Cell Journal (Yakhteh)

1-10

Authors: Mohamadreza Baghaban Eslaminejad , Nesa Fani , Maryam Shahhoseini

Management of mesenchymal stem cells (MSCs) capabilities to differentiate into osteogenic and chondrogenic lineages would be of utmost importance for their future use in difficult to treat cases of destroyed bone and cartilage. Thus, an understanding of the epigenetic mechanisms as important modulators of stem cell differentiation might be useful. Epigenetic mechanism refers to a process that regulates heritable and long-lasting alterations in gene expression without changing the DNA sequence. Such stable changes would be mediated by several mechanisms including DNA methylation and histone modifications. The involvement of epigenetic mechanisms during MSC bone and cartilage differentiation has been investigated during the past decade. The purpose of this review is to cover outstanding research works that have attempted to ascertain the underlying epigenetic changes of the nuclear genome during in vitro differentiation of MSCs into bone and cartilage cell lineages. Understanding such genomic alterations may assist scientists to develop and recognize reagents that are able to efficiently promote this cellular differentiation. Before summarizing the progress on epigenetic regulation of MSC bone and cartilage differentiation, a brief description will be given regarding in vitro conditions that favor MSC osteocytic and chondrocytic differentiation and the main mechanisms responsible for epigenetic regulation of differentiation.

19-28

Authors: Maryam Yadegari , Mahmoud Orazizadeh , Mahmoud Hashemitabar , Ali Khodadadi

Objective: A study of the histological events under interleukin-1α (IL-lα) induction of bovine nasal cartilage (BNC) could result in useful data to better understand the mechanisms involved in tissue breakdown in joint diseases. The aim of this study was to investigate the effects of IL-lα on chondrocyte phenotype and extracellular matrix (ECM) changes in BNC explants. Materials and Methods: In this experimental study, samples were divided into two groups. Group I (control group) BNC explants were cultured only in Dulbecco’s modified Eagle’s medium (DMEM). In group II, BNC explants were treated with IL-lα (10 ng/ml) for 28 days. Then, samples were harvested on culture days 3, 7, 14, 21 and 28 and chondrocyte morphology and ECM alterations were assessed by invert microscopy and histology by hematoxylin and eosin (H&E) and Alcian blue. Cell viability was evaluated by the lactate dehydrogenase (LDH) assay test. Data were analyzed by the t test and p<0.05 was considered significant. Results: IL-lα induced significant morphological changes in cartilage. In the presence of IL-lα, most chondrocytes transformed into a fibroblast-like morphology with a granular black point appearance. An increase in the cell: matrix ratio was observed and there were decreased numbers of chondrocytes.IL-lα induced breakdown of ECM. We observed partial degradation of ECM between days 7-14 and complete degradation occurred between days 21-28 of culture. The LDH levels increased. Conclusion: IL-1α induced morphological changes in chondrocytes and increased destruction of cartilage ECM. There was a parallel correlation between proteoglycan degradation and changes in chondrocyte morpholgy.

37-44

Authors: Mohaddeseh Larypoor , Mansour Bayat , Zuhair Mohammad Hassan , Abbas Akhavan Sepahy , Masoud Amanlou

Objective: Aflatoxin B1 (AFB1) suppresses the immune system. To decrease such suppressive effects on the immune system, a wide range of herbal medicines like garlic are utilized. Biological activities of garlic in vitro and in vivo have also been verified. Our previous studies demonstrated that aged garlic (dry garlic bulbs preserved in the freezer for six months at -20˚C) have increased immunostimulator fractions and reduced immunosuppressor fractions. This study focuses on the immunosuppressor activity of AFB1 and immunostimulator activity of aged garlic extract (AGE) through the evaluation of CD4+ CD25+ FoxP+ regulator cell (Treg) counts and the pattern of cytokine production in Balb/c normal mice. Materials and Methods: In this experimental research, AFB1 was separated from Aspergillus flavus (PTCC 5004) by HPLC and AGE prepared using the Mantis method. The Delayed-Type Hypersensitivity (DTH) test was carried out to determinate the effectiveness of different doses of AGE and AFB1, which can both have an effect on the immune system. Subsequent experiments were carried out on 20 Balb/c mice to estimate the effects of AGE and AFB1 on the number of Treg cell in 4 groups: 10 μl/kg/day of AFB1 and AGE diluents were administered for 4 consecutive days to group 1. AFB1, 2. control, 3. AGE + AFB1 and 4. AGE via intraperitoneal (IP) route, respectively. Mice were sacrificed and splenocytes harvested and the percentage of splenic Treg cells was measured by flow cytometry analysis. The ELISA method was utilized to measure Cytokine production. Results: The findings reveal that AGE increased the level of IFN-γ and IL-4 cytokines produced by splenocytes stimulated by specific tumor antigen and decreased the number of Treg cells in the spleen (p<0.05). AFB1 increased the number Treg cells in the spleen and decreased cytokine production (p<0.05). In groups 2 (control) and 4 (AGE) the number of Treg cells decreased (p value<0.05) whereas in groups 1 and 3 the number of Treg cells increased (p<0.05). Conclusion: This study indicated that AGE is able to alter the cytokine production in normal mice into a Th1 protective pattern which is beneficial to the immune system in general and anti-tumor immunity in particular. AFB1 is able to alter the cytokine production into a Th2 protective pattern. Therefore, AGE might be used as herbal medicine with few side effects as compared to chemotherapy in treating cancers caused by substances like AFB1.

