In vivo vascularization of endothelial cells derived from bone marrow mesenchymal stem cells in SCID mouse model

Objective: In vivo and in vitro stem cell differentiation into endothelial cells is a promising area of research for tissue engineering and cell therapy

Materials and Methods: We induced human mesenchymal stem cells [MSCs] to differentiate to endothelial cells that had the ability to form capillaries on an extracellular matrix [ECM] gel. Thereafter, the differentiated endothelial cells at early stage were characterized by expression of specific markers such as von Willebrand factor [vWF], vascular endothelial growth factor [VEGF] receptor 2, and CD31. In this experimental model, the endothelial cells were transplanted into the groins of severe combined immunodeficiency [SCID] mice. After 30 days, we obtained tissue biopsies from the transplantation sites. Biopsies were processed for histopathological and double immunohistochemistry [DIHC] staining

Results: Endothelial cells at the early stage of differentiation expressed endothelial markers. Hematoxylin and eosin [H and E] staining, in addition to DIHC demonstrated homing of the endothelial cells that underwent vascularization in the injected site

Conclusion: The data clearly showed that endothelial cells at the early stage of differentiation underwent neovascularization in vivo in SCID mice. Endothelial cells at their early stage of differentiation have been proven to be efficient for treatment of diseases with impaired vasculogenesis

[Effects of stem cell factor on activity of glutathione stransferase and recovery from acetaminophen-induced hepatotoxicity in mice]

Objective: Acetaminophen [APAP] overdose causes acute liver injuries. Studies show that stem cell factor [SCF] and its receptor, c-Kit, enhance liver recovery from APAPinduced injuries in mice. In this study we explore the effect of SCF on activity of glutathione S-transferase [GSTs] enzymes which are considered to be important in APAP metabolism

Methods: We divided 45 Balb/c mice into three groups. Within each group there were three sub-groups of five mice per subgroup. The groups included: 1. APAP [300 mg/kg B.W., i.p.]; 2. SCF [40 microg/kg B.W., i.p.] given.30 minutes after APAP [300 mg/kg B.W., i.p.], and 3.control mice treated with normal saline. The mice were sacrificed at 1, 12 and 24 hours, respectively. Hepatotoxicity was evaluated in the 24 hour group by histopathology and assessment of biochemical serum markers [ALT and AST]. We assessed the levels of SCF receptor [c-Kit] protein and GST enzyme activities in the liver tissues

Results: Hepatotoxicity was induced by APAP [300 mg/kg, B.W] as evident by both histopathological observations and a significant [p<0.05] increase in serum ALT and AST levels, which were reversed by SCF administered post-APAP. SCF administration after APAP administration significantly increased GSTs enzyme activity levels by 24 hours, however it led to a significant decrease in c-Kit protein level compared to the control and APAP groups

Conclusion: Our data suggest that SCF binding to its receptor [c-Kit] on liver cells may attenuate APAP-induced liver injuries by increasing GST activities in the livers of mice

association of -475 and -631 interleukin-2 gene polymorphism with multiple sclerosis in Iranian patients

