Journal of Pathobiology Reaearch
Volume:10 Issue: 3, 2008

β-thalassemia is caused by absence or reduction of β-globin chain synthesis. One of the effective therapeutic methods for this disease can be gene therapy by viral vectors. The capacity of lentiviral vectors is approximately 8 kb, we designed a 6 kb construct containing mini LCR and β-globin gene instead of LCR region. The aim of this study is to make a recombinant lentiviruses containing miniLCR and β-globin gene for transfer to the target cells for gene therapy of β-thalassemia. HS2, HS3, HS4 segments (mini LCR) and β-globin gene with 5΄ and 3΄ UTR were amplified from the genomic DNA of a normal individual by PCR. Each segment was cloned in pTZ57R/T vector and then sub cloned first into the pBGGT vector and finally into the pLenti-Dest vector. Final transfer vector and the three helper packaging plasmids (Plp1, Plp2, Plp/VSVG) were cotransfected into 293T packaging cells using lipofectamine 2000. Harvested viruses were confirmed by RT-PCR on extracted RNA of these recombinant lentiviruses. The titer of lentiviral stock determined in a K562 cell line and compared with COS-7 cell line. The titer in both cell lines was the same. Optimum MOI for COS-7 cell line was 5 and when polybrene was used transduction increased by 2 fold. The remaining transduced COS-7 colonies were expanded and DNA was extracted. By PCR, random integration of construct into the genome was evaluated. The produced lentiviruses can be an appropriate means for effective transfer of the designed construct into dividing and non-dividing cells such as hematopoetic stem cells for transplantation of beta thalassemia patients. Efficiency of transduction by leniviruses is more than the gene targeting technique. Also units of HS2, HS3 and HS4 regions in mini LCR and selection of larger HS3 unit may increase the expression of beta globin gene.

This study was conducted to evaluate the frequency of human hydatidosis in Kurdestan province by ELISA technique. In this study the sera of 1979 individuals were collected from different area (cities and villages) of Kordestan province. The serum dilution of 1/400 was selected for ELISA test. The results indicated that 1.12% of the individuals from Kordestan province showed positive sera. The results also showed that in Kordestan 0.9 % and 1.42% of the people who live in the cities and villages had positive sera respectively. In this study 1.65% of female and 0.45 % of male were positive. From the obtained result we found maximum number of infected people were in the range of 30-40 years (1.59%). According to the results obtained from this study the highest percent of infection was found in the city of Ivandarre, the reasone for this defference (1.69%) is due to the fact that most of the people who are involved in animal husbendary in the province live in this city.

Clinical application of embryonic stem (ES) cells faces difficulties regarding tissue rejection as well as ethical limitations. One solution for these issues is to reprogram somatic cells by the injection of their nucleus into an enucleated oocyte or zygote. However, technical complications and ethical considerations have impeded the therapeutic implications of this technology. An approach which is most recently developed is in vitro induction of reprogramming in adult cells. This was first achieved by using four transcription factors, including Oct4, Sox2, c-Myc and Klf4. Subsequently, many ongoing efforts were performed for enhancing this method, also for making it compatible with clinical applications. However, there is still a long road ahead. In this paper we review strategies to reprogram somatic cells to embryonic state and discuss about the recent strategy and the relevant developments.

The global HIV epidemic continues to expand and exceeding previous predictions. An effective vaccine represents the best hope to curtail the HIV epidemic. DNA vaccines induce humoral and cellular responses and mimic live vaccines without their pathogenic potential. The importance of CD8+ CTL responses in controlling HIV and SIV viremia has led to production of a series of vaccine candidates that effectively induce these responses. It is now widely believed that an HIV vaccine strategy must stimulate both a strong humoral (antibody) as well as cell-mediated (CTL) immune response.The p24 and gp41 play many important roles in host-virus interaction and pathogenesis. These proteins are considered as attractive vaccine candidate in which their immunogenecity and immunomodulatory effects have been confirmed. In this study, a construct, pcDNA3.1Hygro- (p24-gp41), was evaluated as a DNA vaccine candidate in Balb/C mice for generation of effective cellular immune responses. For immunizing, we used dendrosome, a novel family of vehicles for transfection and therapy. IFN-γ cytokine production and total antibody were detected by ELISA. Lymphoprolifration assay was performed by MTT test. ELISA and MTT assays confirmed that the cited p24-gp41 fusion gene is able to enhance immune responses in mice. The construct that was used in this research can be a good candidate for DNA vaccine against HIV-1, if the future complementary tests demonstrate the same trends of immunogenic responses shown in this study.