55-64

Authors: Maryam Haji Ghasem Kashani , Mohammad Taghi Ghorbanian , Leili Hosseinpour

Objective: There is longstanding experimental and clinical evidence that supports the idea that replacement of dopaminergic (DAergic) neurons can ameliorate functional disabilities of Parkinson’s disease (PD). The purpose of the present study is to examine the efficacy of transplantation of rat bone marrow stromal cell (BMSCs)-derived tyrosine hydroxylase-positive (TH+) cells induced by deprenyl into 6-hydroxydopamine (6-OHDA)-lesioned rat models, using behavioral tests and immunohistochemical evaluations. Materials and Methods: In this experimental study, undifferentiated BrdU-labeled BMSCs were incubated in serum-free medium that contained 10-8 M deprenyl for 24 hours. Afterwards, BMSCs were cultured for 48 hours in α-minimal essential medium (α-MEM) supplemented with 10% FBS, then differentiated into TH+ neurons. We randomly divided 24 hemiparkinsonian rats as follows: group 1 (control) received only medium, while groups 2 and 3 were injected with 2×105 BMSCs and deprenyl-treated cells in 4 μl medium. Injections were made into the injured strata of the rats. Rotational behavior in response to apomorphine was tested before transplantation and at 2, 4, and 6 weeks post-graft. Animals were then sacrificed, and the brains were extracted for immunohistochemical and electron microscopic studies. Results: Apomorphine-induced rotation analysis indicated that animals with grafted cells in groups 2 and 3 exhibited significantly less rotational behavior than those in the control group at 2, 4, and 6 weeks after transplantation. Immunohistochemical analysis demonstrated that BrdU-labeled cells expressed specific neuronal markers, such as NF 200 and TH, at the implantation site. The presence of TH+ cells in conjunction with the reduction in rotation might show the capacity of grafted cells to release dopamine. Ultrastructural analysis revealed the presence of immature neurons and astrocyte-like cells at the graft site. Conclusion: TH+ neurons induced by deprenyl can be considered as a cell source for PD autograft therapy.

75-82

Authors: Farkhondeh Pouresmaeili , Javad Jamshidi , Eznollah Azargashb , Shahdokht Samangouee

Objective: Osteoporosis is a bone disorder that reduces bone mineral density (BMD) and leads to bone fracture. In addition to different factors, gene polymorphisms have been revealed to be associated with osteoporosis. In this study, we investigated the association between the BsmI polymorphism of vitamin D receptor (VDR) gene (rs1544410) and BMD in a population of Iranian women. Materials and Methods: In this case control study, clinical risk factors for osteoporosis were obtained from the participants through a questionnaire for a case-control study. The World Health Organisation (WHO) criteria were applied for the diagnosis of the disease. Peripheral blood samples were obtained from 146 pre- and or postmenopausal Iranian women aged between 35 and 71 years (53.53 ± 9.8). The study population was classified for BMD into normal and osteoporotic groups, who matched for age, pregnancy status, menstrual condition, and body mass index (BMI). The BMD of the lumbar spine (L1-4) and femoral neck was measured. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was performed to detect and analyze the genotype. Results: The frequencies of AA and GG were significantly different between the two groups (p value<0.05), with the first genotype being higher in the patients and the second being higher in the normal group. The GG genotype was significantly associated with increased BMD in the lumbar spine (p value<0.05) but non-significant in the femoral neck (p value>0.05). Conclusion: BsmI polymorphism of VDR gene has a significant association with BMD in the lumbar spine and may have a minor effect on the proximal femur BMD in Iranian women.

98-107

Authors: Samira Abbasi , Mehdi Edrisi , Amin Mahnam , Mahyar Janahmadi

Objective: 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. Materials and Methods: 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. Results: 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. Conclusion: 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.

116-123

Authors: Zahra Esfahanian , Mandana Behbahani , Mehrnaz Shanehsaz , Mohammad Javad Hessami , Mohammad Ali Nejatian

Objective: 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). Materials and Methods: 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). Results: 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. Conclusion: Grapevine fanleaf virus (GFLV) can potentially increase the cytotoxicity of grape cultivars.

