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

In vitro cytotoxic and anti-cancer effects of body wall for sea cucumber (Holothuria leucospilota)

In recent years efforts to find bioactive compounds from live organisms especially marine animals have been increased. In the present study, the anticancer and cytotoxic effects of sea cucumber body walls (Holothuria leucospilota) were investigated. For this purpose, sea cucumbers were collected from Larak Island at depths of 10 to 30 m and extraction process was done with methanol and diethyl ether solvent which then concentrated by rotary evaporator (40℃) following lyophilization with vacuum freeze dryer. XTT method was used to investigate anticancer and cytotoxic effects of body wall extracts. The results showed that the methanolic extract could prevent proliferation of human oral epidermoid carcinoma cells (KB) at concentrations of 100 and 500 μg/ml. The diethyl etheric extract also could prevent proliferation of KB at 500 μg/ml concentration. Overall result showed that sea cucumber body wall had a strong cytotoxic effect on normal cell line (Human embryonic kidney cell [HEK]) which can be used as potent cytotoxic material. However these extracts did not show significant therapeutic value against KB cells

Optimization of transfection methods for Huh­7 and Vero cells: a comparative study

Availability of an efficient transfection protocol is the first determinant in success of gene transferring studies in mammalian cells which is accomplished experimentally for every single cell type. Herein, we provide data of a comparative study on optimization of transfection condition by electroporation and chemical methods for Huh-7 and Vero cells. Different cell confluencies, DNA/reagent ratios and total transfection volumes were optimized for two chemical reagents including jetPEI™ and Lipofectamine™ 2000. Besides, the effects of electric field strength and pulse length were investigated to improve electroporation efficiency. Transfection of cells by pEGFP-N1 vector and tracking the expression of GFP by FACS and Fluorescence Microscopy analysis were the employed methods to evaluate transfection efficiencies. Optimized electroporation protocols yielded 63.73 ± ± 2.36 and 73.9 ± 1.6 % of transfection in Huh-7 and Vero cells respectively, while maximum achieved level of transfection by jetPEI™ was respectively 14.2 ± 0.69 and 28 ± 1.11 % for the same cells. Post transfectional chilling of the cells did not improve electrotransfection efficiency of Huh-7 cells. Compared to chemical based reagents, electroporation showed the superior levels of transfection in both cell lines. The presented protocols should satisfy most of the experimental applications requiring high transfection efficiencies of these two cell lines.Наличие эффективного протокола трансфекции является первым условием успешных исследований по переносу генов в клетки млекопитающих, что достигается экспериментально для каждого конкретного типа клеток. Здесь мы приводим данные сравнительного исследования по оптимизации условий трансфекции клеток Huh-7 и Vero с помощью электропорации и химическими методами. Для двух химических соединений, jetPEI™ и Lipofectamine™ 2000, были оптимизированы сочетания различных клеток, соотношения ДНК/реагент и общие объемы трансфекции. Кроме того, для улучшения эффективности электропорации было изучено влияние силы электрического поля и длины импульса. Трансфекция клеток с помощью вектора pEGFP-N1, определение экспрессии GFP с помощью FACS и флюоресцентная микроскопия были использованы для оценки эффективности трансфекции. В оптимизированных протоколах достигалась трансфекция на уровне 63.73 ± 2.36 и 73.9 ± 1.6 % в клетках Huh-7 и Vero соответственно, в то время как максимальный уровень трансфекции с помощью jetPEI™ составлял 14.2 ± 0.69 и 28 ± 1.11 % для тех же клеток. Охлаждение клеток после трансфекции не улучшало эффективность электротрансфекции клеток Huh-7. В обеих клеточных линиях электропорация позволила достичь более высокого уровня трансфекции по сравнению с использованием химических реагентов. Представленный протокол может быть пригодным для большинства экспериментальных манипуляций, которые требуют высокого уровня трансфекции исследуемых клеточных линий

High resolution melting curve assay for detecting rs12979860 IL28B polymorphisms involved in response of iranian patients to chronic hepatitis C treatment