Multiple sclerosis [MS] is a chronic autoimmune disease due to demyelination of the central nervous system. It is believed that cytokines are involved in the pathogenesis of MS. The interleukin-2 [IL2] gene is powerful functional candidate that is involved in immune regulation and operation. In this study, for the first time, we investigated the effect of -475 A/T and -631 G/A IL2 polymorphisms on MS disease in Iranian patients. In this case-control study, 100 MS patients [mean age: 32.95 +/- 6.51 years, age range: 20-42 years] selected according to McDonald criteria, and 100 ethnically, sex and age matched healthy controls [mean age: 29 +/- 7.8 years, age range: 20-52 years] with no personal or family history of autoimmune diseases were studied. The restriction fragment length polymorphism-polymerase chain reaction [RFLP-PCR] method was applied to define different alleles and genotypes of IL2 promoter single nucleotide polymorphism -475 A/T as well as -631 G/A among individuals. chi [2] was calculated and Fisher's exact test was applied to analyze the obtained data. The value of p <0.05 was considered significantly. Evaluation of the -475 IL2 revealed that T allele and A/T genotype are present in 2% and 4% of MS patients, respectively, whereas T allele was absent in control samples. The comparison between alleles and genotypes in MS patients and healthy controls was not significant [p=0.1]. For the -631 position, 1% and 2% of MS patients carried A allele and A/G heterozygote genotypes, respectively. All control samples had G allele and G/G genotype. The differences between patients and controls were not significant [p=0.4]. Moreover, our results showed a very low frequency of T at -475 and A at -631 IL2 position in each of the two groups. Both -475 and -631 IL2 polymorphisms were higher in MS patients as compared to controls, but the frequency differences were not significant. Based on these data, it is suggested that the -475 and -631 IL2 polymorphisms as functional promoter position may be involved in IL2 expression and regulation. To find out the exact effect of the mentioned SNPs on susceptibility to MS, study on a larger sample size is suggested

The infl uence of -330 IL-2 gene polymorphism on relapsing remitting and secondary progressive multiple sclerosis in Iranian patients

Multiple sclerosis (MS) is a chronic infl ammatory demyelinating disease of the central nervous system. Interleukin-2 (IL-2) is identifi ed as the crucial and main immunoregulatory cytokines. Previously, we showed signifi cant association between -330 T/T IL-2 genotype and relapsing remitting MS among Iranian population. In this study we investigated 100 relapsing remitting, 30 secondary progressive MS and 125 healthy controls to compare the relapsing remitting and secondary progressive course MS in association to -330 IL-2 polymorphism. Our results showed that the -330 T/T IL-2 genotype was signifi cantly more frequent in relapsing remitting and secondary progressive MS than controls. The signifi cant increased frequency of -330 T/T IL-2 genotype in secondary progressive than relapsing remitting MS, imply -330 T/T IL-2 genotype can cause higher susceptibility to secondary progressive MS than relapsing remitting.

Immunoregulatory effects of glutathione during mesenchymal stem cell differentiation to hepatocyte-like cells

The role of mesenchymal stem cell in cellular therapy is the subject of interest for many researchers. The differentiation potential of MSCs and abilities in modulations of the recipient's immune system makes them important cells in tissue regenerative studies. MSCs by releasing the proinflammatory cytokines play important role in immunomodulatory systems; however the signaling pathways for releasing of these mediators are not well understood. Glutathione has been shown to play a role in modulation of cytokines in hepatogenic differentiation. In the current study we aimed to investigate the effects of buthionine sulfoximine [BSO, inhibitor for glutathione synthesis] and N-acetylecystin [NAC, an inhibitor for ROS generation] on proinflammatory cytokines production in a hepatogenic differentiation model. BSO and NAC significantly decreased IL-6 and TNF-alpha levels at 14 days of differentiation, whereas, NAC decreased the levels of IL-8 at days 2 and 14 of differentiation. Moreover, intracellular glutathione level during the differentiation was depleted. Our current study suggests a novel role of GSH as an immunopharmacological regulatory molecule during hepatogenic differentiation. Finally, this information may shed some light on the understanding of MSCs responses in transplantation and cell therapy in diseases such as chronic hepatic diseases.