130-135

Authors: Asghar Abdoli , Hoorieh Soleimanjahi , Masoumeh Tavassoti Kheiri , Abbas Jamali , Azam Jamaati

Objective: 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. Materials and Methods: 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. Results: 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. Conclusion: 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.

142-151

Authors: Azim Hedayatpour , Iraj Ragerdi , Parichehr Pasbakhsh , Laya Kafami , Nader Atlasi , Vahid Pirhajati Mahabadi , Soudabeh Ghasemi , Reza Mahmoudi

Objective: 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. Materials and Methods: 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). Results: 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. Conclusion: Our results indicate that ADSCs may provide a feasible, practical way for remyelination in diseases such as MS.

160-165

Authors: Qi Kong , Fangyun Sun , Xiuping Chen

Objective: 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. Materials and Methods: 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. Results: 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. Conclusion: 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.

176-181

Authors: Mohammad Sadraeian , Mohammad Javad Khoshnood Mansoorkhani , Milad Mohkam , Sara Rasoul-Amini , Mahdi Hesaraki , Younes Ghasemi

Objective: 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. Materials and Methods: 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. Results: 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. Conclusion: 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.

190-197

Authors: Hassan Rassouli , Shiva Nemati , Siamak Rezaeiani , Ali Sayadmanesh , Mohammad Reza Gharaati , Ghasem Hosseini Salekdeh , Hossein Baharvand , Hamid Gourabi

Objective: 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. Materials and Methods: 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. Results: 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. Conclusion: Our results showed no significant differences in function between laboratory produced and commercialized hLIF.

198-205

Authors: Zahra Armand , Gholamreza Najafi , Farah Farokhi , Ali Shalizar Jalali

Objective: Cyclosporine (Cs), a cyclic undecapeptide with potent immunosuppressive activity, causes several adverse effects including reproductive toxicity. This study aims to examine the ability of Crataegus monogyna aqueous fruit extract as an antioxidant to protect against Cs-induced reproductive toxicity. Materials and Methods: In this experimental study, 32 adult male Wistar rats were divided into four groups of eight animals each. Rats in two groups received 40 mg/kg/day Cs for 45 days by oral gavage. In addition, one of the two groups received Crataegus monogyna aqueous extract at a dose of 20 mg/kg/day orally four hours after Cs administration. The remaining two groups consisted of a vehicle treated control (Cont) group and a Crataegus monogyna control (Cr) group. Differences between groups were assessed by analysis of variance (ANOVA) using the SPSS software package for Windows. Results: Cs treatment caused a significant decrease in sperm count and viability with an increase in DNA damage and protamine deficiency of the sperm cells. We observed significant decreases in fertilization rate and embryonic development, in addition to an increased rate of embryo arrest in Cs-treated rats. Crataegus monogyna co-administration attenuated all Cs-induced negative changes in the above-mentioned parameters. Conclusion: Supplementation with Crataegus monogyna a queous fruit extract could be useful against reproductive toxicity during Cs treatment in a rat model.

212-217

Authors: Hamid Reza Momeni , Malek Soleimani Mehranjani , Mohammad Ali Shariatzadeh , Mahnaz Haddadi

Objective: Sensory neurons in dorsal root ganglia (DRG) undergo apoptosis after peripheral nerve injury. The aim of this study was to investigate sensory neuron death and the mechanism involved in the death of these neurons in cultured DRG. Materials and Methods: In this experimental study, L5 DRG from adult mouse were dissected and incubated in culture medium for 24, 48, 72 and 96 hours. Freshly dissected and cultured DRG were then fixed and sectioned using a cryostat. Morphological and biochemical features of apoptosis were investigated using fluorescent staining (Propidium iodide and Hoechst 33342) and the terminal Deoxynucleotide transferase dUTP nick end labeling (TUNEL) method respectively. To study the role of caspases, general caspase inhibitor (Z-VAD.fmk, 100 μM) and immunohistochemistry for activated caspase-3 were used. Results: After 24, 48, 72 and 96 hours in culture, sensory neurons not only displayed morphological features of apoptosis but also they appeared TUNEL positive. The application of Z-VAD.fmk inhibited apoptosis in these neurons over the same time period. In addition, intense activated caspase-3 immunoreactivity was found both in the cytoplasm and the nuclei of these neurons after 24 and 48 hours. Conclusion: Results of the present study show caspase-dependent apoptosis in the sensory neurons of cultured DRG from adult mouse.

