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1.  Molecular Characterization of QDPR Gene in Iranian Families with BH4 Deficiency: Reporting Novel and Recurrent Mutations 
JIMD Reports  2015;21:123-128.
Newborn screening for PKU has been in practice in Iran since 2007. Some hyperphenylalaninemia cases have tetrahydrobiopterin (BH4) biosynthesis deficiency/disorder. Several genes including QDPR (encodes DHPR enzyme, the necessary cofactor for PAH activity) have been associated with the BH4. Mutations have been previously described in the QDPR gene. The incidence of BH4 deficiency is expected to be higher in Iran due to high rate of consanguineous marriages.
We identified a total of 93 BH4-deficient families. A multiplex set of STR markers linked to 4 genes responsible for the BH4 deficiency (i.e., GCH1, PCBD1, PTS, and QDPR genes) was used to quickly determine which gene may be responsible to cause the disease. Mutation analysis of QDPR gene revealed some known and novel mutations. Our findings show that no common mutation predominates, and they are scattered in the gene in our population.
PMCID: PMC4470949  PMID: 26006720
2.  The Usefulness of Anti-HCV Signal to Cut-off Ratio in Predicting Viremia in Anti-HCV in Patients With Hepatitis C Virus Infection 
Hepatitis C Virus (HCV) infection is diagnosed by antibody and RNA based methods. Patients with anti-HCV sample rate/cutoff rate (S/CO) ratios > 1 are reported as anti-HCV positive. RNA based methods are introduced to confirm positivity in seropositive samples.
The current study aimed to assess relationship between S/CO rates and HCV-RNA levels in the laboratory to identify HCV viremia in patients with a positive anti-HCV.
Patients and Methods:
All serum samples were assayed for anti-HCV by ELISA method. A total of 265 anti-HCV positive patients were tested for HCV-RNA testing by quantitative method using Artus HCV RG Real-time Polymerase Chain Reaction (RT- PCR) kit. Statistical analysis was done by SPSS version 16.
Of the 265 patients with HCV infection, 204 (77%) were male and the mean age was 43.53 ± 13.17 years, ranging 1 - 81 years. No correlation was found between S/CO ratios and HCV-RNA levels. There was significant difference in S/CO ratio between viremic and non-viremic subjects. The sensitivity, specificity, negative predictive value, and positive predictive value were 100%, 81.4%, 100%, and 77.2%, respectively in the S/CO ratio of 2.7.
The present study indicated that anti-HCV S/Co ratio is useful to predict non-viremic patients. A cut-off value of 2.7 can determine the usefulness of HCV-RNA testing. Patients with S/CO < 2.7 are not viremic; therefore, HCV-RNA testing is not recommended. It is suggested that laboratories report S/CO ratio along with anti-HCV results to manage HCV infection better, especially in countries that quantitative HCV testing is expensive or not available.
PMCID: PMC4449862  PMID: 26034549
Hepatitis C Antibodies; Hepatitis C Infection
3.  The Effects of Ultraviolet Light and Riboflavin on Inactivation of Viruses and the Quality of Platelet Concentrates at Laboratory Scale 
This study investigated the effects of Riboflavin (RB) combined with different doses of UV on Platelet Concentrate (PC) which was infected by three models of virus. Platelet quality after treatment was also assessed.
Three models of virus used in this study were Vesicular Stomatitis Virus (VSV), Herpes Simplex Virus (HSV), and Polio virus, which were added to PC. After photochemical treatment with RB and UV light, residual viral infectivity was titrated using 50% Tissue Culture Infective Dose (TCID50)/ml. This treatment was done with concentration of 50 μM of RB and different doses of UV light (0.24, 0.48, 0.97, 1.29 J/cm2). Platelet quality was assessed by measuring pH, Lactate Dehydrogenase (LDH), MTT assay and cell count after treatments and during 4 days of storage against control groups.