Expression of cytochrome P450 and glutathione S-transferase in human bone marrow mesenchymal stem cells

Currently several studies are being carried out on various properties of mesenchymal stem cells [MSCs] however there are a few investigations about drug metabolizing properties of these cells. The aim of this study was to measure the key factors involved in drug metabolism in human bone marrow MSCs. For this purpose, cellular glutathione [GSH], glutathione Stransferase [GSTs] and cytochrome P450 class 3A4 [CYP3A4] were detected in these cells. Results showed that CYP3A4 and GSTA1-1 mRNA are not detectable in MSCs however mRNA specific for GSTP1-1 was considerably expressed in MSCs. GSH content and GST activity was also detected in MSCs. These data suggest that human bone marrow MSCs possess the drug metabolizing activity which may be useful in handling drugs and chemotherapeutic agents passing to the bone marrow

Estimation of atlatoxin-albumin adduct in serum sample by optimized HPLC-fluorescence

With consideration of lethal effects of aflatoxins specially Bl on human health. Estimation of aflatoxin-albumin adduct, as an important marker of aflatoxin exposure, seems essential. The aim of this study is optimization of HPLC-fluorescence method for measurement of this important marker in blood serum. In this study, blood serum of three groups of rats as A] positive controls [treated with AFB1], B] negative controls [without treatment] and standard rats [treated with radiolabeled AFBl] were used. After albumin isolation using ammunium sulphate and acetic acid, purity of albumin was tested by SDS-PAGE electrophoresis and albumin concentration was quantified by bradford method. Then albumin was hydrolysed by pronase and aflatoxin bound to albumin was released as aflatoxin-lysine. Pronase was precipitated and albumin was digested by aceton in cold, the volume of supernatant was reduced by freeze-drier and injected into HPLC system. Aflatoxin was quantified in comparison to standard rats samples. The purity of this isolated albumin was confirmed by SDS-PAGE electrophoresis. Albumin concentration in positive, negative and standard samples were 10, 13 and 12.5 mg/ml, respectively. Detection limit [20 pg/mg Alb] for measurement of aflatoxin was determined by HPLC method, specificity and sensitivity of method were 92% and 100% respectively. The mean concentration of AF-Alb adducts in serum of positive control rats was 10 ng/mg Alb and the reproducibility of the method after several repeat was very good. In this study, for AF-Alb adduct quantification by HPLC method, mobile phase, I percentage of solvents and run time were changed and the affinity chromatography before HPLC, was deleted. Therefor HPLC- fluorescence which is a precise and specific method, and since it is fast, highly reproducible and cost effective, also with improvement made, could easily be used for the quantification of this important marker in serum

Heterologous immunization, a way out of the problem of monoclonal antibody production against carcinoma antigen 125

Monoclonal antibodies [mAbs] are essential tools for many molecular immunology investigations, epitope mapping and molecular modelling, clinical laboratory diagnostic tests and immunotherapy. Humoral immune response of immunized animals largely depends on the nature of antigen and the immunization technique. Polysaccharides and heavily-glycosylated proteins are very elusive targets incapable of mounting long-lasting, high affinity antibody responses. Carcinoma antigen 125 [CA 125], a well known tumor marker of ovarian cancer, is a mucin type antigen consisting of repetitive units of heavily glycosylated moieties which render production of mAbs very difficult. To evaluate the efficacy of heterologous antigen preparations as a way of mouse immunization in the production of anti-CA 125 mAb. Two different protocols of immunization were used for priming of NMRI mice. In the first method, mice conventionally immunized by three intraperitoneal injections of purified CA 125 and boosted by the antigen three days before fusion. In the second approach, mice were primed by three intraperitoneal injections of living CA 125 positive cells of OVCAR-3 cell line, and boosted by intravenous injection of the purified extracellular domain of CA 125. Production of mAb was performed by standard hybridoma technology and mAbs were characterized by different immunoassays. The first method failed to produce stable clones despite six time fusion. A total of ten stable clones, however, were produced in the second approach. Some of the clones were characterized and found to have excellent immunoreactivity when tested by ELISA assay, western blotting, intracellular and surface immunofluorescent staining of OVCAR-3 cell line and immunohistochemical staining of ovarian cancer tissues. Altogether the results of the present study clearly showed that heterologous antigen preparation is the method of choice for immunization when production of monoclonal antibody against highly glycosylated poorly immunogenic antigens is concerned