224-229

Authors: Ali Neshasteh-Riz , Ali Mahmoud Pashazadeh , Seyed Rabie Mahdavi

Objective: To assess relative biological effectiveness (RBE) of 131I radiation relative to 60Co gamma rays in glioblastoma spheroid cells. Materials and Methods: In this experimental study, glioblastoma spheroid cells were exposed to 131I radiation and 60Co gamma rays. Radiation induced DNA damage was evaluated by alkaline comet assay. Samples of spheroid cells were treated by radiation from 131I for four different periods of time to find the dose-response equation. Spheroid cells were also exposed by 200 cGy of 60Co gamma rays as reference radiation to induce DNA damage as endpoint. Results: Resulted RBE of 131I radiation relative to 60Co gamma rays in 100 μm giloblastoma spheroid cells was equal to 1.16. Conclusion: The finding of this study suggests that 131I photons and electrons can be more effective than 60Co gamma rays to produce DNA damage in glioblastoma spheroid cells.

238-243

Authors: Mohammad Reza Bonyadi , Parvin Bastani

Objective: Toxoplasma gondii infection, an intracellular parasite, is often asymptomatic or is caused by different clinical diseases without being detected. Evaluation of IgG, IgA, and IgM in order to diagnose the pending Toxoplasmosis may confront some problems. Several researches has showned that Toxo IgG avidity can be useful in the recent active Toxoplasmosis. In current study, modification and importance of improved Toxoplasma Avidity IgG testing has been evlauated for differentiating Toxoplasma gondii infection in early stage of pregnancy. Materials and Methods: This experimental study included 300 pregnant women with risk of Toxoplasmosis in their initial months of pregnancy. We randomly divided 300 serum samples into A group (n=60) with high avidity and B group (n=40) with borderline avidity. The samples with Toxo IgG levels were classified to four subgroups. IgG avidity was evaluated by enzyme-linked immunosorbent assay (ELISA) method. Results: The mean absorbance of 100 samples in two groups was calculated, and then, two dilutaion curves with plotted absorbance against dilution were drawn for each serum sample. The results of this study showed that in groups with different concentrations of toxo IgG, appropriate dilution of serum is suitable for testing of Avidity. Our findings revealed the subgroups of 1, 2, 3, and 4 with serum dilutios of 1/3 , 1/6, 1/9, and 1/18 respectively, had real and good avidity. Conclusion: One of the issues affectig the results of avidity is high concenteration of Toxo IgG in serum sample. As shown in this study, the best points of dilution for well avidity in both high and borderline avidities are marked with arrows in figures 1-8. This study confirmed that improved methods of measuring Toxo Avidity IgG are very important.

250-257

Authors: Maryam Shahali , Maryam Kabir-Salmani , Karim Nayernia , Hamid Reza Soleimanpour-Lichaei , Mohammad Vasei , Seyed Javad Mowla , Ehsan Ranaie , Mehdi Shakibaie , Mohammad Hossein Modaresi

Objective: Piwil2, a member of Ago/Piwi gene family containing Piwi and PAZ domains, has been shown to be ectopically expressed in different cancer cells, especially its remarkable expression in cancer stem cells (CSCs), and is also known to be essential for germ line stem cell self-renewal in various organisms. The hypothesis that CSC may hold the key to the central problem of clinical oncology and tumor relapse leads to more anticancer treatment studies. Due to emerging controversies and extreme difficulties in studying of CSC, like the cells using in vivo models, more attempts have expended to establish different in vitro models. However, the progress was slow owing to the problems associated with establishing proper CSC cultures in vitro. To overcome these difficulties, we prompted to establish a novel stable cell line over-expressing Piwil2 to develop a potential proper in vitro CSC model. Materials and Methods: In this experimental study, mouse embryonic fibroblasts (MEFs) were isolated and electroporated with a construct containing Piwil2 cDNA under the control of the cytomegalovirus promoter (CMV). Stable transfectants were selected, and the established MEF-Piwil2 cell line was characterized and designated as CSC-like cells using molecular markers. Functional assays, including proliferation, migration, and invasion assays were performed using characterized CSC like cells in serum-free medium. Additionally, MEF-Piwil2 cell density and viability were measured by direct and indirect methods in normoxic and hypoxic conditions. Results: The results of reverse transcriptase-polymerase chain reaction (RT-PCR), western blot, and immunocytochemistry revealed an overexpression for Piwil2 in the transfected Piwil2 cells both in the RNA and protein levels. Furthermore, analysis of the kinetic and stoichiometric parameters demonstrated that the specific growth rate and the yield of lactate per glucose were significantly higher in the MEF-Piwil2 group compared to the MEF cells (ANOVA, p<0.05). Also, analysis of functional assays including migration and invasion assays demonstrated a significantly higher number of migrated and invaded cells in the MEF-Piwil2 compared to that of the MEF cells (ANOVA, p<0.05). The MEF-Piwil2 cells tolerated hypoxia mimetic conditions (CoCl2) with more than 95% viability. Conclusion: According to the molecular and functional studies, it has been realized that Piwil2 plays a key role(s) in tumor initiation, progression and metastasis. Therefore, Piwil2 can be used not only as a common biomarker for tumor, but also as a target for the development of new anticancer drug. Finally, the main outcome of our study was the establishment of a novel CSC-like in vitro model which is expected to be utilized in understanding the complex roles played by CSC in tumor maintenance, metastasis, therapy resistance or cancer relapse.

