معصومه بخشایش

The protozoa of leishmania lead to cutaneus and visceral leishmaniasis. Activation macrophages phagocyte parasites and this mechanism can be done with production of active radicals oxygen and nitric oxide، That is done through immunomodulator، as a basic process for production of the new drug. In this study، the effect of garlic extract as an immunomodulator on J774 cells infected with Leishmania major and iNOS gene expression and IFNγ has been investigated. Leishmania major promastigotes (MRHO/IR/75/ER) were added to the in-vitro cultured macrophages (J774 cells) and these cells were incubated for 72 hours. Various concentrations of garlic extract (9. 25-18. 5-37-74-148 mg/ml) were added to the infected cells. MTT assay was applied for cellular prolifration. After 72 hours of incubation، supernatants were collected and total RNA was extracted from the infected cells. The expression of IFNγ and INOS genes were studied by RT-PCR method. Untreated sample with garlic was used as control. All the experiments were repeated at least three times، and representative results were analyzed. Statistical significance (P < 0. 05) was analyzed by Student’s t-test using SPSS version16. The result of this study، using MTT method showed that، the IC50 concentration of garlic extract was 37 mg/ml. Also the expression of IFNγ and iNOS genes by RT-PCR indicated that garlic extract lead to rise of expression of these genes in the J774 cells infected with L. major. Based on the findings and importance of the cellular immunity cytokines in developing and importance of the responses، the hypothesis that the effect of AGE in cellular immunity is due to rising the IFNγ cytokines and expression of iNOS genes is confirmed.

Multiple sclerosis(MS) is the most common inflammatory disease of central nervous system which is caused by an autoimmune process leading to destruction of myelin sheath. In this disease, CD4+ T–lymphocytes, mostly of TH1 phenotype, play important roles in destruction of neuronal tissues. Because of the probable etiologic participation of EBV virus in immunopathogenesis of MS, the aim of this study was to evaluate the relationship between serum levels of anti-Epstein Barr virus antibodies and production of TH1 and TH2 cytokines.Patients and A descriptive study was performed on 68 MS patients in different stages of the disease and 20 healthy individuals. Blood samples were taken and plasma levels of anti-EBNA-1 and VCA antibodies were determined by ELISA method. Then, peripheral blood mononuclear cells (PBMC) were isolated by Ficoll Hipaque separation and stimulated with PHA in optimal culture conditions. Concentrations of IFN-γ, IL-12 and IL-4 in culture supernatants were then measured by ELISA. Statistical analysis was performed by using SPSS version 16. Mann-Whitney, t-test,and Pearson correlation coefficient were used to compare the variables and find the correlation between them. P<0.005 was considered statistically significant. The mean levels of anti EBNA-1 and VCA antibodies were significantly higher in patients compared to controls (P=0.04, P=0.001 respectively). Concentrations of IFN-γ, IL-4 and IL-12 were also significantly higher in MS patients than healthy individuals (P=0.001, P=0.005, p=0.002 respectively). Although the ratios of IFN-γ to IL-4 and IL-12 to IL-4 were higher in MS patients than healthy individuals, this increase did not reach a significant level (P>0.05). A statistically significant correlation was found between anti EBNA-1 and VCA antibodies and IL-12 production (P =0.02, r=0.27& P=0.04, r=0.25 respectively) whereas no significant correlation was found between these antibodies and production of IFN-γ and IL-4 in MS patients. The significant correlation between the level of anti-Epstein Barr virus antibodies and IL-12 production in MS patients implies the probable role of this cytokine in skewing immune responses toward TH1 phenotype. It is possible that this putative viral agent in combination with other etiological agents is involved in the etiology and progression of the disease.

Discovering the drug mechanisms in visceral leishmaniasis is very important to confirm treatment methods. The direct effect on the cell membrane and induction of necrosis are the causes of side-effects in conventional drugs like Glucantime. Therefore, if a drug induces apoptosis but not necrosis in leishmania, we could conclude that the drug is more effective. The aim of this study was the assessment of miltefosine on cell death mechanisms and the apoptosis of standard strain of lishmania infantum in Iran.

The cytotoxic effect of miltefosine was studied with colorimetric assay (MTT) and the promastigotes apoptosis was studied with flow cytometry. In summary, 105 leishmania infantum promastigotes treated with IC50 dose of miltefosine, and control cells were added to binding buffer with FITC conjugate, annexin V and propidum iodide (PI). The cells were studied with flowcytometry and the results were analyzed with cell quest program. Morphology and cell size of promastigote were studied.

IC50 of Miltefosine were calculated 7-8 μmol. After a 24-hour incubation of treated cells with miltefosine, 22% were annexin-V FITC positive but only 2% of control cells were annexin-V FITC positive. After 48 hours the percent of apoptotic cells increased (80% annexin-V FITC) whereas no change was detected in control group.

Initially, it was assumed that apoptosis emerged with metazoan. Recently, several studies have demonstrated that a process of apoptosis also operates in eukaryotic organisms including various species of leishmania. Studying the precise mechanism of cell death pathway and induction of death in leishmania treated with miltefosine is very important for planning effective treatment strategies.