Tehranolide could shift the immune response towards Th1 and modulate the intra-tumor infiltrated T regulatory cells

Artemisia diffusa contains a new type of sesquiterpene lactone with an endoperoxide group [Tehranolide]. Due to the existing similarity between the structures of Tehranolide and Artemisinin, it was hypothesized that Tehranolide would have similar effects as Artemisinin. In this study, the immunotherapeutic effectiveness of Tehranolide was investigated by direct intra-tumoral injection. Tehranolide was purified from Artemisia diffusa, and its effect on the tumor volume was investigated. The splenocyte proliferation, shifting of cytokine profile, and the presence of naturally-occurring CD4+CD25+Foxp3+ Treg cells were assessed to describe the anti-tumor immune response. Analysis of immune response showed that, intra-tumoral injection of Tehranolide decreased the rate of tumor growth compared to control group. Furthermore, the proliferative response of mice treated with Tehranolide was enhanced. In comparison with the control group, production of both IL-4 and IFN-gamma was induced [p<0.05]. The results indicated a decrease in tumor CD4+CD25+Foxp3+ T lymphocytes in the Tehranolide-treated group compared to the control group. Treatment of tumors with Tehranolide attenuated CD4+CD25+Foxp3+ Treg cell-mediated immune suppression and elicited a persistent anti-tumor immunity against cancer

Comparative study of the major Iranian cereal cultivars and some selected spices in relation to support Aspergillus parasiticus growth and aflatoxin production

Aflatoxins are toxic fungal metabolites enable to contaminate a wide range of natural substrates. This contamination can be host-specific for different plant species. In this study, the ability of a toxigenic Aspergillus parasiticus to produce various aflatoxins on major Iranian cereals was evaluated with special focus on plant susceptibility to toxin production at cultivar level. Aspergillus parasiticus cultured on major Iranian cereal cultivars and some selected spices was incubated in shaking condition at 28?C for 6 days. The concentration of aflatoxins B1 and total [B1, B2, G1 and G2] was measured by thin layer chromatography. The amounts of aflatoxin B1 produced on maize, wheat and rice cultivars were in the ranges of 1.0-33.9, 41.9-193.7, and 39.1-82.3 micro g/g fungal weight, respectively. Interestingly, genetically modified Bacillus thuringiensis rice [GM rice] of Tarom Molaii cultivar examined for the first time in this study showed less susceptibility to aflatoxin production in comparison with its normal counterpart [P < 0.05]. The mean of aflatoxin production on maize cultivars was less than both wheat and rice cultivars that indicates considerable resistance of maize to aflatoxin compared with two other cereals. Unlike to Cuminum cyminum, both Helianthus annuus and Carum carvi seeds were highly resistant to aflatoxin production. These results indicate that inter- and intra-species differences exist in susceptibility of the major Iranian cereals as well as spices tested to A. parasiticus growth and aflatoxin production. Further studies are recommended to determine resistance markers of selected cultivars of Iranian cereals

Capillary network formation by endothelial cells differentiated from human bone marrow mesenchymal stem cells

Human bone marrow derived mesenchymal stem cells [HBMSCs] have the potential to differentiate into cells such as adipocyte, osteocyte, hepatocyte and endothelial cells. In this study, the differentiation of hBMSCs into endothelial like-cells was induced in presence of vascular endothelial growth factor [VEGF] and insulin-like growth factor [IGF-1]. The differentiated endothelial cells were examined for their ability to express VEGF receptor-2 [VEGFR2] and von willebrand factor [vWF]. Then the cells were adopted to grow and develop capillary network in a semisolid gel matrix in vitro. The capillary network formation in a well of 24-well plate was found to be 85% in presence of VEGF [50ng/ml] and IGF-1 [20ng/ml] of the culture media. These data may suggest that the expression of endothelial markers in endothelial like-cells derived from hBMSCs is associated with their ability to form capillaries

