Cell Journal (Yakhteh)
Volume:15 Issue: 2, Summer 2013

Intra-peritoneal administration of riluzole has been shown to preserve the membrane properties and firing characteristics of Purkinje neurons in a rat model of cerebellar ataxia induced by 3-acetylpyridine (3-AP). However, the exact mechanism(s) by which riluzole restores the normal electrophysiological properties of Purkinje neurons is not completely understood. Changes in the conductance of several ion channels, including the BK channels, have been proposed as a neuro protective target of riluzole. In this study, the possible cellular effects of riluzole on Purkinje cells from 3-AP-induced ataxic rats that could be responsible for its neuro protective action have been investigated by computer simulations. This is a computational stimulation study. The simulation environment enabled a change in the properties of the specific ion channels as the possible mechanism of action of riluzole. This allowed us to study the resulted changes in the firing activity of Purkinje cells without concerns about its other effects and interfering parameters in the experiments. Simulations were performed in the NEURON environment (Version 7.1) in a time step of 25 μs; analyses were conducted using MATLAB r2010a (The Mathworks). Data were given as mean ± SEM. Statistical analyses were performed by the student’s t test, and differences were considered significant if p<0.05. The computational findings demonstrated that modulation of an individual ion channel current, as suggested by previous experimental studies, should not be considered as the only possible target for the neuro protective effects of riluzole to restore the normal firing activity of Purkinje cells from ataxic rats. Changes in the conductance of several potassium channels, including voltage-gated potassium (Kv1, Kv4) and big Ca2+-activated K+ (BK) channels may be responsible for the neuro protective effect of riluzole against 3-AP induced alterations in the firing properties of Purkinje cells in a rat model of ataxia.

Type 1 diabetes mellitus (T1D) is an autoimmune and multifactorial disorder. Subsequent analysis on human leukocyte antigen (HLA) region shows that HLA-DRB1 and -DQB1 genes have the strongest association with T1D. In this study, for the first time, we investigated the influence of gender on the HLA-DRB1 and -DQB1 association with type 1 diabetes mellitus in Iranian patients in order to determine gender dependent HLA heterogeneity in Iranian T1D patients. In this case control study, the HLA-DRB1 and -DQB1 typing were performed on 105 Iranian T1D patients and 100 healthy controls. The data were evaluated by using Fisher exact test. Our results indicate that DRB1*04:01, DQB1*03:02 alleles and DRB1*04:01-DQB1*03:02 haplotype were significantly more frequent in male T1D patients than females. Also, DRB1*03:01, DRB1*15:01, DQB1*06:01 alleles, DQB1*03:01/05:01 genotype, DRB1*03:01-DQB1*02:01 and DRB1*15:01-DQB1*06:01 haplotypes were significantly higher in female T1D group than males. Furthermore, our results showed that DRB1*04:01 and DQB1*03:02 alleles were significantly more frequent in male T1D patients 1-5 years old at onset than females with similar condition. The DRB1*03:01 allele and DRB1*03:01-DQB1*02:01 haplotype were significantly higher in female T1D patients 6-10 years old at onset than males with similar condition. The DRB1*15:01 allele and DRB1*15:01-DQB1*06:01 haplotype were significantly more frequent in female T1D patients 16-20 years old at onset than males with similar condition. Our findings suggest that gender has a significant influence on the distribution of HLA-DR and -DQ alleles, genotypes and haplotypes. Also, distribution of the HLA-DRB1 and -DQB1 alleles, genotypes and haplotypes vary based on the gender of T1D patients in different age at onset.

Grape virus diseases are a serious problem in Iran. Leaves and fruits of grape have been used for different purposes like cooking in Iran. The present investigation was carried out to study on the cytotoxic-activities of extracts of fruits and leaves of Vitis vinifera from both virus-free and virus-infected grape cultivars against breast cancer cell line (MDA-MB-231) and human embryonic kidney normal cell line (HEK 293). In this experimental study, the considered grape cultivars were as follows: Rish Baba Sefid, Shahani Ghasre Shirin, Rotabi Zarghan, Asgari Najaf Abad, Fars, Kaj Angor Bojnord, Sarkesh Shiraz and Siahe Zarqan. A real-time multiplex polymerase chain reaction (real-time Multiplex PCR) assay was applied to detect virus infected cultivars. The cytotoxic effect of the methanol extracts of different Vitis vinifera varieties on cultured cells was monitored using (3- (4, 5-Dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide (MTT) assay at different concentrations (62.5, 125, 250, 500, 750, 1000 μg mL-1). Among these cultivars, Grapevine fanleaf virus (GFLV) along with related symptoms was detected in Siahe Zarqan and Fars. Methanolic extracts of leaves and fruits of Vitis vinifera from both virus free and virus infected cultivars showed a range of limited to moderate cytotoxic activity. However, methanol extract of leaves belonged to virus infected cultivars was found to have strong cytotoxic effect against MDA-MB-231 at different concentrations. Grapevine fanleaf virus (GFLV) can potentially increase the cytotoxicity of grape cultivars.