In peripheral blood polymorphonuclear and mononuclear cells Nitric Oxide (NO) could be synthesized by an enzyme called inducible NO synthase (iNOS). iNOS gene (NOS2A) is located on chromosome 17 at position 17q11.2-q12. NO is released during inflammatory responses. In the present studies the frequency of NOS2A gene polymorphisms and their effects on NO production were investigated in the Peripheral Blood polymorphonuclear and mononuclear cells of normal individuals. In this study the frequency of NOS2A gene polymorphisms at positions -1659 C/T and +150 C/T of 232 normal subjects were investigated using PCR-Allele specific and PCR-RFLP methods, respectively. To study the effect of -1659 C/T and +150 C/T polymorphisms on NO production, polymorphonuclear and mononuclear cells of peripheral blood of 92 normal subjects were isolated and then stimulated by E.coli culture supernatants (ATCC 25922) for induction of iNOS enzyme and NO production. After 24h, the level of NO production in the culture supernatant were measured by Griess reaction. Polymorphisms as mentioned above were also studied in these normal cases. The results showed no significant difference in the level of NO production of various genotypes in the polymorphonuclear and mononuclear cells of peripheral blood of normal subjects. Our results indicated no significant correlation between NOS2A genotypes and NO production. No significant difference was observed between Gambia and China normal population and normal subjects of this study in NOS2A -1659 C/T and +150 C/T polymorphisms. In Iran these differences are due to the genetic and ethnic differences among the studied populations, which indicates the importance of NOS2A polymorphism in the NO production, suggesting further studies in other ethnic groups.

Chronic Myeloid Leukemia (CML) is a malignant clonal disorder of hematopoietic stem cells which results in increase of myeloid cells, erythroid cells and platelets in the peripheral blood and hyperplasia in bone marrow. Investigations have shown different types of BCR- ABL variants in these patients. In Iran only 3 types of these variants have been identified because most of the clinical laboratories usually use only few sets of primers which can not detect all types of the variants simultaneously. In this study we developed a method with which all types of variants in chronic Myeloid leukemia patients can be recognized. blood samples from 100 persons who were under treatment or diagnosis for CML were received from Clinical laboratory. RNA was extracted from 600 µl of each sample using Roche Commercial kit and converted to cDNA by reverse transcriptase enzyme the cDNAs were analyzed for BCR- ABL variants using two sets of primers. All samples were also studied by RT- Multiplex Nested PCR method. RT-Multiplex PCR could detect BCR-ABL in all samples which were positive for these Fusion Gene mRNA. From 100 collected samples 46 percent were positive and 54 percent were negative by RT-Multiplex PCR method and 44 percent were positive and 56 percent were negative by RT-Multiplex Nested PCR method. By using one step PCR we detected more variants of BCR- ABL in one tube at shorter time and lower cost. This method showed 100 percent specificity and can further be improved by taking more samples and also real time PCR for quantitative analysis.

Anti-cyclic citrullinated peptide (anti-CCP) antibodies are highly specific markers for rheumatoid arthritis (RA) and useful for predicting rheumatoid arthritis (RA) development and progression. We assessed the diagnostic value of anti-CCP antibodies in our patients with RA. Anti-CCP antibodies and rheumatoid factor (RF) titers were determined in 247 serum samples: 128 from RA patients, 119 from control group (48 from healthy controls, 71 from patients with rheumatic disease other than RA or hematologic malignancies). There were 128 (93 females, 35 males) patients with rheumatoid arthritis and 119 (78 females, 41 males) controls. The sensitivity of Anti-CCP was 66.40% for diagnosis of RA with a specificity of 94.11%. The sensitivity of RF was 69.53% and its specificity was 81.51%. In our patients Anti-CCP has a moderate sensitivity but high specificity for diagnosis of RA.

An entomological survey was carried out for Leishmania vector incrimination of sand flies in northwestern Iran. Among other specimens, 358 sand flies belong to the Sergentomyia Genus were tested for leptomonad infection using semi-nested PCR method as well as sequence anlalysis of ITS-rDNA fragment. Results of semi-nested PCR against kietoplast DNA showed reptile leptomonad infection in two specimens of S.dentata. The ITS2 sequence analysis of the specimens revealed 76% identity with those of Leishmania (sauroleishmania) adleri of Genbank. However, further studies need to clarify the species identity of the leptomonads. Interestingly, blood meal analysis of the sand flies determined an S.sintoni specimen with mammalian hemoglobin. This reptile related sauroleishmania parasites lacks the Lipophosphoglican (LPG) necessary for entrance to human phagocytes cells, and hence are not human pathogen. However, the GlycoInositoPhosphoLipid (GIPL) molecules of this parasite reacts with sera of kala-azar patients and may cause false positive scores in sero-epidemiological surveys for kala-azar. Sauroleishmania can be transmitted to human by infected bite of some Sergentomyia subgenera that show intermediate