Background: A recent genome-wide association study (GWAS) on patients with chronic hepatitis C (CHC) treated with peginterferon and ribavirin (pegIFN-α/RBV) identified a single nucleotide polymorphism (SNP) on chromosome 19 (rs12979860) which was strongly associated with a sustained virological response (SVR). The aim of this study was twofold: to study the relationship between IL28B rs12979860 and sustained virological response (SVR) to pegIFN-α/RVB therapy among CHC patients and to detect the rs12979860 polymorphism by high resolution melting curve (HRM) assay as a simple, fast, sensitive, and inexpensive method. Materials and Methods: The study examined outcomes in 100 patients with chronic hepatitis C in 2 provinces of Iran from December 2011 to June 2013. Two methods were applied to detect IL28B polymorphisms: PCR-sequencing as a gold standard method and HRM as a simple, fast, sensitive, and inexpensive method. Results: The frequencies of IL28B rs12979860 CC, CT, and TT alleles in chronic hepatitis C genotype 1a patients were 10 (10/100), 35 (35/100), and 6 (6/100) and in genotype 3a were 13 (13/100), 31 (31/100), and 5 (5/100), respectively. In genotype 3a infected patients, rs12979860 (CC and CT alleles) and in genotype 1a infected patients (CC allele) were significantly associated with a sustained virological response (SVR). The SVR rates for CC, CT and TT (IL28B rs12979860) were 18, 34 and 4, respectively. Multiple logistic regression analysis identified two independent factors that were significantly associated with SVR: IL-28B genotype (rs 12979860 CC vs TT and CT; odds ratio ORs, 7.86 and 4.084, respectively), and HCV subtype 1a (OR, 7.46). In the present study, an association between SVR rates and IL28B polymorphisms was observed. Conclusions: The HRM assay described herein is rapid, inexpensive, sensitive and accurate for detecting rs12979860 alleles in CHC patients. This method can be readily adopted by any molecular diagnostic laboratory with HRM capability and will be clinically beneficial in predicting treatment response in HCV genotype 1 and 3 infected patients. In addition, it was demonstrated that CC and CT alleles in HCV-3a and the CC allele in HCV-1a were significantly associated with response to pegIFN-α/RBV treatment. The present results may help identify subjects for whom the therapy might be successful

Biological and immunological characteristics of Brucella abortus S99 major outer membrane proteins

Introduction and objective: Outer membrane proteins (OMPs) of Brucella are considered as immunogenic structures which can be used to design and develop a subunit vaccine for human brucellosis. Brucella abortus S99 OMPs promote the synthesis of high levels of specific anti-Brucella IgG molecules in rabbits when administrated with lipopolysaccharide (LPS). The objective of this study is evaluation of the efficacy of B. abortus major OMPs with LPS in the induction of immune response against brucellosis. Materials and methods: OMPs were derived from B. abortus by sequential extraction of sonicated cells with ultracentrifugation and predigestion with lysozyme. Proteins could be separated by anion-exchange chromatography and gel-filtration. Based on SDS-PAGE profiles, porins have been dominantly purified among three different classes of B. abortus OMPs. Sera of immunized rabbits against B. abortus porins were analyzed by enzymelinked immunosorbent assay (ELISA). LPS of B. abortus and complete Freud's adjuvant (CFA) were also applied to elicit higher levels of anti-Brucella antibodies. Results: ELISA confirmed the potency of porins and porins combination with CFA and LPS to promote humoral specific response. Among the above-mentioned compounds, a combination of porins + LPS or porins + CFA has been the most potent immunogenic compound to induce higher titer of antibody against B. abortus S99 in the animal model. Conclusion: The application of a complex of Brucella LPS and porins as an effective method to elicit protective and long-lasting immunity against Brucella infection and would be studied to design and develop a subunit vaccine for human brucellosis

Preparation and Evaluation of a New Lipopolysaccharide-based Conjugate as a Vaccine Candidate for Brucellosis

