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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-α 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.

Asthma and Chronic Obstructive Pulmonary Disease (COPD) are two important lung and airways diseases which affect the lives of ~500 million people worldwide. Asthma is a heterogeneous disease that is broadly defined as a clinical syndrome characterized by altered lung function, mucus hypersecretion, peribronchial inflammation and hyperresponsiveness In contrast, the effect of inhalation of toxic particles and gases on the innate and adaptive inflammatory immune systems underlie the pathogenesis of COPD. In the last decade, knowledge concerning the pathophysiologic mechanisms underlying asthma and COPD has risen tremendously and current dogma suggests that the pathogenesis of both diseases is driven by the chronic inflammation present in the airways of these patients. Thus, understanding the mechanisms for the persistence of inflammation may lead to new therapeutic approaches. In this review, we provide an overview of the main signal transduction pathways implicated in asthma and COPD pathophysiology focusing on inflammasome signaling in various cells types which result in altered inflammatory mediator expression.

Aflatoxins are primarily produced by the fungi Aspergillus flavus and Aspergillus parasiticus, which contaminate a wide variety of food and feed commodities including maize, oilseeds, spices, groundnuts, tree nuts, milk and dried fruits [Strosnider et al., 2006]. Presence of aflatoxins in food chain is associated with decrease in quality and quantity of food and feed materials. In addition, consumption of aflatoxin-contaminated products can pose a risk of development of various diseases in human and animals. Aflatoxins are produced in toxigenic fungi after undergoing biosynthesis pathway involving several enzymes and reactions. Upon consumption of aflatoxin contaminated products by human and animals, the toxin undergoes metabolism via cytochrome P450 enzymes in the liver. Aflatoxin metabolism in mammalian organs is a committed process and different metabolites are produced which can exert adverse effects of toxic metabolites. Aflatoxin epoxide (8,9-epoxide) is the major toxic metabolite which can bind to DNA and induce hepatocellular carcinoms. The extent of aflatoxin toxicity and carcinogenicity in human and animals depends on several factors including the metabolic capacity of the organism. Aflatoxin contamination of food products is associated with health and socioeconomic costs which is difficult to valuate in the developing countries. Moreover, the current regulations do little to help reduce aflatoxin and related health effects. Therefore the focus should be on promoting the adaptation of strategies that can control aflatoxin and its associated health risks. According to Wu and Khlangwiset (2010), interventions to reduce aflatoxin-induced illness can be grouped into three categories; agricultural, dietary and clinical. Agricultural interventions are methods that can be applied either in the field (preharvest) or in drying, storage and transportation (postharvest) to reduce aflatoxin levels in food. The dietary and clinical interventioans are considered as secondary interventions by which the aflatoxinrelated illness can be reduced. These two types interventions are associated with advantages and disadvantages. Due to concern for the potential effects of aflatoxins on human health, most countries have legislation that restricts marketing of aflatoxin-contaminated grains [Van Egmond, 1989]. The United States Food and Drug Administration has set an aflatoxin limit of 20 μg/kg for

There are several issues to be considered to reduce the risk of rejection and minimize side effects associated with liver cell transplantation in chronic liver diseases. The source and the condition of stem cell proliferation and differentiation ex vivo and the transplantation protocols are important safety considerations for cell based therapy. The biochemical and molecular markers are important tools for safety evaluation of different processes of cell expansion and transplantation. Studies show that hepatocytes differentiated from adult and embryonic stem cells exhibit biochemical and metabolic properties resembling mature hepatocytes. Therefore these assays can help to assess the biological and metabolic performance of hepatocytes and progenitor stem cells. The assays also help in testing the contribution of transplanted hepatocytes in improving the repair and function of damaged liver in the recipient. Here we review the biochemical and metabolic markers, which are implicated in evaluation of safety issues of stem cells used for therapeutic purposes in chronic liver diseases and regeneration of damaged liver. We also highlight application of biochemical tests for assessment of liver cell transplantation.

