Effect of pentoxifylline on bcl-2 gene expression changes in hippocampus after ischemia-reperfusion in wistar rats by a quantitative RT-PCR method

Ischemia-reperfusion injury is the tissue damage caused when blood supply returns to the tissue after a period of ischemia or lack of oxygen. Ischemia-reperfusion brain injury initiates an inflammatory response involving the expression of adhesion molecules and cytokines. Twenty-four male Wistar rats [250-300 g body wt] were used in this study. The animals were divided into four groups of 6 rats each: I: Control group that was subjected to ischemia-reperfusion, II: Ischemia-reperfusion group that was subjected to all surgical procedures, III: Drug group that received pentoxifylline [200, 400 and 600 mg/kg] 60 min before and after ischemia and IV: Vehicle group that received saline. Seventy two h after ischemia-reperfusion, the hippocampus was taken for studying the changes in bcl-2 gene expression. We used quantitative real-time PCR for the detection of bcl-2 gene expression in ischemia and drug groups and then compared them to normal samples. The results showed the gene dosage ratio of 0.66 and 1.5 for ischemia group and the drug groups, respectively. The results also showed the bcl-2 gene expression declined in ischemia group as compared to the drug group. Furthermore, we observed a significant difference in the bcl-2 gene expression between ischemia and drug groups. These findings are consistent with anti-apoptotic properties of bcl-2 gene. Furthermore this method provides a powerful tool for the investigators to study brain ischemia and respond to the treatment drugs with anti-apoptotic agents

[Formulation of povidone iodine 7.5% antiseptic foam and evaluation of its antimicrobial effect]

Iodine and Iodophore have been longley used as antiseptic to prevent infections with good efficacy and safety. The aim of this study was to formulate new povidone iodine 7.5% antiseptic foam and evaluate its antibacterial effect. In this experimental study, in the first step, the effects of the foaming agents including Sodium Lauryl Ether Sulfate [SLES], Sodium Lauryl Sulfate [SLS] and Nonoxynol 9 were studied and then the effect of pH and different buffer formulations including citrate, phosphate citrate and phosphate buffers on the organoleptic properties and the amount of the free iodine were evaluated according to the USP 32 criteria. The effects of the emollient such as glycerin and glycerox HE were evaluated and the accelerated stability test was taken for optimum formulations. Finally, the antimicrobial effect of the proper formulation was evaluated. SLES 3% in Citrate buffer with the pH 3.5 showed the best results for the quality of the foam and glycerin 1% was selected as proper emollient agent in presence of poloxamer 407 as foam stabilizer. The stability test was done on the superior formula and it proved its antiseptic properties. The foam formulation contained povidone iodine 7.5%, SLES 3%, Poloxamer [407] 2%, citrate buffer and glycerin 1% has passed stability as well as and antimicrobial effects tests; therefore, it can be used as a new antiseptic product and an appropriate detergent to prevent spreading of infectious disease

Cloning, expression and purification of penicillin binding proteinla [PBP2A] from methicillin resistant staphylococcus aureus: a study on immunoreactivity in BALB/C mouse

Staphylococcus aureus [S. aureus] is a major nosocomial pathogen and the infection with this organism in human is increasing due to the spread of antibiotic resistant strains. One of the resistance mechanisms of 5. aureus comprises modification in binding proteins to penicillin. Vaccine strategy may be useful in controlling the infections induced by this organism. This study aimed at developing and producing the recombinant protein PBP2a as a vaccine candidate and evaluating the related humoral immune response in a murine model. A 242 bp fragment of mecA gene was amplified by PCR from S. aureus COL strain and then cloned into prokaryotic expression vector pET-24a. For expression of recombinant protein, pET24alpha-mec cplasmid was transformed into competent E.coll BL2l [DE3] cells. Recombinant protein was over expressed with 1 mM isopropythio-beta-D-galctoside [IPTG] and purified using Ni-NTA agarose. SDS-PAGE and western blotting were carried out to confirm protein expression. For immunization of experimental groups, Balb/c mice were injected subcutaneously with 20 pg of recombinant PBP2a three times with three weeks intervals. The sera of experimental groups were collected three weeks after the last immunization and then specific antibodies were evaluated by ELISA method. Successful cloning of mecA was confirmed by colony-PCR, enzymatic digestion, and sequencing. SDS-PAGE and western blot analysis showed that recombinant protein with molecular weight of 13 kDa is over expressed. In addition, high titer of specific antibody against PBP2a in vaccinated mice was developed as compared with the control group and confirmed the immunogenicity of the vaccine candidate. Results suggest that PBP2a recombinant induced specific antibodies and can be used as Staphylococcal vaccine candidate after further studies