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. χ2 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.

Monitoring of influenza virus shedding and optimization of multiplicities of infection (MOI) is important in the investigation of a virus one step growth cycle and for obtaining a high yield of virus in vaccine development and conventional basic diagnostic methods. However, eluted infectious viruses may still be present immediately after virus inoculation and when cells are washed following virus cultivation which may lead to a false positive virus infectivity assay. In this experimental study, we investigated influenza virus progeny production in Madin-Darby canine kidney (MDCK) cells with five different MOI at determined time points. The results were analyzed by end point titration tests and immunofluorescence assay. Higher titers of eluted virus were observed following a high MOI inoculation of virus in cell culture. Most probably, this was the result of sialic acid residues from viral hemagglutin in proteins that were cleaved by neuraminidase glycoproteins on the surface of the influenza virus, which promoted viral spread from the host cell to the culture supernatant or during endocytosis, where viruses recycle to the cell surface by recycling endosomes which culminated in virus shedding without replication. We demonstrated that the pattern of influenza virus progeny production was dose-dependent and not uniform. This production was influenced by several factors, particularly MOI. Understanding the exact features of viral particle propagation has a major impact in producing high virus yields in the development of vaccines. Use of lower MOI (0.01) could result in accurate, precise quantitative assays in virus diagnosis and titration methods.

Tooth loss is a common problem and since current tooth replacement methods cannot counter balance with biological tooth structures, regenerating natural tooth structures has become an ideal goal. A challenging problem in tooth regeneration is to find a proper clinically feasible cell to seed.This study was designed to investigate the odontogenic potential of human bone marrow mesenchymal stem cells (HBMSCs) for seeding in tooth regeneration. In this experimental study, three pregnant Sprague Dawley (SD) rats were used at the eleventh embryonic day and rat fetuses were removed surgically using semilunar flap under general anesthesia. The primary mandible was cut using a stereomicroscope. The epithelial and mesenchymal components were separated and the dissected oral epithelium was cultured for 3 days. We used flow cytometry analysis to confirm presence of mesenchymal stem cells and not hematopoietic cells and to demonstrate the presence of oral epithelium. Bone marrow mesenchymal stem cells (BMSCs) and cultured oral epithelium were then co-cultured for 14 days. BMSCs cultured alone were used as controls. Expression of two odontogenic genes Pax9 and DMP1 was assessed using quantitative reverse transcription- polymerase chain reaction (RT-PCR). Expression of two odontogenic genes, Pax9 and DMP1, were detected in BMSCs co-cultured with oral epithelium but not in the control group. Expression of Pax9 and DMP1 by human BMSCs in the proximity of odontogenic epithelium indicates odontogenic potential of these cells.

Multiple sclerosis (MS) is an immune-mediated demyelinating disease of the central nervous system (CNS). Stem cell transplantation is a new therapeutic approach for demyelinating diseases such as MS which may promote remyelination. In this study, we evaluate the remyelinating potential of adipose mesenchymal stem cells (ADSCs) and their effect on neural cell composition in the corpus callosum in an experimental model of MS. This experimental study used adult male C57BL/6 mice. Cultured ADSCs were confirmed to be CD73+,CD90+, CD31-,CD45-, and labeled by PKH26. Animals were fed with 0.2% w/w cuprizone added to ground breeder chow ad libitum for six weeks. At day 0 after cuprizone removal, mice were randomly divided into two groups: the ADSCs-transplanted group and the control vehicle group (received medium alone). Some mice of the same age were fed with their normal diet to serve as healthy control group. Homing of ADSCs in demyelinated lesions was examined by fluorescent microscope. At ten days after transplantation, the mice were euthanized and their cells analyzed by luxol fast blue staining (LFB), transmission electron microscopy and flow cytometry. Results were analyzed by one-way analysis of variance (ANOVA). According to fluorescent cell labeling, transplanted ADSCs appeared to survive and exhibited homing specificity. LFB staining and transmission electron microscope evaluation revealed enhanced remyelination in the transplanted group compared to the control vehicle group. Flow cytometry analysis showedan increase in Olig2 and O4 cells and a decrease in GFAP and Iba-1 cells in the transplanted group. Our results indicate that ADSCs may provide a feasible, practical way for remyelination in diseases such as MS.