266-271

Authors: Hussain Habibi , Saeid Abroun , Abbas Hajifathali , Masoud Soleimani , Saeid Kaviani , Nasim Kalantari , Susan Eslahchi

Objective: Multiple myeloma (MM) is a plasma cell malignancy where plasma cells are increased in the bone marrow (BM) and usually do not enter peripheral blood, but produce harmful factors creating problems in these patients (e.g. malignant plasma cells over activate osteoclasts and inhibit osteoblasts with factors like RANKL and DKK). These factors are a main cause of bone lesion in MM patients. Recently SOST gene which responsible to encodes the sclerostin protein was identify. This protein specifically inhibits Wnt signaling in osteoblasts (inhibition of osteoblast differentiation and proliferation) and decrease bone formation and can also cause bone lesion in MM patients. Materials and Methods: In this experimental study, human myeloma cell lines (U266 b1) were purchased from Pasteur Institute of Iran. Samples consisted of BM aspirates from the iliac crest of MM patients. BM with more than 70% plasma cell were selected for our study (6 patients) and one healthy donor. RNA extraction was done with Qiagen kit. was undertaken on mRNA of samples and cell lines. Also we purchased unrestricted somatic stem cells from Bonyakhte Company to evaluate the effect of soluble factors from myeloma cell lines on osteogenic differentiation medium. Results: Our results showed that SOST is expressed significantly in primary myeloma cells derived from MM patients and myeloma cell lines. In other words, patients with more bone problems, express SOST in their plasma cells at a higher level. In addition, myeloma cells inhibit osteoblast differentiation in progenitor cells from umbilical cord blood stem cell (UCSC) in osteogenic inducing medium. Conclusion: There are many osteoblast maturation inhibitory factors such as DKK, Sfrp and Sclerostin that inhibit maturation of osteoblast in bone. Among osteoblast inhibitory agents (DKK, Sfrp, Sclerostin) sclerostin has the highest specificity and therefore will have less side effect versus non-specific inhibitory agents. Our results also show that based on SOST expression in MM, there is a potential to inhibit sclerostin with antibody or alternative methods and prevent bone lesion in MM patients with the least side effect.

1-6

Authors: Yousef-Reza Yousefnia Pasha , Seyed Gholam-Ali Jorsaraei , Mahtab Zeinalzadeh , Masoumeh Golsorkhtabar Amiri , Arsalan-Ali Ramaji

Objective: In sever oligospermia; one of the paths used for surgical sperm retrieval (SSR) is to extract sperm via a testicular biopsy. The aim of our study is to determine the reliable time interval between testicular biopsy and intracytoplasmic sperm injection (ICSI) procedure in order to obtain optimumsperm parameters (Count, motility and normal morphology). Materials and Methods: This cohort study was carried out on 30 patients which were candidates for ICSI. After collection and keeping the samples obtained from the testicular biopsy in Ham’s F10 environment, the concentration, motility and morphology of the sperm in each sample was evaluated immediately as well as 2 and 4 hours after processing. The Data were then compared with each other. For the statistical analysis, Friedman, Willcoxon and Cochran tests were used. Results: The mean of sperm concentration was 5.69 ± 6.14 million and the motility was10.83 ± 12.63% at 2 hours following biopsy which was significantly higher than those obtained after 0 and 4 hours of the biopsy (p <0.05). Conclusion: The reliable preincubation time which resulted in the highest rate of spermatozoa parameters after testicular biopsy and before incubation was 2 hours.

19-24

Authors: Mehri Hajikhan Mirzaei , Mehrdad Noruzinia , Hamid Karbassian , Yousef Shafeghati , Mousa Keyhanee , Ali Bidmeshki-Pour

Objective: Breast cancer is one of the most common malignancies in women worldwide. It is caused by a number of genetic and epigenetic factors. Aberrant hypermethylation of the promoter regions in specific genes is a key event in the formation and progression of breast cancers as well as the DBC2 gene, as a tumor suppressor gene. Different studies show that the DBC2 gene is inactivated through epigenetic mechanisms such as methylation in its promoter region. In this study, authors have tried to analyze methylation status in the promoter region of DBC2 gene in affected women and healthy controls. Materials and Methods: In this experimental study, we evaluated the methylation status of DBC2 gene with nested methylation-specific PCR (MSPCR) using specific methylated and unmethylated primer sets, in three separate PCR reactions. We used 50 tissue and blood samples of patients with breast cancer, 5 normal tissues and also 30 normal blood samples. Results were evaluated by the Mann-Whitney test, SPSS 16.0 statistical software. Results: Nested MSPCR results demonstrated that the frequency of the DBC2 promoter region methylation status in tumor and blood samples of the affected patients was significantly higher than that of the corresponding normal controls. Conclusion: DBC2 gene inactivation by methylation of CpG islands in the promoter region probably is a crucial step in the process of cell proliferation and susceptibility to different cancers, including breast cancer. Our study provides new evidence that aberrant methylation of the DBC2 gene is involved in the tumorigenesis of breast cancer. DNA methylation in this gene is proven to be a potential marker for tumor diagnosis and prognosis, as well as a novel therapeutic target.