Study of 5 HT[3] and 5 HT[4] receptors expression in HT29 cell line and human colon adenocarcinoma tissues

Serotonin [5HT] has been shown to be a mitogenic factor in several carcinomas. Its mitogenic effect is elicited through a wide range of 5HT receptor subtypes. In this study, the effects of 5HT, 5HT[3] [1-phenylbiguanide hydrochloride] and 5HT[4] [cisapride] agonists in promoting the growth of the HT29 cell line and the growth-inhibition effect of the 5HT[3] receptor antagonist [Y-25130 hydrochloride] and 5HT4 receptor antagonist [RS 23597-190] were investigated. The expressions of 5HT[3] and 5HT[4] receptors in human colon cancer tissues and the HT29 cell line were studied. The growth-promoting and growth-inhibition effects of 5-HT, 5HT[3] and 5HT[4] agonists and antagonists on the HT29 cell line were studied using MTT assay. Receptor expression has been demonstrated by western blotting. The results showed that 5HT, 5HT[3], and 5HT[4] agonists caused significant proliferation of HT29 cells. 5HT[3] and 5HT[4] receptor antagonists had an inhibitory effect on the growth of these cells. Western blot analysis gave bands from colon tissue extracts and the HT29 cell line. The results indicate which 5HT[3] and 5HT[4] receptors are significantly expressed in both colon cancer tissue and the HT29 cell line. Expression for the 5HT[3] receptor is more potent. Furthermore, 5HT plays a mitogenic role in colon cancer cells and antagonists of 5HT[3], and 5HT[4] receptors can inhibit cancer cell growth

Evaluation of potency of measles vaccine used in Iran: comparison of WHO and NIBSC method in cell culture

Measles has been a major cause of illness and death in children and vaccination against the disease is part of the WHO global immunization program. A suitable vaccine should create maximum immune response against the pathogen and must be safe for the user. Thus, after production, vaccines must be analyzed and controlled by the producer and confirm by relevant governmental organizations. The Food and Drug Control Lab [FDCL], Ministry of Health, is the secondary control center on potency of vaccines in Iran. In this study, we have set up the WHO and NIBSC methods in FDCL and compare these methods on determining the potency of measles vaccine. Measles vaccines were obtained from Razi Institute Iran. Nine dilutions of vaccine [10[-1] to 10[-5]] in 0.5 log interval were mixed with Vero cell suspension and seeded. In WHO method, the cells were incubated at 36°C for 10 days, during which the cells were checked for cytopatic changes everyday. To set up the assay, we tested the vaccine dilution with four different cell suspensions [2x10[5]-5x10[4]/well] and four different concentration of serum [2.5-10%]. Based on our results, in the assays, 5% serum and 1x10[5] cells were used. The potency assay was performed with six different vaccines produced in one batch and the mean potency for Measles was 10[4.32 +/- 0.24] CCID[50]/vial for a ten-dose vial. In NIBSC method following seeding of Vero cells, the medium was removed after 3 hours and overlay was added. Then the plates were incubated at 35°C for 10 days. After incubation period, the overlay was removed, the plaques were stained with methyl violet and counted. This assay was repeated three times and the mean of the results was 5.83 +/- 0.03 log[10] PFU/dose. In this study, we have set up the WHO and NIBSC methods and results indicated that the potency of the vaccine is in acceptable range in either method. Furthermore, the WHO method is simple and less time consuming compared to NIBSC which is complicated and requires more effort to produce reproducible results