Pathobiology Research
Year:2011 | Volume:13 | Issue:4|Papers Count:9

Objective: Enterotoxigenic Escherichia coli is considered as the most important agent of children diarrhea and mortality in developing countries. This bacterium causes 300-600 thousands of deaths in the children under 5 years of age per year. With difficulties in treatment as well as its wide prevalence, designing an effective vaccine against this microorganism is the objective of world Health Organization (WHO). The CfaB protein as immunogen and major subunit of fimberia has a critical role in the bacterial attachment to small intestine epithelium and the produced antibody against this protein can prevent attachment of bacterium to epithelial surface. Hence, this molecule alone or with other virulent factors has been considered by many researchers in vaccine designing. In this study, expression of colonization factor B with the aim of studying the immunogenesity of this protein as a component of vaccine candidate was performed.Materials and Methods: cfaB gene was amplified by PCR and cloned into pET28a and its expression was evaluated. Since there was no expression, which was due to presence of rare codon, the cfaB gene was again cloned into pET28a using codon bias in E. coli and subsequently expressed.Results: Presence of a 20KD band on SDS-PAGE gel indicated the expression of CfaB protein, which was later confirmed by immunoblotting with anti-His tag and anti CfaB antibodies and purified on Ni-NTA column.Conclusion: Codon optimization and expression in heterologous hosts is a useful approach for obtaining large quantities of recombinant protein.

Objective: Osteon has been introduced as a bone substitute material. It is biphasic calcium phosphate (BCP) that use in dentistry. The aim of the present study was to evaluate the effect of osteon on the proliferation, cell viability and differentiation of saos-2 cells in vitro. Also it was compared with cerasorb bone graft.Materials and Methods: Two different bone grafts materials, osteon and cerasorb, were used to evaluate the effect on proliferation and differentiation rate of saos-2 cells. On day 15 the cell proliferation and cell viability was measured by MTT assay. For determination of differentiation, alkaline phosphatase and alizarin red test was used.Results: Osteon and cerasorb groups showed significantly higher alkaline phosphatase activity and cell differentiation. Cell viability of both bone grafts was significantly lower than control group and cell proliferation was higher in osteon group. Osteon has more suitable biological property compare to cerasorb.Conclusions: The results of the present study showed that osteon and cersorb bone grafts allow proliferation and differentiation of saos-2 cells in vitro.

Objective: In this project, our aim was to construct a novel expressing vector harboring a new sequence from overlapping region of NS3 gene of HCV from infected Iranian patient.Materials and Methods: The partial NS3 (pNS3) gene was amplified by Nested-RT-PCR method using sera of HCV infected patients harboring genotype 1a. After purification and cloning the pNS3 into TA-cloning vector, the best colony was selected based on Blue/White screening and colony-PCR following by confirmation with sequencing and restriction digestion with BglII. The sequenced gene was compared with other reference sequences using alignment softwares. The resultant pNS3 gene subcloned into the expression vector, IRES vector, followed by selection the suitable clones by 2 different colony-PCRs. The gene expression was evaluated using GFP detection, RT-PCR and western blotting techniques after transfection of the IRES-pNS3 vector into the 293 cell line.Results: After pNS3 sequence amplification by RT-PCR, sequencing results showed high homology among the sequences with other reference sequences. This result also showed that it belonged to genotype 1 of HCV. Colony-PCR showed the insertion of gene into expressing vector with the right orientation. GFP expression, RT-PCR and western blotting confirmed transfection of vector, expression of pNS3 gene and production of its protein in 293 cells respectively.Conclusion: This novel expressing vector harboring partial region of NS3 gene in compare to full NS3 gene maybe more useful in immune induction by antigen presenting cells due to absence of genes responsible for protease activity of the protein in the setting of HCV vaccine.

Objective: Cell vital function has correlation with mechanical loadings that cell experiences. Here, effects of in-vitro combined cyclic-static stretch on proliferation of human mesenchymal stem cell (HMSC) were evaluated.Materials and Methods: HMSCs were cultured on gelatin coated elastic membranes, and exposed to stretch loading. Four different regimes of cyclic, static, combined cyclic-static, and cyclic with a period of unloading were exerted on the elastic membrane. Duration of cyclic loading and static loading was 5 and 12 hours respectively.Results: The results illustrate that 10% cyclic stretch causes cell alignment but there were no significant proliferation differences between control and test group. Combined cyclic-static stretch reduced proliferation significantly while cyclic stretch with an unloading period increased cell proliferation significantly. At last, static stretch did not affect cell proliferation significantly.Conclusion: Cell stretching regimes and post-loading duration are effective factors on cell proliferation.