Concentration of 50 μM RB with combination of 1.29 J/cm2 dose of UV resulted in the highest titer reduction of VSV (4 log 10) and HSV (4.26 log10) and lowest titer reduction of Polio virus (2.6 log10). No significant difference was observed between different doses in comparison with control groups. In all treatment groups, the storage stability of platelets in PC was in the acceptable range in comparison with control group.
This study indicated that RB/UV treatment was a promising pathogen reduction technique in PC and had limited effects on platelet quality. However, further optimization of this method is necessary to deal with blood-borne viruses like non-enveloped viruses.
PMCID: PMC4483315  PMID: 26140182
Blood platelet; Riboflavin; Ultraviolet light; Virus inactivation
4.  Cloning and expression of NS3 helicase fragment of hepatitis C virus and the study of its immunoreactivity in HCV infected patients 
Hepatitis C is a major cause of liver failure worldwide. Current therapies applied for this disease are not fully effective and produce side effects in most cases. Non-structural protein 3 helicase (NS3) of HCV is one of the key enzymes in viral replication and infection. Therefore, this region is a promising target to design new drugs and therapies against HCV infection. The aim of this study was cloning and expression of HCV NS3 helicase fragment in Escherichia coli BL21 (DE3) using pET102/D-TOPO expression vector and studying immunoreactivity of the expressed antigen in Iranian infected with hepatitis C.
Materials and Methods:
The viral RNA was extracted from the serum of HCV infected patient. The NS3 helicase region was amplified by RT-PCR. The PCR product was directionally cloned into the expression vector pET102/D-TOPO and transformed into the BL21 strain of E. coli (DE3). The transformed bacteria were then induced by adding 1mM isopropyl-β-D-thiogalactopyranoside (IPTG) into the culture medium to enhance the protein expression. SDS-PAGE and western blotting were carried out to identify the protein under investigation, and finally purified recombinant fusion protein was used as the antigen for ELISA method.
The insertion of the DNA fragment of the NS3 region into the expression vector was further confirmed by PCR and sequencing. SDS-PAGE analysis showed the successful expression of the recombinant protein of interest. Furthermore, immunoreactivity of fusion NS3 helicase was confirmed by ELISA and western blotting.
It seems that this recombinant protein could be a useful source of antigen for future studies on HCV diagnosis and therapy.
PMCID: PMC4366727  PMID: 25810890
HCV; Molecular cloning; NS3 helicase; Recombinant protein
5.  Interferon-Gamma and Interlukin-4 Patterns in BALB/c Mice Suffering From Cutaneous Leishmaniasis Treated With Cantharidin 
Cutaneous leishmaniasis is a health problem in the world. Lesions should be treated on cosmetically or functionally important sites, such as the face and hands. Cantharidin is a terpenoid compound produced naturally by beetles of Meloidae and Oedemeridae families.
The current study aimed to investigate the effect of cantharidin on Cutaneous Leishmaniasis (CL) lesions and IFN-γ and IL-4 patterns in infected BALB/c mice.
Materials and Methods:
Infected BALB/c mice were divided into five groups as: untreated (control group), eucerin-treated and 0.05%, 0.1% and 0.5% cantharidin-treated. Lesions diameter was measured by Vernier caliper every three days for four weeks. Cytokines levels were measured by enzyme-linked immunosorbent assay (ELISA) using U-CyTech kit.
The results indicated that treatment with cantharidin exacerbates lesions compared with the controls, except for 0.05% cantharidin dose that restrained lesion growth significantly. Interferon gamma level in cantharidin-treated groups was significantly less than that of the control group. But interlukin-4 level was similar among the groups.
The current study results indicated that high doses of cantharidin exacerbates leishmaniasis lesion, but low dose of cantharidin inhibits lesion growth.
PMCID: PMC4217669  PMID: 25371808
Mice, Inbred BALB/c; Cantharidin; Leishmaniasis, Cutaneous; Interferon-gamma
6.  Distinct Toll-like Receptor 3 and 7 Expression in Peripheral Blood Mononuclear Cells From Patients with Chronic Hepatitis C Infection 
Hepatitis Monthly  2014;14(4):e16421.