Silymarin, a mixture of flavonolignans, is extracted from milk thistle (Silybum marianum) and has a strong antioxidant activity and exhibits anticarcinogenic, anti-inflammatory, and cytoprotective effects. In this study we attempted to determine whether silymarin and the glutathione modifiers, buthionine sulfoxamine (BSO) and N-acetylcysteine (NAC), are involved in regulation of cyclooxygenase (COX)-2 and inducible nitric oxide synthase (iNOS) in proliferating mesenchymal stem cells (MSCs). Cellular glutathione was manipulated during a 14-day culture using BSO, NAC and silymarin. At intervals of 2, 7 and 14 days, cells were collected and COX-2 and iNOS levels were measured. In parallel, generation of cellular H(2)O(2) and glutathione were measured. Supplementation of the culture media with BSO caused a dose-dependent decrease in MSC proliferation, whereas NAC or silymarin elevated the proliferation (p0.05). Treatment of MSC with NAC or silymarin caused a significant decrease in COX-2 levels. However, COX-2 levels in cells treated with high levels of NAC (1.0 mM) were significantly lower than those in MSCs treated with high levels of silymarin (100 μM). BSO (1.0 and 5.0 μM) caused a significant increase in COX-2 on days 2, 7 and 14. BSO caused a significant increase in iNOS, whereas NAC or silymarin decreased cellular iNOS. Overall result show that glutathione, iNOS and COX-2 in proliferating MSCs are affected by silymarin treatment. It appears that glutathione is the main target of silymarin, and in consequence iNOS and COX-2 are affected in response to silymarin treatment.

The effects of dietary essential oils prepared from caraway seeds on colon carcinogenesis induced by 1,2-dimethylhydrazine (DMH) in rats has been studied. The number of aberrant crypt foci (ACF) and aberrant crypt (AC) induced by DMH were found to be significantly inhibited in colon of rats treated with essential oils in diet (0.01 and 0.1%). To find out the mechanism(s) by which the essential oils reduced colon premalignancies, plasma, liver, and colon tissues were collected and analyzed for parameters related to oxidative stress and xenobiotic metabolizing enzymes. Lack of influence of caraway extracts on hepatic lipid peroxidation products, superoxide dismutase (SOD), catalase (CAT) and ferric reducing ability of plasma (FRAP) may suggest that the oils do not interfere with these factors. However, it was clearly shown that DMH-related changes in hepatic and colonic cytochrome P4501A1 (CYP1A1) and glutathione S-transferae (GST) activities were recovered in liver but not in colon tissue in animals treated with caraway oil preparations. In conclusion, histopathological and biochemical data clearly showed that inhibition of colon premalignant lesions induced by DMH is mediated by interference of caraway oil components in the activities of the main hepatic xenobiotic metabolizing enzymes.

Carcinoma antigen 125 (CA 125) is overexpressed in ovarian cancer and antibodies against it are widely employed for diagnostic purposes. The rarity of CA 125 antigenic domains and its highly glycosylated structure, however, is a problem that may prevent immunized mice from developing a diversified population of anti-CA 125 antibodies. In this study a prime-boost strategy, which potentially could augment the humoral immune responses against rare and poorly immunogenic determinants, was used for immunization of mice and monoclonal antibodies (mAbs) were produced by hybridoma technology. Reactivity of mAbs was then assessed by ELISA, western blotting, immunoprecipitation, immunohistochemistry and immunofluorescence staining of OVCAR-3 cell line. Altogether, 10 clones were produced, 3 of which had IgG isotype and the rest were IgM. Two-third of clones recognized cognate antigen in fixed and living cells and had strong immunoreactivity in IHC staining. In Western blotting, our antibodies recognized CA 125 as high molecular weight antigen mostly migrated in the 3% stacking gel. Immunoprecipitation of OVCAR-3 cell lysate by mAbs resulted in a very similar migration pattern that reconfirmed their specificities. The mAbs produced in this study are invaluable tools in diagnosis and research fields for assessment of CA 125 expression in cancerous ovarian tissues.

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 (p0.05). 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.

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.

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 masculineC 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 microg/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 less than 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.

