Effect of Multiple Intraperitoneal Injections of Human Bone Marrow Mesenchymal Stem Cells on Cuprizone Model of Multiple Sclerosis

Background: Bone marrow mesenchymal stem cells (BM-MSCs) elicit neuroprotective effects, and their repair ability has been investigated in different experimental models. We aimed to investigate the effect of multiple i.p. BM-MSCs injections in the cuprizone model of multiple sclerosis in mice. Methods: Adult male C57BL/6 mice (n = 40) were fed a regular diet or a diet containing cuprizone (0.2% w/w) for 6 six weeks. Bone marrow samples were taken from patients with spinal cord injury. BM-MSCs (2 × 106 in 1 milliliter medium) were administered intraperitoneally for two consecutive weeks at the end of the forth weeks of cuprizone administration. Animals (n = 12) were perfused with 10% paraformaldehyde at the end of sixth week. The brains were sectioned coronally in 6-8-µm thickness (-2.3 to 1.8 mm from bregma). The sections were stained by luxol fast blue-cresyl violet, and images were captured via a microscope. Demyelination ratio was estimated in corpus callosum in a blind manner. A quantitative real-time PCR was used to measure the myelin basic protein gene expression at sixth week. Results: Histologically, cuprizone induced demyelination in the corpus callosum. Demyelinated area was diminished in the corpus callosum of cell-administered group. Cuprizone could decrease myelin-binding protein mRNAs expression in corpus callosum, which was significantly recovered after BM-MSCs injections. Conclusion: Our data indicated a remyelination potency of multiple i.p. BM-MSCs in the cuprizone model of multiple sclerosis in mice.

The Mediating Role of A2A Adenosine Receptors in the Mitochondrial Pathway of Apoptotic Hippocampal Cell Death, Following the Administration of MDMA in Rat.

INTRODUCTION: The 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) is a popular recreational drug and a major source of substance abuse, which ultimately leads to sensations of well-being, elation and euphoria, moderate derealization/depersonalization, and cognitive disruptions, as well as intense sensory awareness. The mechanisms involved in memory impairment induced by MDMA are not completely understood. METHODS: The current study used 40 Sprague-Dawley rats, weighted 200 to 250 g. Experiments were performed in four groups, each containing 10 rats. The first group of rats was used as the control, treated with dimethyl sulfoxide (DMSO). The second group was treated with MDMA. The third group was treated with MDMA and CGS (the adenosine A2A receptor agonist, 2-[p-(2-carboxyethyl) phenethylamino]-5'-N-ethylcarboxamidoadenosine) (CGS 21680) and the fourth group was treated with MDMA and SCH (the A2A receptor antagonist [7-(2-phenylethyl)-5-amino-2-(2-furyl-) pyrazolo-[4, 3-e]-1, 2, 4 triazolo [1,5-] pyrimidine]) (SCH 58261). The drugs in all groups were administrated intraperitoneally (i.p.) once a day for 7 days. In 5 rats of each group, following perfusion, samples were taken from hippocampi to investigate apoptosis. Accordingly, the samples were stained using the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay kit, and studied by light microscopy. In other rats, fresh tissue was also removed to study the expression of bax and bcl-2 by Western blotting technique. RESULTS: It was observed that the coadministration of MDMA with CGS reduced bax expression and prevented apoptosis of hippocampal cells. The coadministration of MDMA and SCH increased bax expression, and also increased the frequency of hippocampal cell apoptosis. CONCLUSION: The results of the current study showed that administration of CGS with MDMA decreased the common side effects associated with MDMA.

Direct immunomodulatory influence of IFN-ß on human astrocytoma cells.

Astrocytes actively play a pivotal role in inflammatory disease intensity of central nervous system especially multiple sclerosis (MS). Although IFN-ß is a selective therapy for MS but the role of IFN-ß in stimulating the astrocytes to produce cytokines is not clearly revealed. Therefore, it is encouraging to assess the modulatory role of IFN-ß on astrocytes of brain tissue. The aim of our study was to analyze the molecular mechanisms of recombinant IFN-ß 1a directly affecting IL-10, iNOS, MMP-9 and TIMP-1 expression in central nervous system for the first time. In this way, in vitro procedures conducted by human astrocytoma A172 and 1321N1 cell lines as a model system. The total RNA from A172 and 1321N1 cells treated with IFN-ß and LPS/IFN-γ/IFN-ß and untreated cells were extracted and evaluated for IL-10, iNOS, MMP-9 and TIMP-1 expression by real-time RT-PCR. We found a significant dose-dependent increase in IL-10 gene expression in A172 and 1321N1 cells treated with IFN-ß or LPS/IFN-γ/IFN-ß. Moreover, a significant decrease was observed in iNOS expression suggesting a similar mechanism of action for both cells. Eventually there were no significant changes concerning the modulation of the MMP-9 and TIMP-1 in response to IFN-ß treatment. In part, the immunomodulatory effect of IFN-ß may be due to increase of IL-10 and suppression of iNOS expression in astrocytes of brain tissue.

The in vitro effects of sodium salicylate on von Willebrand factor and C-reactive protein production by endothelial cells.