Familial Mediterranean fever (FMF) is an autosomal recessive disorder characterized by recurrent febrile attacks accompanied by serosal and synovial membrane inflammation. FMF is caused by mutations in the MEFV gene and are found usually among Mediterranean populations, Armenians, Turks, Arabs and Jews. The aim of this study was to determine the frequency of MEFV gene mutations among FMF patients in the Azeri Turk population in North-West of Iran. In this descriptive study, 130 FMF patients with Azeri Turk origin were screened for mutations in four exons (2, 3, 5 and10) of MEFV gene. Genomic DNA was extracted from whole blood and entered in ARMS-PCR and PCR-RFLP reactions. When cases were negative in ARMS-PCR and PCR-RFLP, the exons were amplified and subjected to direct sequencing. Our results showed that the most common mutations in this study population was M694V (40.19%) followed by E148Q (17.64%), V726A (13.72%), M680I (12.74%) and M694I (2.94%) mutations. Four new mutations including K618N, K716M, S614F and G136E were identified in our study. The prevalence of five common mutations in our study was highly similar to previous studies analysing the Mediterranean basin populations. Investigation by sequencing also revealed four new variants in the study population. The main genotype- phenotype correlation finding was the presence of M694V mutation in homozygote or compound heterozygote state in the patients with renal manifestations.

Herb combination has been very popular in traditional medical prescriptions such as Traditional Chinese Medicine (TCM). Persistent efforts and attempts have been made to dissect the action mode of TCM in recent years, which has provided certain evidence for inter-herbal interactions. However, the interactions among different components in a single herb have been largely neglected. In this experimental study, the interactions among different components of a single herb were explored. The effect of three main sesquiterpenes (germacrone, curdione, furanodiene) isolated from Curcuma WenyujinY.H.Chenet C Ling on MDA-MB-231 and MCF-7 breast cancer cell proliferation alone or in combination with a fixed-dose-combination was investigated. Furanodiene significantly inhibited cancer cell proliferation while germacrone and curdione showed no effect. Germacrone enhanced furanodiene’s anti-proliferative effect. Curdione showed no effect on furanodiene’s anti-proliferative effect but partly reversed the anti-proliferative effect of germacrone and furanodiene combined. The morphological and mitochondrial membrane potential (Δψm) changes showed similar results. However, they demonstrated complicated interactions on the expression of apoptotic-related proteins and key signal transduction proteins. Unpredictable and complex interactions among different components in Curcuma WenyujinY.H.Chenet C Ling may exist. The intra-herb interactions should be taken into consideration when attempts are made to interpret the art of TCM formulation or other similar recipes.

We studied both the presence of some carbohydrate compounds in a three-dimensional (3D) matrix harvested from human gingiva and the cell behavior in this matrix. In this experimental research, in order to prepare 3D scaffolds, human palatal gingival biopsies were harvested and physically decellularized by freeze-thawing and sodium dodecyl sulfate (SDS). The scaffolds were placed within the rings of blastema tissues obtained from a pinna rabbit, in vitro. We evaluated the presence of glycoconjugatesand cellular behavior according to histological, histochemical and spectrophotometry techniques at one, two and three weeks after culture. One-way analysis of variance (ANOVA)comparedthe groups. Extracellular matrix (ECM) remained after decellularization of tissue with 1% SDS. Glycoconjugate contents decreased meaningfully at a higher SDS concentration (p<0.0001). After culture of the ECM scaffold with blastema, we observed increased staining of alcian blue, periodic acid-Schiff (PAS) and toluidine blue in the scaffold and a number of other migrant cells which was caused by cell penetrationinto the scaffold. Spectrophotometry results showed an increase in glycosaminoglycans (GAGs) of the decellularized scaffolds at three weeks after culture. The present study has shown that a scaffold generated from palatal gingival tissue ECM is a suitable substrate for blastema cell migration and activity.This scaffold maypotentially be useful as a biological scaffold in tissue engineering applications.

