Tehran University Medical Journal

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Background: Rabies is an acute encephalitis that causes more than 60,000 deaths worldwide. The only way to save individuals bitten by a rabies-infected animal is the timely use of effective vaccines. Treatment with new generation vaccines is expensive. Therefore, there is a global movement towards the production of less expensive vaccines which retain and improve upon the quality and effectiveness of the vaccine. Production and evaluation of non-classical vaccines is one of the approaches taken in this regard. In this study, we describe a new eukaryotic expression system to express the nucleoprotein N gene of rabies virus which, if suitable, may be evaluated for anti-rabies vaccine production. Methods: The complete sequence of the N gene of rabies virus PV subtype was amplified by real-time polymerase chain reaction and cloned into the pCDNA3.1(+) vector. The cloned gene was excised from the vector by restriction enzyme digestion and sequenced. Due to mutations detected in the N gene, the gene coding sequence was purchased as a recombinant pGH/N vector. Vector pGH/N was amplified and following enzymatic digestion, the excised N gene was once again cloned into vector pCDNA3.1(+). Successful cloning was confirmed using restriction digests and quick check. The recombinant vector pCDNA3.1(+)/N was transformed into cultured BSR cells and protein N expression was analyzed using fluorescent antibody test (FAT). Results: Electrophoresis confirmed amplification of the nucleoprotein N gene and subsequent restriction enzyme digestion showed that the N gene had been successfully cloned into the recombinant pCDNA3.1(+)/N vector. However, DNA sequencing revealed the presence of mutations within the N gene. Restriction digest of the commercial pGH/N vector showed that the N gene had been excised from the vector. Successful cloning of the N gene into the pCDNA3.1(+) expression vector was confirmed using restriction digests and quick check. Protein expression in BSR cells was assayed by immunostaining with anti-ribonucleocapsid FITC-conjugated antibody and visually confirmed by fluorescence microscopy. Conclusion: This study showed that the protein N of rabies virus subtype PV can be expressed in a eukaryotic expression system using the pCDNA3.1(+) expression vector.

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Background: Human leukocyte antigen E is a member of non-classical HLA class I. Interaction between HLA-E molecule on the target cells and inhibitory CD94/NKG2A receptor on the cell surface of natural killer (NK) cells has an important role in the regulation of immune system against pathogens; therefore different cell surface expression of HLA-E molecule plays an important role in host resistance against viral infections as well as host response to treatment. Considering this fact, we analyzed the frequency of different HLA-E genotypes (HLA-E*01010101, HLA-E*01030103, HLA-E*01010103) in major thalassemic patients who underwent frequent transfusion therapy and are thus more susceptible to infectious diseases.

Methods: This study was a cross-sectional study of 104 major thalassemic patients who referred to Tehran Thalassemia Clinic between the years 2015 to 2016. Blood DNA was extracted and proliferated by sequence-specific primer polymerase chain reaction (SSP PCR). The PCR product was subjected to electrophoresis on 1.5 percent agarose gel then DNA fragment bands on the gel were detected by exposing to UV light. Furthermore, PCR products were also subjected to sequencing analysis for further confirmation.

Results: From 104 patients in this study, 49 (47.1%) were man and 55 (52.9%) were women. These patients were in the age range of 16 to 43 years (mean+SD; 31.03±4.7 year). The frequency of HLA-E*01010103 genotype (64.4 percent) was significantly (P= 0.001) higher than the genotypes of HLA-E*01010101 (15.4%) and HLA-E*01030103 (20.2%) whereas there was no difference between the frequency of HLA-E*0103 allele (52.4%) and HLA-E*0101 (47.6%).

Conclusion: This is the first study that examined the HLA-E polymorphisms in Iranian thalassemic patients referred to Tehran Thalassemia Clinic. This study has shown that the frequency of HLA-E*01010103 genotype was significantly higher than other genotypes of HLA-E whereas there was no difference between the frequency of HLA-E*0103 allele and HLA-E*0101 allele. Whether different frequencies of HLA-E genotype may affect thalassemic patients’ susceptibility to blood-borne infections will be of interest for future studies.

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Results: RNA sequencing results showed the number of PIK3CA (1 vs 3), FGFR3 (7 vs 6) and FGFR1 (9 vs 12) isoforms and their expression were different in bladder normal tissues in comparison to tumor tissues. Overexpression of PIK3CA gene have been observed in 42% of tumor samples but statistically was not significant. Increased FGFR3 gene (P=0.01) and decreased FGFR1 (P=0.01) expression were significant. There was an association with overexpression of FGFR3 and cigarette smoking ((P=0.037) and family history (P=0.004).

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