Background: There is significant interest in using nanofibers in tissue engineering from stem cells. The transdifferentiation of mesenchymal stem cells into the hepatic lineage in a nanofibrous structure has not been reported. In this study, a three dimensional nanofibrous scaffold is introduced for differentiation of human bone marrow derived mesenchymal stem cells (hBMSCs) into hepatocytes. Methods: A scaffold composed of Poly (e-caprolactone), collagen and polyethersulfone was fabricated by the electrospinning technique. After characterization of isolated hBMSCs, the performance of the cells on the scaffold was evaluated by Scanning Electron Microscopy (SEM) and MTT assay. Cytological, molecular and biochemical markers were measured to confirm differentiation potential of hBMSCs into hepatocytes. Results: The isolated cells possessed the basic properties of mesenchymal stem cells (MSCs). Based on scanning electron microscope (SEM) analysis and MTT assay, it was shown that the cells adhere, penetrate and proliferate on the nanofibers. Cultured cells on the nanofibers differentiated into hepatocyte-like cells and expressed hepatocyte specific markers such as albumin, α-fetoprotein, cytokeratin-18, cytokeratin-19 and cytochrome P450 3A4 at mRNA levels. Appearance of a considerable number of albumin-positive cells cultivated on the scaffold (47 ± 4%) as compared to the two-dimensional culture system (28 ± 6%) indicates the supporting role of the scaffold. The efficiency of the cells to produce albumin, urea, transferrin, serum glutamic pyruvic transaminase and serum oxaloacetate aminotransferase in hepatocytes on the scaffold further attest to the functionality of the cells. Conclusion: The data presented in this study show that the engineered nanofibrous scaffold is a conductive matrix which supports and enhances MSC development into functional hepatocyte-like cells.

The aim of this study was to find out substitution effect of fetal bovine serum (FBS) with human platelet releasate (HPR) as a major growth factor source during expansion and differentiation of human bone marrow-derived mesenchymal stem cells (hBMSC) into hepatocytes.Propagation and differentiation potential of hBMSCs into hepatocyte-like cells in a medium fortified with HPR instead of FBS were investigated with morphological, cytochemical and molecular experiments.Multiplex analysis showed that HPR was more efficient than FBS in supporting hBMSC outgrowth. The proliferation rate of MSC in presence of HPR (derived from 10(9) platelets/ml) was about threefold greater than that of FBS (P0.001). Despite the differences in MTT value, hBMSCs-driven HPR or FBS did not differ in terms of gross morphology, immunophenotype and osteogenic differentiation potential. Hepatic differentiation of hBMSCs was successfully performed in the media enriched with HPR. Immunoreactivity of cells with monoclonal antibodies against for albumin and alpha-fetoprotein (AFP) was even more positive in hepatocytes differentiated in presence of HPR as compared to that of FBS. The gene expression of albumin, AFP and cytokeratin-18 at mRNA levels in differentiated cells attest to supporting role of HPR in hepatic differentiation media. These findings were further confirmed with greater urea production (approximately twofold) in the culture media of cells differentiated under HPR compared to that in FBS (P0.001).Human platelet releasate is an efficient and safe substitute for FBS in culture media used for expansion and differentiation of hBMSCs to hepatocyte.

This study was to investigate the effect of sodium selenite (SS) on in vitro maturation of mouse preantral follicles.The isolated preantral follicles were cultured in TCM 199 medium supplemented with different concentrations (0, 5, 10, 15 ng/ml) of SS and 3 mg/ml bovine serum albumin (BSA) or 5% Fetal Bovine Serum (FBS). The ovulation was induced by addition of 1.5 IU/ml human chorionic gonadotropin. The size and development of follicles and oocytes were assessed by calibrated eyepiece.The survival rates of follicles in FBS supplemented groups containing 5 and 10 ng/ml SS (88.23%, 90.83%) were higher than other groups (P0.05 and P0.001 respectively). The mean diameter of follicles (199.84 +/- 15.58 microm) and the percentage of MII oocyte (33.08%) were higher in FBS supplemented group containing 10 ng/ml SS (P0.001).The sodium selenite and FBS improve the in vitro growth and maturation of mouse preantral follicles.

Aflatoxins are secondary metabolites that are produced by toxigenic strains of some Aspergillus species on foods. Neem plant is a known inhibitor of aflatoxin production. We studied the effects of different concentrations of aqueous neem leaf extract on fungal growth and aflatoxin production by Aspergillus parasiticus (NRRL 2999) at different incubation times. The toxigenic fungus was cultured on sucrose low salts medium in the presence of various concentrations of extracts (0.2, 0.8, 3.12, 12.5 and 50% v/v). After shaking incubation of cultures for 2, 4, 6, 8, 10 and 12 days at 28 degrees C, the fungal mycelia was collected and processed for determination of dry weight. Mycelia and culture media were assayed by TLC method to detect aflatoxin B(1) (AFB(1)). The extracts did not have any obvious effect on fungal growth. AFB(1) production in the control samples increased to the maximum level on the 8th day. The inhibition of aflatoxin synthesis by plant extracts was found to be time- and dose-dependent. The maximum inhibitory effect was 80-90% in the presence of 50% concentration that when compared with control samples was significant (P < 0.05). AFB(1) secretion/production ratio in all of control and treated samples, other than 2nd day, approximately stayed and neem had no effect on it.