Objective: Survey of molecular characterization of nucleoprotein gene of H9N2 avian influenza viruses and determination of the genetic relationship of Iranian H9N2 viruses and other Asian viruses.Materials and Methods: The nucleoprotein (NP) genes from 4 isolates of H9N2 viruses isolated from commercial chickens in Iran during 2008-2009 were amplified by RT-PCR method and sequenced. Nucleotide sequences (orf) of the NP genes were used for phylogenetic tree construction.Results: Nucleotide sequence analysis of the NP gene showed that the Iranian virus isolates did not exhibit insertions or deletions within nucleoprotein (NP) gene as compare with their prototype A/turkey/winconsin/66; however numerous point mutations were occurred in the gene length of these viruses similar to the previous Iranian strains. Nucleotide sequence analysis showed that these isolates are very closely related (96/7-99/6) and shared a homology of 91/9-92/3% and 91/5-92% with 2 human isolates A/HK/1073/99 and A/Hk/2108/2003, respectively. Phylogenetic analysis of the NP gene showed that all the NP genes of the Iranian H9N2 viruses fall into a single group within a G1-like sublineage which had contributed as donor of six internal genes to H5N1 a highly pathogenic avian influenza.Conclusions: The current study indicated that the NP gene of H9N2 influenza viruses circulating in Iran during the past years were well conserved. It seems that the differences between these Iranian virus isolates are the result of accumulation of point mutations among them.

Objective: Evaluation of the effects of different doses of antioxidant Taurine on oxidative stress and human sperm parameters following cryopreservation.Materials and Methods: The semen of 20 fertile men were divided to 5 aliquot, one part considered as a fresh after analysis of standard semen parameters (Motility, Abnormal Morphology, Viability) and Protamine deficiency, DNA damage and measurement of ROS and RNS. The other part was loaded on to a 80% and 40% Allgrad gradient and centrifuged. The pellet was washed and divided into 4 separate fractions for control (non-frozen) and cryopreservation groups in absence or presence of 0.25 and 50 mM Taurine. The frozen specimen were thawed and then examined. Sperm Motility evaluated using a CASA software. The Viability of spermatozoa was assessed by the Trypan-Blue stain method. Levels of ROS determined by spectroflorometry assay using DCFH-DA. DNA fragmentation examined by SCD test and Protamine deficiency examined by CMA3 + staining. At the end results were analyzed using ANOVA test.Result: Cryopreservation procedure increased the amount of ROS and adding 25 mM of Taurine improved post-thaw motility, progressive motility and sperm protamine deficiency. However, different doses of Taurine (25 and 50 mM) had no significant effects in total abnormalities, viability, DNA fragmentation and ROS reduction.Conclusion: Antioxidant Taurine has no significant effects on ROS production following human sperm cryopreservation. But with dose of 25mM could improve the quality of spermatozoa due to the assessment of motility and protamine deficiency.

Objective: Helicobacter pylori is to blame for one of the most common chronic infections in humans and can cause peptic ulcer, gastritis, duodenitis and non-ulcer dyspepsia. Developing a reliable test to detect the infection is of great importance. For a variety of reasons, the existing tests including the stool culture, biopsy, PCR, and urea breath test are not up to par. The PCR-ELISA test is a preferably specific and sensitive approach to detect Helicobacter pylori in stool specimen and biopsy.Materials and Methods: Sixty-seven stool samples were collected from 127 patients who had undergone gastrointestinal endoscopy and stomach biopsy at Hazrat Rasool Akram Hospital, of Iran University of Medical Sciences based in Tehran. DNA was extracted from the samples before they were subjected to PCR of ureC gene. PCR-ELISA test was conducted and the results were compared.Results: Stool-PCR test 31 (46.1%) of the 67 patients in question were positive and the same test on biopsies showed that 34 patients (50.7%) were infected while the rest tested negative. But the PCR-ELISA test on stool samples suggested that 42 patients (62.6%) were infected, and the same test on biopsies showed that 47 patients (70.1%) were positive.Conclusion: The results of these tests showed that detection of Helicobacter pylori through PCR-ELISA test on stool samples has high specificity and sensitivity and could be used as initial test to detect Helicobacter pylori infection.

Objective: Toxoplasmosis infection has spread throughout the world, it is created by the intracellular protozoan Toxoplasma gondii. Toxoplasma cause changes in the behavior of the rodents. Rodents play an important role in the life cycle of the Toxoplasma gondii, they are the main infection reservoir of the domestic cats. The purpose of this study was to investigation Toxoplasma infection in the Tehran's Rats.Materials and Methods: In this study, 150 mice were collected from all 5 regions of Tehran (North, South, East, West, Center) by live traps over 8-months period. For infection detection, anti-rat conjugate was used in ELISA method.Results: The results showed that 36/7 percent of rats in Tehran have Toxoplasma infection. Maximum number of infected rats were found in the South and Central parts of Tehran with 11.7 percent and with minimum of 1.47 percent in the West of Tehran.Conclusion: The extent of infection indicates the importance of these wild rats in the persistence and life cycle this parasite.