OBJECTIVE: The role of inflammatory and endothelial dysfunction markers in the atherogenic process has been well recognized. The data have made both C-reactive protein (CRP) and von Willebrand factor (vWF) promising targets for the cardiovascular disease research and drug development. Inhibition of CRP and vWF synthesis, therefore, might be a potential therapeutic strategy. METHODS: The effect of sodium salicylate on vWF production by human umbilical vein endothelial cells (HUVECs) using enzyme-linked immunosorbent Assays (ELISA) and real-time PCR was examined. In addition, small interfering RNA (siRNA) against NF-κB was used to investigate the existence of a role for this signaling pathway. RESULTS: Our findings demonstrated that sodium salicylate decreased vWF, but not CRP production at both mRNA and protein levels significantly and this might not occur via nuclear transcription factor (NF-κB) inhibition. CONCLUSION: Our results indicated a further rationalization of the effects of sodium salicylate on atherothrombotic events by attenuation of vWF production.

TGF- ß1-mediated apoptosis associated with SMAD-dependent mitochondrial Bcl-2 expression.

BACKGROUND: Transforming growth factor (TGF) ß1 can elicit various cellular responses, including inhibition of cell growth, migration, differentiation, and apoptosis. In addition, TGF-ß1 is able to induce apoptosis in certain lymphomas. METHODS: In the present study, the role of SMADs, Bax, Bcl-xl, and Bcl2 was characterized in 2 B-lymphoma cell lines, Burkitt and pre-B cell. RESULTS: Apoptosis was detected after exposure of TGF-ß on Raji and Nalm 6 cell lines and was evaluated by flow cytometry by using annexin V, reverse transcriptase-polymerase chain reaction, and Western blot analysis. Flow Cytometry With Cell Sorting analysis showed that apoptosis could be observed after 24 hours of TGF-ß treatment and was continued after 48 hours. TGF-ß downregulated the Bcl-xl and Bcl-2, whereas the Bax was upregulated. Furthermore, messenger RNA of SMAD6 and SMAD7 showed the significant upregulation. CONCLUSION: The results indicated that alteration in gene expression and protein level may determine the induction of apoptosis pathway in these lymphoma cell lines exposed to TGF-ß.

Effects of TGF-ß and b-FGF on the potential of peripheral blood-borne stem cells and bone marrow-derived stem cells in wound healing in a murine model.

Peripheral blood fibrocytes make up a newly identified leukocyte subpopulation that displays fibroblast-like properties. These blood-borne cells can rapidly enter the site of injury at the same time as circulating inflammatory cells. Marrow stroma includes a subpopulation of undifferentiated cells that are capable of becoming one of a number of phenotypes, including chondrocytes, osteoblasts, adipocytes, and fibroblasts. Adult human bone marrow contains a minority population of bone marrow mesenchymal stem cells (BMSCs) that contribute to the regeneration of tissues such as bone, cartilage, muscle, ligaments, tendons, fat, and stroma. Evidence that these BMSCs are pluripotent, rather than being a mixture of committed progenitor cells each with a restricted potential, includes their rapid proliferation in culture. We hypothesized that peripheral blood mesenchymal stem cells (PBMSCs) and BMSCs have an effective role in wound healing. In this study, we identified and quantified the marrow stem cells (MSCs) derived from blood and bone marrow recruited and migrated to the wound site. Our results show that the synergistic effects of transforming growth factor-beta (TGF-ß) and basic fibroblast growth factor (b-FGF) lead to a significant increase in migration and recruitment of both PBMSCs and BMSCs to the wound site, with more potent effects on PBMSCs as compared with BMSCs. Reverse transcription polymerase chain reaction of collagen type I (COL1A1) transcripts (348 bp) confirmed that TGF-ß and b-FGF activate collagen I (production in marrow stem cells at higher transcription levels), with more vigorous effects of TGF-ß on PBMSCs as compared with the same condition on BMSCs.

NPHS2 mutations in children with steroid-resistant nephrotic syndrome.

INTRODUCTION: Congenital nephrotic syndrome may be caused by mutations in NPHS1 and NPHS2, which encode nephrin and podocin, respectively. Since the identification of the NPHS2 gene, various investigators have demonstrated that its mutation is an important cause of steroid-resistant nephrotic syndrome. We aimed to evaluate frequency and spectrum of podocin mutations in the Iranian children with steroid-resistant nephritic syndrome. MATERIALS AND METHODS: We examined 20 children with steroid-resistant nephritic syndrome referred to Ali Asghar Children's Hospital, in Tehran, Iran. Mutations in the 5th and 7th exons of NPHS2 were assessed. The mutational analysis of NPHS2 was performed by DNA sequencing. RESULTS: The mean age at the onset of proteinuria was 6.4 +/- 3.6 years. None of the children had mutations in the exons 5 or 7. CONCLUSIONS: Our study suggests that NPHS2 mutations in exons 5 and 7 are not seen in our children. Therefore, we cannot recommend NPHS2 (exons 5 and 7) mutation for screening in Iranian children with steroid-resistant nephritic syndrome. Other exons of podocin or other podocyte proteins in Iranian children may play a role in pathogenesis of steroid-resistant nephritic syndrome.