For immunotherapy of human papillomavirus (HPV) -16-associated cervical cancers the E7 protein is considered a prime candidate. However it is a poor inducer of cytotoxic T-cell response, when being used as a singular antigen in protein vaccination. Hence, in this study we focused on the utilization of a vaccine delivery system for prevention or treatment of cervical cancer. In this experimental study, we designed and evaluated a novel fusion protein comprising HPV16 E7 antigen fused to Shiga toxin B-subunit (STxB) as both an antigen vector and an adjuvant. Then we designed two preventive and therapeutic tumor models to investigate the prevention and inhibition of TC-1 cell growth in female C57BL/6 mice, respectively. In each model, mice were immunized with the recombinant protein of E7-STxB or E7 without any adjuvant. We demonstrated that prophylactic immunization of E7-STxB protected mice against TC-1 cells. Also in the therapeutic model, E7-STxB inhibited TC-1 tumor growth inlungs. The results were significant when compared with the immunization of E7 singularly. We concluded that immunization with the E7-STxB protein without any adjuvant could generate anti-tumor effect in mice challenged with TC-1 cells.This research verifies the clinical applications and the future prospects of developing HPV16 E7 therapeutic vaccines fused to immunoadjuvants.

Different studies have shown an association of TLR4 polymorphisms with susceptibility/resistance to malaria disease. In the current immunogenetic study, we assessed the TLR4 genotypes formed by the two common single nucleotide polymorphisms (SNPs) (Asp299Gly and Thr399Ile) in the co-segregate state in Baluchi Plasmodium falciparum infected and healthy populations from malaria hypoendemic areas of Iran. The study was performed to evaluate the distribution and correlation of TLR4 co-segregating genotypes in patients with mild malaria. Moreover, the frequency of these genotypes was compared with reported results from other populations in similar or contrasting malaria settings around the world. In this case control study, the presence of 2 SNPs in the TLR4 gene (Asp299Gly and Thr399Ile) were analyzed in 350 Baluchi patients with mild malaria and 350 unrelated healthy controls by using polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) techniques followed by sequencing analysis. Differences in the TLR4 co-segregate genotype frequencies among the studied group were determined by Fisher’s exact test. Although the distribution of the two commonly co-segregating TLR4 genotypes presented a diverse and distinct pattern in the Baluchi population, no significant difference was detected between the cases and controls (p>0.05). A lower frequency of TLR4 Asp299Gly/Thr399Thr was observed in Baluchis with mild malaria compared to African populations (p<0.05). Differences in the co-segregation patterns of TLR4 Asp299Gly/Thr399Ile genotypes in the Baluchi population compared to other malaria endemic populations may suggest different local evolutionary pressure on TLR4 polymorphisms by malaria in this region. The higher frequency of Asp299Gly/Thr399Ile genotypes among the Baluchi population compared with the African population (p<0.05) which suffers from a larger number of severe cases might suggest that this genotype has a role in protecting against severe malaria. These findings are useful for further understanding the pathogenesis of severe malaria.

Leukemia inhibitory factor (LIF) plays important roles in cellular proliferation, growth promotion and differentiation of various types of target cells. In addition, LIF influences bone metabolism, cachexia, neural development, embryogenesis and inflammation. Human LIF (hLIF) is an essential growth factor for the maintenance of mouse embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) in a pluripotent, undifferentiated state. In this experimental study, we cloned hLIF into the pENTR-D/TOPO entry vector by a TOPO reaction. Next, hLIF was subcloned into the pDEST17 destination vector by the LR reaction, which resulted in the production of a construct that was transferred into E. coli strain Rosetta-gami™ 2(DE3) pLacI competent cells to produce the His6-hLIF fusion protein. This straightforward method produced a biologically active recombinant hLIF protein in E. coli that has long-term storage ability. This procedure has provided rapid, cost effective purification of a soluble hLIF protein that is biologically active and functional as measured in mouse ESCs and iPSCs in vitro. Our results showed no significant differences in function between laboratory produced and commercialized hLIF.