Hepatitis C virus (HCV) is a major cause of chronic liver disease, with around 130 million infected people worldwide. HCV is recognized by Toll-like receptors (TLRs), which are key mediators of innate immune response. Up on activation of TLRs, anti-viral cytokines and pre-inflammatory are produced.
In this study, we compared the expression levels of two members of the TLR family (TLR3 and TLR7) that recognize viral RNA in peripheral blood mononuclear cell (PBMC) of patients with chronic HCV infection and healthy controls.
Patients and Methods:
In this case-control study, blood samples were collected from patients admitted to Blood Transfusion Research Center, Tehran, Iran. PBMC was isolated from blood of chronic HCV patients (n = 25) and age and sex-matched healthy controls (n = 25). RNA was extracted from PBMC and cDNA was synthesized from total RNA templates using reverse transcriptase. The relative level of expression was quantified by real-time PCR using Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as reference gene and the results were compared by Pfaffl method. Data were analyzed using non-parametric Wilcoxon test. P < 0.05 was considered significant.
In both groups, we had 13 males and 12 females with a mean age of 48.7 ± 16. TLR3 (6.23 ± 0.91 vs. 3.89 ± 0.85, P < 0.001) and TLR7 (1.48 ± 0.82 vs-1.33 ± 1.18, P < 0.001) expressions were significantly lower in patients with chronic HCV infection when compared with healthy controls.
This study suggests that decrease in levels of TLR3 and TLR7 expression is a mechanism that may enable HCV to evade the host innate immune response.
PMCID: PMC3989766  PMID: 24748896
Hepatitis C, Chronic; Toll-Like Receptor 3; Toll-Like Receptor 7; Immunity, Innate; Gene Expression
7.  Th1 Platform Immune Responses Against Leishmania major Induced by Thiol-Specific Antioxidant-Based DNA Vaccines 
The Thiol-specific antioxidant (TSA) is an antigen of Leishmania major which is believed to be the most promising molecule as a vaccine candidate against leishmaniasis.
In this study, we investigated the protective efficacy of TSA-based DNA vaccine against L. major infection.
Materials and Methods:
Recombinant plasmid construction TSA (pcTSA) was prepared and transfected into eukaryotic cells and expression was confirmed with western blot and RT-PCR. The mice were assigned to six different groups and DNA immunization was performed with 100 µg intramuscular recombinant plasmid with a two-week interval. Cytokines and lymphocyte proliferation assay, antibody responses and determination of parasite burden were performed following immunization and the challenging infection with L. major.
The antibody and IFN-γ titers were higher in pcTSA + AlPO4 group the immunized mice with pcTSA alone, but there was no statistically significant difference between the two groups. Additionally the IL-4 titer was not statistically different between the groups following immunization and challenge. After infection with L. major promastigotes, the immunized mice with pcTSA and the one immunized with both pcTSA + AlPO4 presented a considerable reduction in diameter of lesion but there was no statistical difference between the two groups. The immunized mice had significantly lower parasite loads. No significant differences were observed between the two vaccinated groups. However the highest reduction in parasite burden was observed in the group immunized with pcDNA + AlPO4. No significant differences were observed in survival rate of the immunized mice after the challenge with L. major.
In conclusion, TSA-based DNA vaccine induced Th1 platform immune response and aluminum phosphate could improve the efficacy of these vaccines with induction of humoral and cellular immune responses against L. major infection. There were no significant differences observed between pcTSA and pcTSA + AlPO4 groups.
PMCID: PMC4138682  PMID: 25147675
DNA vaccine; Leishmania major; Thiol-S Antioxidant; Aluminum Phosphate
8.  Inactivation of model viruses suspended in fresh frozen plasma using novel methylene blue based device 
Background and objective
There is a concern on safety of human Fresh Frozen Plasma (FFP) as it is a source of some medicinal products. The possibility of transmission of blood-borne are reported often due to emerging viruses. There are some Pathogen Reduction Technologies (PRT) to inactivate viruses. Methylene Blue (MB) based method is one of them. The aim of this study was to examine new designated device to inactivate model viruses.