In this study, two techniques were used to compare the specific activity and total concentration of mycelial glutathione S-transferase (GST) in fungal strains isolated from natural sources. The fungi identified as Aspergillus parasiticus and Aspergillus flavus have been divided into two groups based on their ability to produce aflatoxins. Altogether 26 fungi were isolated, among which 12 were capable of producing varying levels of aflatoxin and 14 were proved to be non-toxigenic. GST specific activity in mycelial preparation was measured spectrophotometrically using 2,1-chloro-2,4-dinitrobenzene as the substrate. The results showed that the mean GST activity in toxigenic isolates was 25.06 +/- 9.8 micromol/mg protein/min which was 2.8-fold greater than that measured in non-toxigenic isolates (8.84 +/- 5.5 micromol/mg protein/min). Moreover, the GST concentration was compared in toxigenic and non-toxigenic isolates using an Enzyme Linked Immunosorbent Assay based on antigen (fungal preparation) and antibody (antibody produced against fungal GST in rabbit). The results of ELISA showed that the mean GST level in toxigenic and non-toxigenic fungi was 1.17 +/- 0.55 and 0.40 +/- 0.24, respectively. These results further confirm that the aflatoxin production in the fungal strains is correlated with GST expression and using ELISA, it is possible to discriminate aflatoxin-producing fungi from their non-toxigenic counterparts.

Neuroserpin, a member of the Serine Proteinase Inhibitor (Serpin) superfamily, is known to be a neuroprotective factor in the focal ischemic stroke followed by reducing the microglial activation. Neuroserpin is a protein rich of methionine residues that can scavenge the free radical species which may increase its neuroprotective effect. On the other hand, the oxidative modifications of the amino acid residues in neuroserpin may lead to changes in its conformation and function. In this study, it was investigated the changes in the conformation and the function of the oxidized neuroserpin.Neuroserpin expressed in E. coli, BL21 or M15 harboring plasmid pQE81L containing neuroserpin cDNA. Expressed neuroserpin was purified by resin sulfopropyl A50 precharged with 0.1 M NiSO4 under denaturing condition. Neuroserpin was oxidized under oxidative stress condition in the presence of different concentration of hydrogen peroxide. The oxidation of neuroserpin was conveniently detected by a carbonyl content assay using 2, 4 dinitrophenylhydrazine. Changes in tertiary structure of neuroserpin were monitored by spectrofluorimeter to study the alteration of intrinsic fluorescence and also fluorescence of 8-anilinonaphthalin-1 sulfonic acid (ANS) in native and oxidized form of neuroserpin.Total expressed neuroserpin was estimated 4-5 mg/lit in 2XYT culture media. SDS-PAGE analysis of purified neuroserpin showed a single band which reflects the efficiency of the resin SP A50 for purification of the proteins containing 6xHis tag. Carbonyl content of oxidized and native neuroserpin was estimated 12.3 +/- 0.3 and 0.45 +/- 0.05, respectively. The inhibitory activity of oxidized neuroserpin decreased up to 40-60% as compared with native form of neuroserpin. Intrinsic fluorescence and also the emission of ANS bind to the hydrophobic region of the protein altered from 380 to 85 and in the case of ANS from 105 to 150 in oxidized and native form of neuroserpin, respectively.The decreased intrinsic fluorescence intensity, an enhancement in the fluorescence of ANS, and loss of the inhibitory activity up to 40-60% in neuroserpin, all suggested a conformational modification in the protein under the oxidative stress condition. Remaining the inhibitory activity of neuroserpin reflects that the protein tolerates the oxidative stress condition effectively.