Among the members of legionellaceae, Legionella Pneumophila is involved in 95% of cases of severe pneumonia. Isolation of the causative agent from bronchoalveolar lavage (BAL) fluid specimen is a delicate process and also time-consuming. Moreover, it has been shown that some Legionella strains may be viable but cannot be cultured. The aim of this study was comparison of culture and PCR for detection of Legionella pneumophila from bronchoalveolar lavage (BAL) fluid specimens In this study, 70 BAL fluid specimens were collected from patients suspected to Legionnaires’ disease. These samples were cultured on selective buffered charcoal-yeast extract agar (BCYE) and then tested with specific L. pneumophila primers for mip gene. Among 70 BAL samples, three (4.2%) were positive with culture and six (8.4%) of specimens were positive by PCR. The three culture positive samples were all positive after specific DNA amplification. Among 63 culture-negative samples, 3 were positive after amplification. The clinical features of the patients were in accordance with legionellosis. The accurate diagnosis of Legionella pneumophila has an important implication for the treatment of infection. Analysis of the results showed that PCR is faster and more sensitive for isolation and identification of L. pneumophila to apply on BAL fluid specimens than culture. Therefore, specific Legionella PCR can be a good option for isolation and identification of Legionella pneumophila from bronchoalveolar lavage (BAL) fluid specimens in patient of severe pneumonia

Nucleostemin (NS) is a recently identified gene that is expressed mainly in the nucleoli of neuronal, embryonic and mesenchymal stem cells which plays a role in their self renewal. Over expression of this gene has been reported in several cancer cell lines tumoral tissues including gastric, hepatic and prostate cancer. In this study, we investigated the effects of suppression of this gene by RNAi technique in a human bladder cancer cell line. After confirmation of expression of nucleostemin in the bladder cancer cell line 5637, 21-mer oligoes against NS were transfected into cells and suppression of NS was confirmed by RT-PCR, immunocytochemistry (ICC) and western blotting. Proliferation assays were done by counting the cell numbers. Changes in cell cycle and apoptosis induction were assessed by staining cells with Propidium Iodide and Annexin V-FITC respectively. Our results revealed that nucleostemin is highly expressed in 5637 carcinoma cells. Expression of NS, by semi-quantitative RT-PCR, after treatment with specific oligoes against it, decreased around 85% in comparison to control groups. In addition, ICC and western blotting further confirmed the suppression of nucleostemin at the protein levels. Analysis of cell proliferation assays showed a considerable decline in the number of cells, specially, 72 h after treatment of cells with oligoes against NS. Cell cycle analysis after NS suppression showed increase of sub-diploid events, characteristic of apoptotic cells that were confirmed by staining cells with Annexin V-FITC dye. It seems that nucleostemin expression plays an important role in the induction of cell proliferation as well as inhibition of apoptosis occurrence in 5637 cells. Further studies focusing on suppression of this gene in vivo, may help to find potential therapeutic strategies to cure bladder cancer.

E1A oncoprotein of adenovirus type 5 is a regulatory factor which controls transcription of other adenovirus genes. This protein promotes both viral genom replication and host cell transformation by altering the function of certain important cellular proteins such as p21 and Rb. The aim of the present study was to constantly reduce the expression of E1A gene in HEK 293 cell line by RNAi technique in order to analyse the effects of this suppression on these cells. The U6 promoter and shRNA regions from the control and E1A specific siRNA coding plasmid as pSP-81 and pSP81-E1A were subcloned into pcDNA3.1. Then these constructs were transfered into the HEK 293 cancerous cells using lipofection method and successfully transfected cell colonies were selected based on neomycin antibiotic resistance. Changes in E1A gene expression were analysed by RT-PCR technique after selection process. Final analysis showed no obvious difference in E1A gene expression level in the suppresed and control groups, upon transfection with the constructed plasmids. In order to examine the possible influence of cloning procedure on the function of U6 promoter, cells were transfected with Dr. Hacker’s original plasmids, but no inhibition of E1A gene expression was observed again. The results of sequencing revealed existence of a mutation in the siRNA target region for the E1A gene sequence. These results illustrated that no considerable suppression has been occurred by repeating Dr. Hacker’s expriment, even with application of very effective lipofection method. To examine the sequence of the E1A gene, the PCR product of the 13s region of the gene was sequenced. Sequencing revealed existence of a point mutaion in the siRNA target region. It seems that observed impaired interference could be attributed to this mutation in the E1A gene of the studied cells.