it is not clear, whether different types of pathogenesis are due to different strains of organism or to variable host factors. The purpose of this study was to investigate possible correlation of clinical symptoms with assemblages among symptomatic and asymptomatic cases collected from southwest of Iran. METHODS: Fecal samples were collected from 100 symptomatic and asymptomatic cases, which were positive for G. duodenalis. The samples were subjected to semi-nested PCR and RFLP for gdh gene. RESULTS: Among symptomatic patients, 54% had mixed genotypes AII and BIII, 28% and 18% of samples indicated assemblages BIII and AII, respectively. In contrast, among asymptomatic cases, 64%, 26% and 10%samples had mixed genotypes, BIII and AII assemblages, respectively. Statistical analysis using Chi- Square test showed that there was no significant correlation between assemblage and clinical symptoms in current study. CONCLUSION: High prevalence of mixed infection in both groups may affect this conclusion, therefore further study in more details are necessary to clarify these finding. Additionally, it is important to carry out investigations regarding human host factors as well.