Production of a soluble and functional recombinant apolipoproteinD in the Pichia pastoris expression system

ApolipoproteinD (ApoD) is a human glycoprotein from the lipocalin family. ApoD contains a conserved central motif of an 8-stranded antiparallel β-sheet, which forms a beta-barrel that can be used for transport and storage of diverse hydrophobic ligands. Due to hydrophobic nature of ApoD, it has been difficult to generate a recombinant version of this protein. In the present work, we aimed at the production of ApoD in the robust Pichia pastoris expression system. To this end, the ApoD gene sequence was synthesized and subcloned for expression in the yeast host cells. Following integration of the ApoD gene into the yeast genomic region using homologous recombination, the ApoD recombinant protein was induced using methanol, reaching its maximum induction at 96 h. Having purified the ApoD recombinant protein by affinity chromatography, we measured the dissociation constant (KD) using its natural ligands: progesterone and arachidonic acid. Our results provide a viable solution to the production of recombinant ApoD protein in lieu of previous obstacles in generating soluble and functional ApoD protein. © 2016 Elsevier Inc. All rights reserved

Nanomolecular detection of human influenza virus type A using reverse transcription loop-mediated isothermal amplification assisted with rod-shaped gold nanoparticles

Reverse transcription loop-mediated isothermal amplification (RT-LAMP) and rod-shaped gold nanoparticles (gold nanorods; GNRs) were employed for nanomolecular detection of human influenza virus type A RNA. After cDNA synthesis from the RNA, the primers targeting the M protein gene were used for LAMP amplification. A blue shift from red to purple from the GNR inserting into the LAMP-DNAs can be seen by the naked eye. Transmission electron microscopy revealed the formation GNR aggregates due to their interactions with LAMP DNA. One pg RNA (10-3 dilution of the viral cDNA) was detected using this colorimetric test. The nanomolecular test showed 100% sensitivity and 95.8% specificity in comparison to results by RT-PCR. Also, the test indicated 100% sensitivity and 100% specificity in comparison to results by RT-LAMP. The described nanomolecular test could detect human influenza virus type A RNA in nearly 1 hour. This journal is © the Partner Organisations 2014

Correlation between hepatitis B G1896A precore mutations and HBeAg in chronic HBV patients

Background: Hepatitis B virus (HBV) infection is an important health concern worldwide, with critical outcomes. Hepatitis B e antigen (HBeAg) negative chronic hepatitis B is frequently caused by a mutation (G1896A) in the hepatitis B virus (HBV) precore (PC) reading frame, which creates a stop codon, causing premature termination of the HBe protein. Objectives: This study aimed to investigate the G1896A PC mutation and its effect on HBeAg detection in chronic HBV patients. Patients and Methods: In this study, 120 chronic HBV patients neither vaccinated or who had benefited from immunoglobulin therapy, were recruited. The HBV-DNA was extracted from plasma and polymerase chain reaction (PCR) was performed. Positive PCR products were subjected to automated sequencing. The HBV serological markers hepatitis B s antigen (HBsAg), HBeAg were tested. Results: One hundred out of 120 (83.3%) patients were HBeAg negative and 100% were HBsAg positive. The comparison of nucleotide sequences with the reference sequence (Accession number: AB033559) in HBeAg negative patients showed that there was a high rate of mutations in G1896A (93.18%). Conclusions: This study indicates that the rate of G1896A mutation at the PC region among HBeAg negative patients, in the Golestan province of Iran, was similar to the average rate encountered in other parts of Iran. The PC stop codon mutation was detected in 93.18% of HBeAg negative patients. Further studies with larger sample sizes are required to elucidate the exact role of these mutations in the clinical course of chronic HBV infection. © 2015, Ahvaz Jundishapur University of Medical Sciences

Human adenoviruses role in ophthalmic pterygium formation

Background: Ophthalmic pterygium is a common benign lesion of unknown origin and the pathogenesis might be vision-threatening. This problem is often associated with exposure to solar light. Recent evidence suggests that potentially oncogenic viruses such as human papillomavirus and Epstein-Barr virus may be involved in the pathogenesis of pterygia. Expression of specific adenovirus genes such as E1A and E1B, which potentially have many functions, may contribute to their oncogenic activity as well as relevance to cellular immortalization. Objectives: For the first time, we aimed to investigate involvement of adenoviruses in pterygium formation. Patients and Methods: Fifty tissue specimens of pterygium from patients undergoing pterygium surgery (as cases), 50 conjunctival swab samples from the same patients and 10 conjunctival biopsy specimens from individuals without pterygium such as patients undergoing cataract surgery (as controls) were analyzed for evidence of adenovirus infection with polymerase chain reaction using specific primers chosen from the moderately conserved region of the hexon gene. Furthermore, ß-globin primers were used to access the quality of extracted DNA. Data was analyzed using SPSS (version 16) software. Results: Of 50 patients, 20 were men and 30 women with mean age of 61.1 ± 16.9 years ranged between 22 and 85 years. All samples of pterygia had positive results for adenoviruses DNA with polymerase chain reaction, but none of the negative control groups displayed adenoviruses. The pterygium group and the control groups were ß-globin positive. Direct sequencing of PCR products confirmed Adenovirus infection. Conclusions: Adenoviruses might act as a possible cause of pterygium formation and other factors could play a synergistic role in the development. However, further larger studies are required to confirm this hypothesis. © 2015, Kowsar Medical Publishing Company. All rights reserved