To develop a RT-PCR technique coupled with Enzyme Linked Immunosobant Assay (ELISA) i.e. RT-PCR-ELISA for measurement of class-Pi glutathione S-transferase (GST-P)-specific mRNA in esophageal diseases.In this study, 66 esophageal tissue biopsies diagnosed as non-erosive reflux disease (NERD), gastroesophageal reflux disease (GERD), adenocarcinoma (ADC), and squamous cell carcinoma (SCC) were used. Standardization of the RT-PCR-ELISA was carried out using specific GST-Pi and beta-actin primers, biotin labeled probe, DIG-labeling RT-PCR and anti-DIG-HRP conjugate.The results of RT-PCR-ELISA based on OD ratio of GST-Pi mRNA/beta-actin showed that there was no significant difference in GST-Pi expression in normal, NERD and GERD samples. Overexpression of GST-Pi in malignant tissues (ADC and SCC) was distinguishable. The OD ratio of GST-Pi mRNA expression to beta-actin mRNA was 1.17+/-0.13 and 1.3+/-0.13 in ADC and SCC samples, respectively, which is significantly higher (P0.05) than matching control (0.78+/-0.06 and 0.85+/-0.07).RT-PCR-ELISA showed that GST-Pi expression was not altered in GERD and NERD esophagus, whereas, in ADC and SCC samples, it was significantly higher (P0.05) as compared to inflamed and normal tissues.

The effect of aflatoxin B1 (AFB1) on the expression of glutathione S-transferase-P (GST-P) which is the major isoform of GST in developmental stages has been investigated in rat liver during prenatal and postnatal stages. Following administration of AFB1 (0, 0.5, 1.0, 2.0, 3.0 or 4.0 mg/kg bw) injected I.P on day 8.5 of gestation the number of dead or reabsorbed fetuses and malformed embryos were recorded. Then the fetal livers were processed for measurement of total GST and GST-P activities, using 1-chloro-2,4-dinitrobenzene (CDNB) and ethacrynic acid (ETA) as substrates respectively. RT-PCR using rat GST-P specific primers was performed on mRNA extracted from livers. Besides, the effects of AFB1 on hepatic GST and GST-P were assessed in groups of suckling rats directly injected with the toxin. The results show that a single dose of AFB1 (1.0 or 2.0 mg/kg bw) caused approximately 50-60% depletion in fetal liver GST towards CDNB. Postnatal experiments revealed that liver GST (using CDNB as substrate) was significantly induced (approximately 40%) in suckling rats injected with a single dose of AFB1 (3.0 mg AFB1/kg) 24 h before killing. Liver GST-P expression was unaffected due to AFB1 exposures of rats before and after the birth. This finding was substantiated by western blotting and RT-PCR techniques. These data suggest that AFB1-related induction in rat liver total GST after birth may be implicated in protective mechanisms against AFB1. In contrast, inhibition of this enzyme in fetal liver following placental transfer of the carcinogen may explain high susceptibility of fetal cells to trans-plancental aflatoxins. Furthermore, lack of influence of AFB1 on GST-P expression in developmental stages can role out the involvement of this class of GST in AFB1 biotransformation.

In contrast to the murine and human VHs, camels' single domain antibodies (sdAb) have sufficient solubility. These antigen-specific fragments are expressed well in Escherichia coli. Here, we report high expression and purification of sdAbs against MUC1 mucin. MUC1 is a high molecular weight glycoprotein with an aberrant expression profile in various malignancies. The sdAb genes were sub-cloned into a pET32a(+) vector to overexpress the protein coupled with fusion tags in E. coli BL21(DE3). The expressed single domain antibodies were purified by immobilized metal affinity chromatography and antigen affinity chromatography. Analysis by SDS-PAGE and Western blotting demonstrated the integrity of the sdAbs-tags, while ELISA results confirm that the activity of these molecules compare favorably with that of the parent recombinant antibodies. Enterokinase treated sdAb showed a band at the molecular weight around 12 kDa which demonstrated the naked protein in its natural structure with activities comparable to that of native protein. The high binding activity to MUC1 antigen purified from ascitic fluid (of patients with small-cell lung aggressive carcinoma and metastasis to peritoneum) and the very close similarity of these molecules to human VHs illustrated the potential application of these novel products as an immunodiagnostic and immunotherapeutic reagent.