31-38

Authors: Hoda Jahani , Saeid Kaviani , Majid Hassanpour-Ezatti , Masoud Soleimani , Zeinab Kaviani , Zahra Zonoubi

Objective: The development of combining mesenchymal stem cells (MSCs) with surface modified three-dimensional (3D) biomaterial scaffold provides a desirable alternative for replacement of damaged and diseased tissue. Nanofibrous scaffolds serve as suitable environment for cell attachment and proliferation due to their similarity to the physical dimension of the natural extracellular matrix. In this study the properties of plasma treated poly-C-caprolactone nanofiber scaffolds (p-PCL) and unaltered PCL scaffolds were compared, and then p-PCL scaffolds were evaluated for MSC culture. Materials and Methods: Aligned and random PCL nanofibrus scaffolds were fabricated by electrospining and their surface modified with O2 plasma treatment to enhance MSC proliferation, adhesion and interaction. Chemical and mechanical characterizations were carried out using scanning electron microscopy (SEM), water contact angle and tensile testing. Cell adhesion and morphology were evaluated using SEM 1 day after culture. Statistical analysis was carried out using one way analysis of variance (ANOVA). Results: The proliferation of MSCs were evaluated using 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide(MTT) assay on day 1, 3, and 5 after cell culture. Results showed that the numbers of cells that had grown on PCL nanofibrous scaffolds were significantly higher than those of control surfaces without nanofibers. Furthermore, the proliferation of MSCs on random nanofiber was significantly higher compared to that on aligned nanofiber. Conclusion: This study showed that while both aligned and random plasma treated PCL nanofibrous scaffold are more suitable substrates for MSC growth than tissue culture plates, random nanofiber best supported the proliferation of MSCs.

47-52

Authors: Atefeh Hemati , Mahnaz Azarnia , Mohammad Nabiuni , Ghadireh Mirabolghasemi , Saeed Irian

Objective: Medicinal plants are widely used throughout the world. Since these plants are known to have minimal side effects, many people embrace them. The golpar plant, scientifically known as Heracleum persicum (H. persicum), is a common Asian and Iranian medicinal plant. The use of golpar is recommended in traditional medicine as a contraceptive medication for females; however, no scientifically documented evidence has been reported. This study investigates the effects of the golpar plant on ovarian tissue and folliculogenesis. Materials and Methods: In this experimental study, H. persicum hydroalcoholic extract (HPHE) was used at 400 mg/kg and 1600 mg/kg doses. Adult female rats were divided into three groups: control, sham, and experimental(I, II). The control group did not receive any injection, the sham group received saline solution, and the experimental group received IP injections of HPHE for 21 days, once every other day, during the sexual cycle. At the end of the injection period, ovarian samples were harvested for histological studies. The FSH assay was performed according to the chemiluminescence immunoassay (CLIA) method. Data were statistically analyzed by the Instat3 program and one-way ANOVA. A p value of <0.05 was considered significant. Results: In the experimental group the numbers of primordial and primary follicles increased (p<0.001), while the number of preantral and antral follicles decreased (p<0.01). The atretic follicles decreased in the experimental group, but this decrease was not significant. There was no statistical difference in FSH concentration when compared with the control group. Conclusion: This report gives primary information on the in vivo effects of the HPHE on the ovarian follicles of the female Wistar rat. The results suggest that administration of HPHE may have inhibitory effects on folliculogenesis and cause infertility in females.

61-66

Authors: Maryam Sadat Nabavinia , Mahboobeh Naderi Nasab , Zahra Meshkat , Mohammad Derakhshan , Mehrangiz Khaje-Karamadini

Objective: Despite using the Bacille Calmette Guerin (BCG) vaccine, tuberculosis (TB) is still a worldwide disease that kills 2-3 million people each year. Developing a new and more effective vaccine is one way to possibly reduce the morbidity and mortality of TB. The Mtb72F vaccine is one of the important subunit vaccines applied in human clinical trials. In this study, we have constructed an expression vector that contains the Mtb72F fragment with some new modifications. Materials and Methods: In this experimental study, Mtb32N and Mtb39 fragments were amplified by polymerase chain reaction (PCR) using specific primers and inserted into pET21b\Mtb32C. Colony-PCR, restriction enzyme analysis, and DNA sequencing were used to confirm the accuracy of the cloning. We used Western blot to verify the desired protein expression. Results: The amplified fragments showed the desired size in PCR and digestion methods, and protein expression was confirmed using a monoclonal antibody. Conclusion: Our modification made it possible to insert another gene or gene fragments into the Mtb72F vector for developing new constructs. In addition, our data has shown that the placement of the histidine tag in the carboxyl- (C-) or amino- (N-) terminal part of a protein may influence protein expression and/or stability.