Materials and Methods
Four model viruses were used in this study:Vesicular stomatitis virus (VSV), Herpes Simplex Virus I (HSV-1), Bovine Viral DiarrheaVirus(BVDV) and Polio Virus.50% Tissue Culture Infective Dose (TCID 50) and Reed–Muench Methods were used to titer the viruses. MB in two final concentration of 0.1 μM and 1 μM and illumination in about 627nm with red LED (Lamp Emitting Diode) for 15, 30, 45 and 60 minutes were used. Three replicates employed for each experiments.
1μMconcentration of MB showed more effective than 0.1μMin all designed illumination period for inactivation of HSV, VSV and BVDV. This method also demonstrated best results for enveloped model viruses. The most Log reduction for HSV, VSV and BVDV were6.28, 5.54 and 6.22, respectively. For HSV and BVDV inactivation, the best illumination period was 45 minutes.
Model viruses showed sensitivity combination of MB and illumination using red LEDs. As results show this device could inactivate model viruses and reduce their titer very close to approved commercial devices, in compare.
PMCID: PMC4419045  PMID: 25954491
Blood-borne Viruses; Methylene Blue; Red LED; Fresh Frozen Plasma
9.  Effect of Hepatitis B Virus X Gene on the Expression Level of p53 Gene using Hep G2 Cell Line 
The HBV-X (HBX) protein is believed to contribute to the development of HCC. However, the molecular mechanisms involved in HBX- mediated hepatocarcinogenesis remain obscure. In this study, the effect of hepatitis B virus X gene and its protein product HBxAg on expression of p53 gene in Hep G2 cell line was investigated.
Viral DNA extracted from HBV-positive serum and HBX gene region was amplified using polymerase chain reaction (PCR). Then, PCR product was cloned into the pcDNA3 vector. After confirmation of cloning, the recombinant plasmid pcDNA3-X was transfected into HepG2 cell line using lipid-mediated DNA-transfection procedure. SDS-PAGE and western blotting methods were used to identify expression of HBX protein. Relative quantification was used to analyze the p53gene expression using the 2-ΔΔ Ct method.
Recombinant plasmid pcDNA3–HBX was confirmed by restriction endonucleases digestion and colony-PCR. The results of SDS-PAGE and western blot assays showed that HBX gene could be expressed in Hep G2 cell line. There was no significant difference between the expression levels of p53 compared with GAPDH gene as housekeeping gene (p < 0.05).
There was no significant difference in the protein levels between the transfected cells with X gene containing HBX130 and HBX131 double mu-tations and p53 gene. It is necessary to do more studies on Hepatitis B virus to understand the role of HBX on the development of liver cancer and its function on p53 tumor suppressor protein.
PMCID: PMC3895577  PMID: 24523952
Hepatitis B virus; Hep G2 cell line; p53 gene; X gene
10.  Correlation Between Viral Load of HBV in Chronic Hepatitis B Patients and Precore and Basal Core Promoter Mutations 
Hepatitis Monthly  2013;13(2):e7415.
More than two billion people have been exposed to hepatitis B virus (HBV) worldwide. Furthermore, four hundred million of them are infected with chronic HBV infection. The predominant mutation of the precore region involves a G to A change at nucleotide1896, which creates a premature stop codon at codon 28. Two mutations of A1762T and G1764A are reported as the most prevalent mutations in the basal core promoter (BCP).
The purpose of this study was to investigate the relationship between mutations in precore (PC) and basal core promoter regions, and the viral load.
Patients and Methods
Fifty serum samples from patients with hepatitis B were used. Levels of liver enzymes alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured at the same time of serological markers of hepatitis B by ELISA. HBV-DNA was extracted from the sera, and then PCR performed on the HBV-DNA extracted with the use of specific primer of gene C. HBV viral load was determined by real-time PCR. The PC/ BCP mutations were determined by applying Line Probe Assay technique. The data were analyzed using SPSS software, version 20.