The stem cells can be considered as the miracle of creation, because, they are a major reservoir of the bioactive factors, molecules and cells. These reservoir cells play a major role in development and growth of the tissues and organs. Regardless of the differences in differentiation capacity of embryonic and adult stem cells, both the cells are rich sources of stem cells with therapeutic application. Moreover, manipulation of adult stem cells and reprogramming or reverting embryonic-like state cells can be considered as additional tools for stem cell applications. Stem cells from different sources with different potential (plasticity) may be used to obtain ideal cell lineage with a defined function. Bone marrow, embryo and cord blood are the major stem cell sources which are believed to act systemically for regeneration of tissues and organs in an organism. In addition to embryonic and adult stem cells, there are other sources such as skin, which is a good source of adult stem cells involved in local repair and regeneration of epidermal and dermal tissues (1). Embryonic stem cells (ES): ES cells are pluripotent stem cells derived from the inner cell mass of a blastocyst. The pluripotency, and their ability to replicate indefinitely are two distinctive properties of the ES. ES cells are readily differentiated into primary germ layers, viz., ectoderm, endoderm, and mesoderm. Pluripotency distinguishes ES cells from adult stem cells which are multipotent cells with the capacity to produce limited number of cell types (2). The application of pluripotent stem cells in treatment of blood and immune-system related genetic disorders such as; juvenile diabetes; Parkinson's; blindness, spinal cord injuries and cancers is well established. The transplantation of oligodendrocytes derived from human ES cells into spinal cord-injured individuals was the world's first human ES cell human trial approved by FDA. The diversity of the stem cells present in different tissues implies that the body reserves all the cell types with stemness properties to guarantee important physiological processes and regeneration of organs. Placental stem cells: The placenta has become useful source of stem cells that offer advantages in terms of proliferation and plasticity when compared with adult cells (3). The epithelia, haematopoietic and mesenchymal stem cells are major stem cells isolated from the placenta. Adult stem cells: The pluripotency of adult stem cells isolated from different tissues was approved by showing their differentiation potential into cell types from different germ layers. For example, neural stem cells derived from ectoderm can differentiate into ectoderm, mesoderm, and endoderm. Bone marrow: Bone marrow is a rich source of two important adult stem cell lineages: Hematopoietic Stem Cells (HSCs) and Mesenchymal Stem Cells (MSCs). Hematopoietic stem cells (HSCs); HSCs can give rise to all the blood cell types. The HSCs with self-renewal capacity can differentiate into different types of blood cells, including those involved in the human immune system. There are three types of HSC transplants; syngeneic, autologous, and allogeneic transplants. Human bone marrow grafting is currently considered as significant progress in HSCs therapy. HSCs are also applied in reconstitution of damaged hematopoietic cells and restoring the immune system following chemotherapy in certain diseases. Mesenchymal Stem Cells (MSCs): There are different sources of MSCs and their isolation is relatively easy. The placenta, adipose tissue (4), lung, bone marrow and blood, Wharton's jelly from the umbilical cord, dental pulp and periodontal ligament are often used to isolate MSCs. These cells can be differentiated into various cell lineages and considering their rapid proliferation, multipotency and immunomodulatory properties they are widely used for treatment of various diseases. So far several cell types have been differentiated from MSCs. Mammary stem cells: These cells provide the source of cells for growth of the mammary gland during puberty and gestation. These cells have been isolated from human and mouse tissue as well as from cell lines originated from the mammary gland that can give rise to both the luminal and myoepithelial cell types of the gland. Neural stem cells: The presence of stem cells in the mature animal brain tissue indicates that during the neurogenesis process generation of new neurons continues into adulthood. The subventricular zone, which lines the lateral ventricles, and the dentate gyrus of the hippocampal formation are two major regions of the brain comprising adult neurogenesis. The generation of new neurons in the hippocampus is well established but under certain conditions such as tissue injuries in ischemia, neurogenesis can be induced in other regions in the brain. Testicular cells or spermatogonial progenitor cells: These multipotent stem cells have been derived from spermatogonial progenitor cells found in the testicles. Also multipotent stem cells have been derived from germ cells found in testicles which are known as Human Adult Germline Stem Cells (GSCs). The stem cells and the cells derived from them are capable of directly contribute to organ and tissue repair and regeneration. Intestinal stem cells, olfactory adult stem cells and hair follicles cells are other sources which have been used to isolate different stem cells. The advantage of these cells is that they can be harvested with ease from the patients. Perhaps, with the advancements made in the stem cell technologies, the scientists are becoming more optimistic about the therapeutic potential and safety of the stem cell therapy. The existence of stem cells in different tissues, with proliferation and differentiation capacity indicates that activation of these cells is regulated via pathways linked to development, repair and regeneration. However despite the advancements made in stem cell differentiation, no studies have succeeded in generating a functional organ such as a human liver. The very recent breakthrough in generation of a three-dimensional vascularized and functional human liver from human pluripotent stem cells (iPSCs) by transplantation liver buds made in vitro-grown organ buds is promising for chronic liver diseases (5). Implication for health policy/practice/research/medical education: The implication of this study is to know recent developments in the stem cell technology and applications. To briefly describe the importance of different types of stem cells with their differentiation potential in cell therapy and regeneration medicine. Authors’ Contribution: All the manuscript prepared by the author. Financial Disclosure: The author declares no competing financial interests.