76-81

Authors: Touraj Farazmandfar , Hossein Khanahmad Shahreza , Mohammad Reza Haghshenas , Ghasem Janbabai , Hossein Azadeh , Nader Mansour Samaei

Objective: Lentivirus-derived vectors are among the most promising viral vectors for gene therapy which is currently available, but their use in clinical practice is limited due to associated risk of insertional mutagenesis. Gene targeting is an ideal method for gene therapy, but it has low efficiency in comparison to viral vector methods. In this study, we are going to design and construct an integrase-minus lentiviral vector. This vector is suitable for transient expression of gene and gene targeting with viral vector. Materials and Methods: In this experimental study, three missense mutations were induced in the catalytic domain of Integrase gene in the pLP1 plasmid and resulted D64V, D116A and E152G changes in the amino acid sequence through site directed mutagenesis. The pLenti6.2-GW/EmGFP transfer vector, associated with native and mutated packaging mix, was transfected into 293T cell line. In order to titer the lentivirus stock, the viruses were harvested. Finally, the viruses transduced into COS-7 cell line to assess green fluorescent protein (GFP) gene expression by a fluorescence microscopy. Results: Recombinant and wild lentiviruses titer was about 5~8×106 transducing units/ml in COS-7 cell line. The number of GFP-positive cells transduced with native viruses was decreased slightly during two weeks after viral transduction. In contrast, in the case of integrase-minus viruses, a dramatic decrease in the number of GFP positive cells was observed. Conclusion: This study was conducted to overcome the integration of lentiviral genome into a host genome. Nonintegrating lentiviral vectors can be used for transient gene expression and gene targeting if a Target gene cassette is placed in the lentivirus gene structure. This combination method decreases disadvantages of both processes, such as random integration of lentiviruses and low efficiency of gene targeting.

98-101

Authors: Majid Farshdousti Hagh , Mehrdad Noruzinia , Yousef Mortazavi , Masoud Soleimani , Saeid Kaviani , Maryam Mahmodinia Maymand

Objective: Mechanism of zoledronic acid on osteoblastic differentiation of mesenchymal stem cells (MSCs) has not fully understood. With the knowledge of some drugs mechanism that alter methylation pattern of some genes, the present research sets out to evaluate osterix (OSX) promoter methylation pattern during zoledronic acid-induced osteoblastic differentiation of MSCs. Materials and Methods: In this experimental study, MSCs were isolated from human bone marrow. For osteogenic differentiation, MSCs were pulse treated with 5μM Zoledronic acid for 3 hours and incubated after a medium change in osteogenic differentiation medium for 3 weeks. DNA and RNA were extracted on days 0, 7, 14 and 21 of MSCs differentiating to osteoblast. After cDNA synthesis, OSX expression was evaluated by RT-PCR and quantitative Real-Time PCR. After multiplicity of infection (MOI) treatment, gene specific methylation of OSX was analyzed by methylation specific PCR (MSP). Results: The mRNA expression of OSX was increased in osteoblast differentiated cells induced by zoledronic acid, especially on days 14 and 21 of differentiation (p<0.05), but expression of OSX didn’t change in undifferentiated MSCs. MSP revealed that, on day 0, undifferentiated MSCs are totally methylated. But, on day 7 of differentiation, MSCs treated by zoledronic acid were totally unmethylated. OSX promoter remained unmethylated, afterwards. Conclusion: MSP revealed that OSX had a dynamic pattern in methylation, while MSCs gradually differentiated to osteoblasts. Our finding showed that promoter region of OSX is hypomethylated independently from zoledronic acid treatment during osteoblastic differentiation. This knowledge is important to understand drug mechanisms and can be useful for developing new therapies to combat against bone diseases.