Antiretroviral drug resistance mutations in naïve and experienced patients in Shiraz, Iran, 2014

Resistance to antiretroviral agents is a significant concern in the clinical management of HIV-infected individuals, particularly in areas of the world where treatment options are limited. In this study, we aimed to identify HIV drug-resistance-associated mutations in 40 drug-naïve patients and 62 patients under antiretroviral therapy (ART) referred to the Shiraz HIV/AIDS Research Center – the first such data available for the south of Iran. HIV reverse transcriptase and protease genes were amplified and sequenced to determine subtypes and antiretroviral- resistance-associated mutations (RAMs). Subtype CRF35-AD recombinant was the most prevalent in all patients (98 of 102, 96 % ), followed by subtype A1, and subtype B (one each, 2 % ). Among the 40 ART-naïve patients, two mutations associated with nucleoside reverse transcriptase inhibitor (NRTI) resistance (two with Y115F and T215I) and three associated with non-nucleoside reverse transcriptase inhibitor (NNRTI) resistance (two with G190S and Y181C, four with V179T) were found. Among ART-experienced patients, four mutations associated with resistance to NRTI, four with NNRTI, and five with protease inhibitors (PI) were found. Twenty patients with high levels of resistance were already on second-line therapy. We document for the first time in this region of Iran high levels of ART resistance to multiple drugs. Our findings call for more vigilant systematic ART resistance surveillance, increased resistance testing, careful management of patients with existing regimens, and strong advocacy for expansion of available drugs in Iran. © 2016, Springer-Verlag Wien

Antitumor effect of therapeutic HPV DNA vaccines with chitosan-based nanodelivery systems

Cervical cancer is the second-most-common cause of malignancies in women worldwide, and the oncogenic activity of the human papilloma virus types (HPV) E7 protein has a crucial role in anogenital tumors. In this study, we have designed a therapeutic vaccine based on chitosan nanodelivery systems to deliver HPV-16 E7 DNA vaccine, considered as a tumor specific antigen for immunotherapy of HPV-associated cervical cancer. We have developed a Nano-chitosan (NCS) as a carrier system for intramuscular administration using a recombinant DNA vaccine expressing HPV-16 E7 (NCS-DNA E7 vaccine). NCS were characterized in vitro for their gene transfection ability. Results: The transfection of CS-pEGFP NPs was efficient in CHO cells and the expression of green fluorescent proteins was well observed. In addition, NCS-DNA E7 vaccine induced the strongest E7-specific CD8+ T cell and interferon γ responses in C57BL/6 mice. Mice vaccinated with NCS-DNA E7 vaccine were able to generate potent protective and therapeutic antitumor effects against challenge with E7-expressing tumor cell line, TC-1. Conclusions: The strong therapeutic effect induced by the Chitosan-based nanodelivery suggest that nanoparticles may be an efficient carrier to improve the immunogenicity of DNA vaccination upon intramuscular administration and the platform could be further exploited as a potential cancer vaccine candidate in humans. © 2014 Tahamtan et al

Group a streptococcal serotypes isolated from healthy schoolchildren in iran

Serotypes of group A streptococci are still a major cause of pharyngitis and some post-infectious sequelae such as rheumatic fever. As part of the worldwide effort to clarify the epidemiological pattern of group A streptococci in different countries, the present study was conducted to assess the prevalence of Streptococcus pyogenes serotypes in Iran. A total of 1588 throat swabs were taken from healthy school children in the city of Gorgan during February and March 1999. Of those isolates, 175 resulted positive for group A streptococci. The distribution pattern was similar for girls and boys, with 10.8 and 11.2, respectively. Urban school children showed a higher rate of colonization compared to those in rural areas. Serotyping was performed on 65 of the positive isolates using standard techniques, and only 21 (32) were M-type isolates. Their profiles fell into four types with M1 predominating, which could reflect the presence of rheumatic fever in the region. However, when isolates were challenged for T-antigen types, nearly all were positive (94). The pattern of T types was diverse (18 types), with the most common T types being T1 (26), TB3264 (15), TB\1-19 & B\25\1-19 (9.2) and T2 & 2\28 (7.7). When isolates were tested for opacity factor, only 23 (35) were positive while 34 (52) responded to the serum opacity reaction test. Although the number of isolates in this study was not sufficient to make any epidemiological conclusions, the scarcity of serotyping studies in Iran could render these data useful for future attempts to develop a streptococcal vaccine