Only 82% of the patients were HBeAb positive and 76% of the patients had basal core/ precore mutations and mean viral load was 3/7 × 106 ± 9/7 × 105 IU/ml. Prevalence of mutations in the precore and basal core promoter regions were 46% and 30%, respectively.
Our data indicated that there is a statistically significant relationship between HBV viral load and mutations in precore region (P < 0.05).
PMCID: PMC3628088  PMID: 23599717
Mutations; Hepatitis B Virus; Viral Load
11.  Torque Teno Virus and Hepatitis C Virus Co-Infection in Iranian Pediatric Thalassemia Patients 
Turkish Journal of Hematology  2013;29(2):156-161.
Objective: Torque teno virus (TTV) infects patients at risk for parenteral exposure and chronic blood transfusion, such as those with β-thalassemic. This study aimed to assess the prevalence of TTV infection and co-infection of TTV and hepatitis C virus (HCV) in pediatric thalassemia patients receiving chronic blood transfusion.
Material and Methods: The study included 90 pediatric thalassemia patients receiving chronic blood transfusion that presented to the Mofid Children’s Hospital, Tehran, Iran. The control group included 90 healthy volunteer children. Serum TTV DNA detection via semi-nested PCR and HCV Ab were performed in all the participants. Demographic characteristics and clinical data were collected from each participant for statistical analysis.
Results: In all, 64.4% of the patients had TTV infection, versus 24.4% of the controls (P < 0.01). The thalassemia patients had a greater probability of having TTV and HCV infections than the controls, with a common OR of 5.60 (95% CI: 2.94-10.69) and 2.15 (95% CI: 1.83-2.50), respectively. In total, 17.2% (10/58) of the patients that were TTV positive were also HCV positive, whereas 6.3% (2/32) of the TTV-negative patients were anti-HCV antibody (Ab) positive (P = 0.14).
Conclusion: The prevalence of TTV and HCV infection was higher in the Iranian thalassemia patients on chronic transfusion therapy than in the controls. The high prevalence of TTV in pediatric thalassemia patients on chromic transfusion therapy may indicate the superiority of the parenteral route compared to other routs of TTV transmission.
PMCID: PMC3986954  PMID: 24744647
Thalassemia; Torque teno virus; Hepatitis C virus
12.  Identification of HCV genotypes in HCV infected blood donors 
Indian Journal of Microbiology  2010;50(3):275-279.
HCV infection is a leading cause of chronic liver disease, including cirrhosis of the liver. There are at least six major genotypes and more than 50 subtypes of HCV. The prevalence and distribution of HCV genotypes depend on geographical location. The aim of this study was to identify and compare the HCV genotypes in HCV infected blood donors and patients. In this cross-sectional study, 167 serum samples from 103 blood donors and 64 patients with hepatitis C were investigated for HCV genotypes. HCV genotyping was carried out using type-specific primers from the core region of the viral genome. The highest frequency was for genotype 1a, with 53 and 34 (51.5% versus 53.1%) of subjects in blood donors and patients respectively. Genotype 3a and 1b were the other frequent genotypes with 4 and 16 (3.9% versus 25%) and 39 and 10 (37.9% versus 15.6%) subjects, respectively. There was not any statistical significant association between the place of infection of the patients and genotype. The results of this study indicate that the distribution of genotypes in the two populations was similar. The dominant HCV genotypes between blood donors and patients were 1a, 3a and 1b respectively.
PMCID: PMC3450055  PMID: 23100841
HCV; Genotypes; Blood donors; Patients
13.  Clinical outcomes of Torque teno virus-infected thalassemic patients with and without hepatitis C virus infection 
The Korean Journal of Hematology  2011;46(2):123-127.
Although a marked proportion of thalassemic patients acquire Torque teno virus (TTV) through blood transfusion, its clinical importance is unclear. This study was designed to investigate the clinical importance of TTV infection in thalassemic patients with and without hepatitis C virus (HCV) co-infection in Iran.