Genotype and allele frequencies of N-acetyltransferase 2 and glutathione S-transferase in the Iranian population.

1. Xenobiotic-metabolizing enzymes constitute an important line of defence against a variety of carcinogens. Many are polymorphic, constituting the basis for the wide interindividual variation in metabolic capacity and possibly a source of variation in the susceptibility to chemical-induced carcinogenesis. The aim of the present study was to determine the frequencies of important allelic variants in the N-acetyltransferase 2 (NAT2) and glutathione S-transferase (GST) genes in the Iranian population and compare them with frequencies in other ethnic populations. 2. Genotyping was performed in a total of 229 unrelated healthy subjects (119 men, 110 women) for NAT2 and 170 unrelated healthy subjects (89 men, 81 women) for GST from the general Tehran population. A combination of polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) was applied for typing of NAT2 polymorphisms. Detection of GSTM1 and GSTT1 null alleles was performed simultaneously using a multiplex PCR assay. 3. The frequencies of specific NAT2 alleles were 0.299, 0.314, 0.380, 0.007 and 0.000 for 4 (wild-type), 5 (C481T, M1), 6 (G590A, M2), 7 (G857A, M3) and 14 (G191A, M4), respectively. The most prevalent genotypes were NAT2 5/6 (29.70%) and 4/6 (21.40%). The GSTM1- and GSTT1-null alleles were detected in 44.7 and 21.2% of subjects, respectively. 4. We found that Iranians resemble Indians with regard to allelic frequencies of the tested variants of NAT2. The predominance of slow (49.36%) and intermediate (41.47%) acetylation status compared with wild-type rapid acetylation status (9.17%) in the study group suggests the significant prevalence of the slow acetylator (SA) phenotypes in the Iranian population. Our data confirmed that Iranians are similar to other Caucasian populations in the frequency of both GSTM1- and GSTT1-null alleles.

Involvement of PPAR-gamma and p53 in DHA-induced apoptosis in Reh cells.

Docosahexaenoeic acid (DHA, 22:6 n-3) is an omega-3 polyunsaturated fatty acid that is found in fish oil and exerts cytotoxic effect on a variety of cell lines. The molecular target, responsible for mediating this effect of DHA, still remains unknown. In this report, we presented experimental evidences for the role of PPAR-gamma in conveying the cytotoxic effect of DHA. We showed that DHA induces apoptosis in Reh and Ramos cells and apoptotic effect of DHA is inhibited by the PPAR-gamma antagonist GW9662, indicating that PPAR-gamma functions as the mediator of the apoptotic effect of DHA. Furthermore, our result showed that DHA induces the PPAR-gamma protein levels in both Reh and Ramos cells. Interestingly, DHA was found to induce the expression of p53 protein in Reh cells in a PPAR-gamma-dependent manner. The up-regulation of p53 protein by DHA kinetically correlated with the activation of caspase 9, caspase 3, and induction of apoptosis, suggesting a role for p53 in DHA-mediated apoptosis in Reh cells. Taken together, these findings suggest a new signaling pathway, DHA-PPAR-gamma-p53, in mediating the apoptotic effect of DHA in Reh cells.

Study of correlation between lead-induced cytotoxicity and nitric oxide production in PC12 cells.

Despite reduction in its exposure, lead remains a major health problem. The primary target of lead toxicity is the central nervous system. The cellular, intracellular and molecular mechanisms of lead neurotoxicity are numerous, such as induction of apoptosis and interfering with Ca2+ dependent enzyme like nitric oxide synthase (NOS). To investigate the cytotoxic effect of lead on rat pheochromocytoma (PC12) cells, as a suitable model for neuroscience study, and possible correlation between lead toxicity and nitric oxide (NO) production, this study was performed. The current results showed that lead could induce cytotoxicity as well as NO production in a dose dependent manner in PC12 cells after 24h. The cytotoxicity was positively correlated with increased NOx (nitrite and nitrate) production in these cells. L-NAME, a NOS inhibitor, treatment (2.5 mM) could reverse this cytotoxicity. It can be concluded that lead-induced cytotoxicity in PC12 cells could partly be mediated by higher NO production.

Investigation of acute lead poisoning on apoptosis in rat hippocampus in vivo.

Despite decades of study, the exact mechanism of action of lead, a potent neurotoxic agent, have not been fully elucidated. One of the suggested mechanism of lead neurotoxicity is apoptotic cell death. The present study sought to examine the effect of acute lead poisoning on apoptosis in rat hippocampus. Two to four and 12-14 week old rats were treated for 7 days with 15 mg/kg daily dose of lead acetate intraperitoneally. Control animals received distilled water. In treated groups, the blood lead levels was increased by about 17-19-folds. Histological study of hippocampus revealed apoptotic cells, using light and electron microscopy. In Western blot analysis, the ratio of Bax/Bcl-2 protein expression in hippocampus was significantly increased compared to controls. In conclusion, the lead induced cell death in hippocampus in vivo may partly be due to apoptosis.