Herpes Simplex virus meningitis in children in South East of Caspian Sea, Iran

Background: Herpes simplex virus (HSV) is a member of Herpesviridae and a leading cause of human viral diseases. Meningitis occurs as a complication of HSV-1 or HSV-2 primary infection. Objectives: We aimed to evaluate HSV meningitis in children in Gorgan province, Iran. Patients and Methods: Forty-five cerebrospinal fluid samples were taken from children referred with meningitis symptoms. Samples with negative bacterial culture results were tested for viral, biochemical and cytological assays. DNA extraction and PCR were performed. Results: HSV-1 detected in 4 (8.8%) samples without any HSV-2 infections. Cases with positive results had fever and CSF pleocytosis. Vomiting, headache and higher count of WBC were observed in 3, 2 and 3 cases respectively. The cerebrospinal fluid (CSF) glucose and protein levels were normal and 3 cases showed positive C-reactive protein (CRP) results. Also erythrocyte sedimentation rate (ESR) was higher than normal in all positive cases. Conclusions: Distribution of HSV types in children with meningitis in our area predominantly was type 1 compared with type 2, which has been reported more in other area. © 2014, Ahvaz Jundishapur University of Medical Sciences; Published by Kowsar Corp

Mutations in the S gene region of hepatitis B virus genotype D in Golestan Province-Iran

Mutations of HBsAg especially within the "a" determinant could alter the antigenicity of the protein causing failure of HBsAg neutralization and escaping from the host's immune system, resulting in active viral replication and liver disease. This project aimed to investigate mutation in the S gene region of HBV infected patients in Golestan Province-Iran. HBV-DNA extractions from plasma and PCR of 100 patients were performed. Direct sequencing and alignment of S gene were applied using reference sequence from Gene Bank database. All isolates were belonged to genotype D, subgenotype D1, subtype ayw2. Overall 92 point mutations occurred. Of them, 40 (43.47%) were missense and 52 (56.52%) were silent. Mutations were detected in 95 cases (95%). Five of 40 mutations (12.5%) occurred in "a" determinant and 13 (32.5%), 17 (42.5%), and 2 (5%) were seen in antigenic epitope regions of B cell, CD4⁺ and CTL, respectively. Frame shift mutations were seen in 22 cases (22%). 14% of mutations occurred at Major Hydrophilic Region(MHR) area which P120T/S and R122K/T substitutions were the most frequent ones (4%). Mutation in G145R of the S gene was observed in one case. A large number of MHR mutants are in association with failure of HBsAg detection, vaccine, and immunotherapy escape. This study showed "a" determinant S gene mutations in HBV infected people with HBsAg positivity in Golestan Province-Iran. The rate of mutation in our study was 95%. Collectively, the results of this project exhibited that most of mutations were clustered in CD4⁺ antigenic epitopes. © Springer Science+Business Media, LLC 2012

Immunogenicity evaluation of a DNA vaccine expressing the hepatitis C virus non-structural protein 2 gene in C57BL/6 Mice

Backgrounds: Most of the hepatitis C virus (HCV) infections elicit poor immune responses and 75% to 85% of cases become chronic; therefore, the development of an effective vaccine against HCV is of paramount importance. In this study, we aimed to evaluate co-administration of HCV non-Structural Protein 2 and IL-12 DNA vaccines in C57BL/6 mice. Methods: A plasmid encoding full-length HCV NS2 protein (non-structural protein 2) was generated and used to vaccinate mice. Negative control (an empty expression vector) was also employed to evaluate the background response. To investigate immune responses against vaccine, C57BL/6 mice received three doses of the vaccine with a two-week interval. Cellular immunity was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay for lymphocyte proliferation, lactate dehydrogenase release for cytotoxic T lymphocyte (CTL) activity and cytokine assay. Results: The findings demonstrated that immunization of mice with plasmid expressing HCV NS2 induced CTL response, interferon gamma production, and lymphocyte proliferation compared to negative control. The results also demonstrated that co-administration of IL-12 with the HCV NS2 plasmid induced significantly better immune response in C57BL/6 mice. Conclusion: DNA vaccine encoding HCV NS2 is an effective candidate that can trigger CTL-based immune response against HCV. In addition, the results suggested that combining the DNA vaccine approach with immune stimulatory cytokines may significantly enhance antigen-specific immune responses