Objectives: Development of an efficacious vaccine against brucellosis has been a challenge for scientists for many years. At present, there is no licensed vaccine against human brucellosis. To overcome this problem, currently, antigenic determinants of Brucella cell wall such as Lipopolysaccharide (LPS) are considered as potential candidates to develop subunit vaccines. Methods: In this study, Brucella abortus LPS was used for conjugation to Neisseria meningitidis serogroup B outer membrane vesicle (OMV) as carrier protein using carbodiimide and adipic acid-mediated coupling and linking, respectively. Groups of eight BALB/c mice were injected subcutaneously with 10μg LPS alone, combined LPS+OMV and conjugated LPS-OMV on 0 days, 14 days, 28 days and 42 days. Anti-LPS IgG was measured in serum. Results: The yield of LPS to OMV in LPS-OMV conjugate was 46.55, on the basis of carbohydrate content. The ratio for LPS to OMV was 4.07. The LPS-OMV conjugate was the most immunogenic compound that stimulated following the first injection with increased IgG titer of ~5-fold and ~1.3-fold higher than that produced against LPS and LPS in noncovalent complex to OMV (LPS+OMV), respectively. The highest anti-LPS IgG titer was detected 2 weeks after the third injection (Day 42) of LPS-OMV conjugate. The conjugated compound elicited higher titers of IgG than LPS+OMV, that showed a 100-120-fold rise of anti-LPS IgG in mice. Conclusion: These results indicate that our conjugated LPS-OMV can be used as a brucellosis vaccine, but further investigation is required. © 2014

Prevalence of Merkel cell polyomavirus in Tehran: An age-specific serological study

Background: Several new types of polyomavirus have been discovered in recent years mainly because of the recent state-of-the-art detection technologies. Among the polyomaviruses, Merkel cell polyomavirus (MCPyV) has attracted the most attention because of its possible role in the etiology of Merkel cell carcinoma, a rare but lethal form of skin cancer. Objectives: This study aimed to determine age-specific seroprevalence of MCPyV in Tehran. Patients and Methods: In this cross-sectional study, we collected 440 serum samples from healthy individuals 2 to 78 years of age who visited the Pasteur Institute�s clinic in Tehran, Iran, using a convenience sampling strategy. We developed a virus-like particle-based enzyme-linked immunosorbent assay that uses VP1, the major capsid protein of MCPyV, to detect and quantitate serum antibodies to MCPyV. We compared the prevalence of MCPyV between males and females and across eight age groups. Results: A total of 255 (57.9) of the serum samples were MCPyV positive. The seroprevalence in children under 10 years of age was 25. The seroprevalence increased to 56 over the next decade of life (10 - 19 years of age). The seroprevalence rate in males and females was 56.1 and 59.7 respectively, and a binary logistic regression showed no significant difference between males and females (P = 0.77). However, the prevalence of MCPyV increased with age (P = 0.012). Conclusions: Our results suggest that human exposure to MCPyV occurs throughout life. The MCPyV antibody levels remained high among older adults in our population, consistent with reports from other populations. © 2016, Iranian Red Crescent Medical Journal

Prevalence and risk factors for HBV and HCV in prisoners in Iran: a national bio-behavioural surveillance survey in 2015

Objectives: To provide more accurate estimates of the prevalence of Hepatitis B (HBV) and Hepatitis C (HCV) and their contributing factors among prisoners in Iran. Methods: Cross-sectional study of 6200 Iranian prisoners in 2015. Data were collected through questionnaires and interviews. HBV infection and HCV exposure status of the participants was determined by HBsAg and HCV antibodies blood tests using enzyme-linked immunosorbent assay (ELISA). Data were analysed in STATA-12. Result: Prevalence of HCV exposure was 9.48 (95 CI: 8.73�10.27), and prevalence of HBV was 2.48 (95 CI: 2.07�2.89) in the general prison population. In multivariate analysis, the most important risk factor for HBV was a history of drug use in lifetime (adjusted odds ratio, AOR: 1.8, 95 CI: 1.17�3.02). The main risk factors for HCV exposure were a history of drug use in lifetime (AOR: 4.08, CI: 2.56�6.27), age over 30 (AOR: 2.68, CI: 2.01�3.56), and having tattoos (AOR = 1.67, CI: 1.35�2.07). Conclusion: Although vaccination is used to control HBV among prisoners, prevalence of HCV exposure is alarming in the prison population of Iran, especially among people who inject drugs. Eliminating viral hepatitis in Iran by 2030 requires a national commitment and rapid measures for targeting this high-risk group. Given the increased efficiency of HCV treatment in recent years, prisons provide an opportunity to access patients for treatment. © 2018 John Wiley & Sons Lt