In this case-control study, 107 thalassemic patients on chronic transfusion and 107 healthy individuals were selected. According to HCV and TTV infection status (detected by semi-nested PCR), patients were categorized into 4 groups: TTV and HCV negative, TTV positive, HCV positive, and TTV and HCV positive. Blood ferritin, alanine aminotransferase (ALT), and aspartate aminotransferase (AST) levels in these 4 groups were assessed.
Approximately half of the thalassemic patients (50.5%) and 27.1% of controls had TTV infection. Thalassemic patients had a greater chance of TTV infection compared to the control group with a sex-adjusted OR of 4.13 (95% CI=2.28-8.13). The increased levels of ALT, AST, and ferritin in the TTV and HCV-infected group were not significantly different from those in the TTV and HCV negative group. Co-infection with TTV and HCV did not significantly increase ALT, AST, and ferritin levels compared to infection with TTV alone.
Although common in thalassemic patients, TTV infection appears to have a negligible role in increasing the severity of liver disease, even when co-infection with HCV occurs.
PMCID: PMC3128893  PMID: 21747885
Torque teno virus (TTV); Hepatitis C virus (HCV); Thalassemia
14.  Study on Efficacy of Hepatitis B Immunization in Vaccinated Beta-thalassemia Children in Tehran 
Iranian Journal of Pediatrics  2010;20(2):211-215.
In thalassemic children, HBV infection is common, thus immunization against HBV will reduce and prevent the rate of infection. The aim of this study was to evaluate the efficacy of HBV immunization and the prevalence of HBV infection in beta-thalassemic children in Tehran.
To assess the efficacy of immunization and determine the immune response of children with beta-thalassemia, sera of 99 children who had received three doses (10/20 µg) of recombinant HBV vaccine in months 0, 1, 6, were selected and tested for HBsAg, HBsAb and anti-HBc by ELISA method. Also, these sera were tested for HBV DNA using nested-PCR method.
In 99 beta-thalassemic children, 89 (89.9 %) were anti-HBs positive (responders) and 10 (10.1%) anti-HBs negative (non-responders). 3 (3.03%) were anti-HBc positive and 1(1.01%) was HBsAg positive. HBV DNA was not detected in any of them.
Our results have revealed that hepatitis B vaccine is highly immunogenic for thalassemic children and particularly well tolerated.
PMCID: PMC3446027  PMID: 23056706
Beta-thalassemia; Vaccination; Immunization Hepatitis B; Iran
15.  Natural History of Chronic Hepatitis B Virus Infection Based on Laboratory Testing 
Iranian Journal of Public Health  2014;43(7):990-993.
Understanding of the natural history of chronic HBV infection is useful for presenting the optimal management of chronic HBV infection. The aim of this study was to evaluate the natural history of chronic hepatitis B infection.
In this cross sectional study, 219 untreated chronic hepatitis B patients from Jan. 2010 to Aug. 2012 were included. The subjects were classified in four groups based on serological, biochemical and molecular testing, Serological tests for HBeAg and HBeAb were performed by ELISA method. HBV DNA viral loads were detected by using Light Cycler instrument and ALT/AST levels were assessed by automatic analyzer.
The majority of subjects (94.1%) were HBeAg negative. Of 13 HBeAg positive patients, 5 (2.3%) and 8(3.7%) were considered as immunetolerant and immune reactive, respectively. Of 206 HBeAg negative patients, there were 142 (64.8%) patients in inactive or low replicative phase and 64(29.2%) were in HBeAg negative chronic hepatitis B phase.
The lowest rate of subjects were in immunetolerant phase and most of them had above 20 years old that confirmed successful neonatal vaccines in our country. The highest rate of chronic HBV infected patients were in low replicative phase of chronic hepatitis B infection. Although, it is not recommended to treat these patients, but liver function and also liver biopsy should be considered in patients above 40 years of age.
PMCID: PMC4401063  PMID: 25909066
Natural history; Chronic Hepatitis B; HBeAg; HBV viral load; Real time PCR

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