Development of a novel three-dimensional biocompatible nanofibrous scaffold for the expansion and hepatogenic differentiation of human bone marrow mesenchymal stem cells

In this present study, we examined the differentiation potential of human bone marrow derived mesenchymal stem cells [hBMSCs] into hepatocytes on a three-dimentional [3D] nanofibrous scaffold formed by Poly [e-caprolactone] [PCL], collagen and polyethersulfone [PES]. The nanofiber was prepared by the electrospining technique. HBMSCs were isolated using combining gradient density centrifugation with plastic adherence. Flow cytometric analysis was used to identify the isolated MSCs. The performance of the cells on the scaffold was evaluated by scanning electron microscopy [SEM] and MTT assay. The hBMSCs were then cultured in a hepatic differentiation medium containing hepatocyte growth factor [HGF], oncostatin M [OSM] and dexamethasone [DEX] for up to 21 days. The results showed that the isolated hBMSCs expressed specific markers such as CD44, CD166, CD105 and CD13. The integrity of the MSCs was further confirmed by their differentiation potential to osteogenic and adipogenic lineages. Scanning electron micrographs and MTT analysis revealed that the cells adhered and proliferated well on the nanofibrous hybrid scaffolds. Immunocytochemical analysis of albumin and a-fetoprotein [AFP] showed the accumulation of these markers in the differentiated cells on the scaffold. Hepatocyte differentiation was further confirmed by showing expression of albumin, AFP and cytokeratin-19 [CK-19] at mRNA levels in differentiated cells. In conclusion, the evidences presented in this study show that the engineered scaffold is promising for maintenance of hepatocyte-like cells suitable for transplantation

Class-Pi of glutathione S-transferases

Class-Pi of glutathione s-transferases [GST-Pi] is the specific form of GSTs that are known to participate particularly in the mechanisms of resistance to drugs and carcinogens. This class of the enzyme is referred to as class-P or class-Pi or class pi. The accepted terminology in this review article is class-Pi. In this article following a brief description of identified molecular forms of GSTs, we focus on GST-Pi. We review new findings about the structure and regulation of GST-Pi gene. Then, the role of GST-Pi in liver damage, oxidative stress, carcinogenesis and drug resistance are discussed. Also, the presence of common genetic polymorphism, hypermethylation in GST-Pi gene and the consequences GST-Pi knock out is regarded

Inhibitory effect of aqueous onion and garlic extracts on growth and keratinase activity in trichophyton mentagrophytes

The effect of onion and garlic extracts on fungal growth and keratinolytic activity was studied in Trichophyton mentagrophytes as one of the major etiologic agents of human and animal dermatophytosis in Iran and other parts of the world. In order to find out the best keratinase producer for further steps, culture conditions for 30 strains of T. mentagrophytes isolated from human dermatophytosis were optimized on specific solid and liquid media. All of the isolates produced the enzyme on both selective culture media. The maximum keratinolytic activity at submerged cultivation was reported for cultures of T. mentagrophytes isolate No. 1 grown for a 12-day period at 32°C. Extracellular keratinase activity was in the range of 0.28 to 2.18 u/mg protein in different isolates at predetermined optimal conditions. The growth of T. mentagrophytes isolate No. 1 was inhibited in the presence of various concentrations of onion and garlic extracts. This inhibition reached to a maximum of 100% for both extracts at 10% v/v concentrations. Keratinase synthesis was also inhibited by two extracts as a dose-dependent manner with maximums about 58.54 and 71.36% at 5% concentrations, accordingly. In contrast to the fungal growth, keratinolytic activity was inhibited more by garlic as compared with onion extract. This is the first report on keratinase inhibition by these two natural compounds. Since fungal growth and keratinolytic activity are important factors in pathogenesis of the dermatophytes, their inhibition by onion and garlic indicate that these substances may have potential values for treatment of human and animal dermatophytosis