Molecular basis of Acute Myeloid Leukemia (AML) involves mutations in regulatory genes of cellular proliferation and differentiation.Mutation in tyrosine kinase receptor gene of FLT3 occurs in high frequency in AML, resulting in proliferation and abnormal survival of leukemia cells. Mutations in Internal Tandem Duplication (ITD) and D835 of FLT3 gene are associated with poor prognosis. The aim of this study was to diagnose and determine the frequency of this mutation in AML. This descriptive - observational study was performed on 101 AML patients for mutations in exon 11,12 and interon 11 of ITD and D835 mutation in exon 20 of FLT3 receptor gene using PCR. Mutation in ITD was observed using PCR products run on acrylamid gel 8% and compared to marker. PCR products of D835 mutation on genomic DNA were studied using ECORV restriction enzyme and RFLP technique. ITD mutation was observed in 18% of AML studied patients with differences in subgroups of FAB. Also 6% of the patients showed D835 mutation with difference in subgroups of FAB.This study revealed that mutations in FLT3 gene occurr in substantial number of AML patients. Therefore, molecular diagnosis of these mutations prior to treatment leads to a better decision making for the therapeutic protocol.

So far there is no effective drug therapy to prevent neuronal loss after brain stroke. In the present study we studied effects of The Mitochondrial K-ATP channel regulators on neuronal cell population and neurological function after ischemia reperfusion in the rat. Rats temporarily subjected to four vessels occlusion for 15 minutes followed by 24 hours reperfusion with or without K-ATP channel regulators. Neurological assessment was conducted after 24h reperfusion by consciousness scoring and motor function according to the methods reported by Lemay and Tarloe.After tissue processing and crysel violet staining total and normal cell number was assessed in parietal cortex.Data was analyzed by ANOVA software. The normal cell count of neuronal population significantly increased in the K-ATP channel opener (Diazoxide) treated ischemia-reperfusion group compared with the control group. Cell count and neurological function scores were dependent to dose of K-ATP channel regulators in vivo. Our results showed that Diazoxide treatment leads to better preservation of cortical neurons in rat.

Apoptosis or programmed cell death (PCD) is an important mechanism in both development and homeostasis in adult tissue for the removal of superfluous cells, while its induction is an effective therapeutic approach for cancer. The aim of the present study was to evaluate the effect of cloned gene-related protein Vp2 of infectious bursal disease virus in human lymphoma B cells. In present study after cloning the Vp2 gene in Pichia pastoris system, the Vp2 protein was expressed. Cellular vital capacity was determined by MTT. Then effect of Vp2 protein in human lymphoma B cells was examined by Hoechst staining and flowcytometry techniques, respectively. During MTT, human lymphoma B cell lines revealed to have a meaningful apoptosis at 1mg and 5mg protein concentrations when compared with controls (p<0.01). Apoptotic bodies appeared by Hoechst staining apoptosis was induced suitably after 48 hours by flowcytometry assay. The present study is the first study that has revealed the gene-related protein Vp2 induced apoptosis in human lymphoma B cells in vitro.

Vision is one of the most important organs in the body. Damage to this organ causes a severe disability in humans. In retinitis pigmentosa, the degeneration of photoreceptors causes blindness. So far, more than 100 mutations have been detected in photoreceptors, which they result in opsin malfunction. The aim of present study is to differentiate the hippocampal stem cells of rat to the rod photoreceptors. Stem cells of the hippocampus were obtained from rat embryos, 18 days of age (E18). Pregnant female rats were killed and the head of their embryos were separated. Then, the embryos’ hippocampus was removed according to Banker’s method. Hippocampal cells were dissociated by Fish Bach’s method. The cells were cultured in flasks (25cm2). After 3 days, the cells were isolated by trypsin, counted using trypan blue and hemocytometer. Cell suspensions were prepared in two cell concentrations; high (2×105cells/l) and low (2×104 cells/l) concentration, then, cultured using DMEM/F12 supplemented with fetal bovine serum 10% (FBS) in six wells plates. Prior to culture of the cells, the first and second row of plates were coated with poly L-lysine and inactivated astrocytes, respectively. Following incubation of the plates at 37C for 4 days, different concentrations of All Transe Retinoic Acid (ATRA) and 9–CIS RA were added daily for 6 days, and finally immunocytochemistery was carried out using anti-opsin monoclonal antibody. The results of current study showed that the plates, which are respectively treated with ATRA and 9-CIS RA in a concentration of 500nM and 100nM for 6 days had the most differentiated cells. In addition, maximum differenced cells were observed with 100nM of 9-Cis RA. The differentiated cells were detected in wells, which were only coated with inactivated astrocytes in either a high or low concentration of cell suspension. These findings indicated that inactivated astrocytes as a feeder layer and extrinsic factors such as (ATRA) and 9–Cis RA increase differentiation of hippocampal stem cells into rod photoreceptors.

In diabetics, hyperglycemia is associated with neuropathy, nephropathy, and retinopathy. However, direct toxic effects of glucose on neurons are still largely unknown. Firstly, cellular vital capacity was determined by MTT. Then, effects of different glucose concentrations and the role of Bax protein-induced apoptosis in PC12 cells was examined by Hoechst staining and western blotting techniques, respectively. During MTT, cells revealed to have a meaningful apoptosis at hours 48, 72, and 96 when compared with controls (p<0.01). Apoptosis was induced suitably at a glucose concentration of 13.5mg/dl after 72 hours. Western blotting showed a significant expression of Bax protein in PC12 cells treated with increased glucose concentrations for 72 hours. Increment in glucose concentration may induce apoptosis in PC12 cells. This process occurred under the intense influence of Bax protein.