102-109

Authors: Pantea Izadi , Mehrdad Noruzinia , Morteza Karimipoor , Mohammad Hamid Karbassian , Mohammad Taghi Akbari

Objective: Breast Cancer is the most common cancer in Iranian women. Breast tumors are classified based on the estrogen receptor alpha (ERα) expression status into ER negative and ER positive tumors. ER negative tumors tend to have worse prognosis and less likely to respond to endocrine therapy. Aberrant methylation of gene promoter is one of the mechanisms for gene silencing in breast tumors. Because of its reversible nature, promoter methylation is a good target for new therapeutic strategies. We aimed to evaluate the frequency of this epigenetic event in ERα gene and its association to clinicopathological features in Iranian breast cancer patients. Materials and Methods: In this case control study the patient series consisted of 100 sporadic primary breast cancer cases (51 ER negative and 49 ER positive tumors). None of the participants had chemo or radiotherapy before surgery. In breast tumors ERα promoter methylation were assessed with methylation specific polymerase chain reaction (MSP). Data was collected on clinicopathological features of the patients. Correlation between ERα methylation and clinicopathological characteristics of the patients was investigated by Pearson Chi-Square and Fisher’s exact test. Results: ERα methylation was detected in 98% of ER negative and 65% of ER positive breast tumors. A strong correlation was found between ERα methylation and ER negativity in tumors (p<0.0001). Also, ERα methylation has associated to progesterone receptor negativity (p<0.008) and double receptor negative status (p<0.0001) in breast tumors. Conclusion: ERα methylation occurs with high frequency in the breast tumors of Iranian breast cancer patients and may play a considerable role in pathogenesis of ERα negative tumors as a poor prognosis and more aggressive category. The reversible nature of DNA methylation may provide new therapeutic possibilities in ER negative breast tumors.

116-121

Authors: Akram Hosseini , Firouz Ghaderi Pakdel , Abbas Ahmadi , Samad Zare Ph.D

Objective: This study aims to evaluate the protective effects of American ginseng administered by gastric intubation on sperm vital quality in adult male rats treated with cyclophosphamide (CP). Materials and Methods: In this experimental study, 28 Adult male Wistar rats were assigned to four groups, seven rats in each. The animals allocated to control, CP treated, Ginseng treated and CP-Ginseng treated groups. Rats were treated with CP (6.1 mg/kg/day, i.p) for 6 weeks. American ginseng was used at a dose of 500 mg/kg/day during treatment. Sperm analysis (motion, count, morphology and viability) were evaluated at the end of the experiments. Sperm motion was assessed by Computer-Assisted Sperm Analysis (CASA). The data were analyzed using GB stat software. Probability values of p<0.05 and p<0.01 were considered significant. Results: The epididymal sperm counts in the groups that received CP showed significant decreases compared to the control group. Also dead and abnormal sperms significantly increased following CP treatment compared with control. The motility of caudal sperm was reduced significantly with CP treatment. Therefore, according to the results of this study, co-administration of CP and American ginseng can improve these parameters. Conclusion: American ginseng can prevent the cytotoxic effects of CP on sperm quality factors.

130-141

Authors: Mohammad Mehdi Hassanzadeh Taheri , Ali Reza Ebrahimzadeh Bideskan , Mohammad Reza Miri

Objective: The development of vertebrae is a complex phenomenon that is correlated with distinct morphological and biochemical alterations in the paraxial mesenchyme and glycoconjugates. The purpose of this study is to investigate the glycosylation pattern in paraxial mesenchyme-forming vertebrae by using the lectin histochemical technique. Materials and Methods: In this descriptive-analytic study, B4G fixed paraffin sections of 9 to 15 day Balb/c mouse embryos were processed for histochemical studies using seven different HRP-labelled lectins: Glycin max (SBA), Maclura pomifera (MPA), Wistaria floribunda (WFA), Vicia villosa (VVA) which all of them are specific for N-acetylgalactosamine (GalNAc), Ulex europius (UEA1, binds to α-L-fucose), wheat germ agglutinin (WGA, binds to sialic acid), and Griffonia simplicifolia (GSA1-B4, binds to galactose terminal sugars). The sections were observed separately by three examiners who were blinded to the lectins. Grading was done according to the intensity of the tested lectins’ reactions with the specimen, from negative (-) to severe (+++). Data was analysed with SPSS software (version 11.5) and the non-parametric Kruskal Wallis test; p<0.05 was considered significant. Results: Our findings showed that among the tested lectins, only GalNAc residue sensitive lectins showed regulated changes in paraxial mesenchyme. Reactions of WFA and MPA lectins with paraxial mesenchyme were severe on GD9. Reactions of WFA continued to GD15 constantly, while MPA reactions continued strongly to GD12, significantly decreased thereafter (p<0.001), and then disappeared. VVA and SBA bindings initiated weakly on GD10 and continued to GD12 without changing. These reactions increased significantly (p<0.001) thereafter, became severe to GD14, and later disappeared. The other tested lectins did not reveal regulated changes. Conclusion: According to these findings it can be concluded that only the GalNAc terminal sugar showed temporally regulated changes during the early embryonic development of vertebrae in mice. Therefore it most likely plays a key role (s) in the development of vertebrae, especially in the conversion of mesenchymal cells into chondroblasts. The other tested terminal sugars may have no role in this phenomenon.