Prevalence and risk factors for HBV and HCV in prisoners in Iran: a national bio-behavioural surveillance survey in 2015

Objectives: To provide more accurate estimates of the prevalence of Hepatitis B (HBV) and Hepatitis C (HCV) and their contributing factors among prisoners in Iran. Methods: Cross-sectional study of 6200 Iranian prisoners in 2015. Data were collected through questionnaires and interviews. HBV infection and HCV exposure status of the participants was determined by HBsAg and HCV antibodies blood tests using enzyme-linked immunosorbent assay (ELISA). Data were analysed in STATA-12. Result: Prevalence of HCV exposure was 9.48 (95 CI: 8.73�10.27), and prevalence of HBV was 2.48 (95 CI: 2.07�2.89) in the general prison population. In multivariate analysis, the most important risk factor for HBV was a history of drug use in lifetime (adjusted odds ratio, AOR: 1.8, 95 CI: 1.17�3.02). The main risk factors for HCV exposure were a history of drug use in lifetime (AOR: 4.08, CI: 2.56�6.27), age over 30 (AOR: 2.68, CI: 2.01�3.56), and having tattoos (AOR = 1.67, CI: 1.35�2.07). Conclusion: Although vaccination is used to control HBV among prisoners, prevalence of HCV exposure is alarming in the prison population of Iran, especially among people who inject drugs. Eliminating viral hepatitis in Iran by 2030 requires a national commitment and rapid measures for targeting this high-risk group. Given the increased efficiency of HCV treatment in recent years, prisons provide an opportunity to access patients for treatment. © 2018 John Wiley & Sons Lt

Designing & producing polytope DNA vaccine containing HBsAg gene for the induction of protective immunity against hepatitis C

Background: Considering the immunosuppressive effects and prevalent mutations in some HCV antigens, induction of CD8+ T cell responses is focused on conserved and critical epitopes which as a multi-epitope vaccine can prevent the chronic nature of the disease. Materials and Methods: Two immunodominant HLA-A2-restricted human epitopes (E2614-622 and NS31406-1415) and two H-2d-restricted mouse epitopes (core132-142 and E2405-414) were designed in a sequential tandem, predicted by immunoinformatic analyses. Following the synthesis, related nucleotide sequence was cloned into the pcDNA3.1 vector with and without the fusion of hepatitis B surface antigen (HBsAg). Two constructed plasmids (pcDNA3.1.HPOL and pcDNA3.1.POL, respectively) were evaluated for the protein expression and secretion in Cos-7 cell line. After the vaccination of BALB/c mice (n=6 in each group) with different DNA and peptide immunization regimens, CD8+ T cell activity as well as the type and protective potency of the induced responses were evaluated. Results: Despite the induction of epitope-specific responses in pcDNA3.1.POL injected mice, the group immunized with pcDNA3.1.HPOL indicated a significant increase in the number and activity of CD8+ T cells (P<0.05). Peptide boosting of this group (formulated in two human-compatible adjuvant) still led to the more activation of CD8+ cells, induction of Th1 response and the inhibition of tumor model growth (P<0.05). Conclusion: Fusion of HBsAg as a particle-forming sequence and the source of helper epitopes along the DNA-prime/peptide-boosting immunization regimen are proposed as two promising strategies to improve the CTL multi-epitope vaccines against HCV