Objectives: Persistent hepatitis B virus (HBV) infection often leads to the development of chronic hepatitis and cirrhosis. The role of host genetic factors in chronic HBV infection is not fully understood. We studied the influence of glutathione S -transferase (GST) M1, T1, and P1 polymorphisms in patients with different stages of HBV infections. Methods: The sample population included 41 HBV normal carriers, 37 patients with chronic hepatitis, and 38 patients with cirrhosis (infected with HBV) compared to a control group ( n = 59). PCR-based procedures were performed in the studied populations to confirm the genotypes of GSTT1, M1, and P1. Odds ratio analysis tests were used for statistical evaluation. Results: We found that the frequency of GSTP1-Val (105)/Val (105) genotype was significantly higher in patients with liver cirrhosis (27%) than HBV normal carriers (2.4%; OR 14.8, 95% CI 1.8–122.5) and the frequency GSTP1-Val (105)/Ile (105) genotype was significantly higher in patients with liver cirrhosis (59.5%) than HBV normal carriers (19.5%; OR 6.1, 95% CI 2.1–16.7). The genotype GSTP1-Val (105)/Val (105) was more frequent in patients with chronic hepatitis (19.4%) than HBV normal carriers (2.4%; OR 9.65, 95% CI 1.1–82.8). Patients with cirrhosis also had a higher frequency of the GSTM1 null genotype (71.1%) than HBV normal carriers (27.5%; OR 6.5, 95% CI 2.4–17.4) and the GSTM1 null genotype was more frequent in patients with chronic hepatitis (64.9%) than HBV normal carriers (27.5%OR 4.9, 95% CI 1.8–12.8). The frequency of GSTT1 genotype was similar in all groups. Conclusion: These results suggest that in HBV infection, inheritance of the null GSTM1 and GSTP1-Val (105) polymorphisms involves a host genetic factor that is relevant to disease progression.

Recently, the existence of "heavy-chain" antibody in Camelidae has been described. However, as yet there is no data on the binding of this type of antibody to peptides. In addition, there was not any report of production of single-domain antibodies in two-humped camels (Camelus bactrianus). In the present study, these questions are addressed. We showed the feasibility of immunizing old world camels, cloning the repertoire of the variable domain of their heavy-chain antibodies, panning and selection, leading to the successful identification of minimum-sized antigen binders. Antigen-specific fragments of the heavy-chain IgGs (V(HH)) are of great interest in biotechnology because they are very stable, highly soluble, and react specifically and with high affinity to the antigens. In this study, we immunized two camels (Camelus dromedarius and Camelus bactrianus) with homogenized cancerous tissues, synthetic peptide, and human milk fat globule membrane (HMFG), and generated two V(HH) libraries displayed on phage particles. Some single-domain antibody fragments have been isolated that specifically recognize the tandem repeat region of MUC1. The camels' single-domain V(HH) harbor the original, intact antigen binding site and reacted specifically and with high affinity to the tandem repeat region of MUC1. Indeed soluble, specific antigen binders and good affinities (in the range of 0.2 x 10(9) M(-1) to 0.6 x 10(9) M(-1)) were identified from these libraries. This is the first example of the isolation of camel anti-peptide V(HH) domains.

The mode of action of the extracts prepared from neem plant i.e., Azadirachta indica on aflatoxin formation in toxigenic Aspergillus species is not well understood. Aflatoxin production by A. parasiticus was suppressed depending on the concentration of the plant aqueous extract (0, 1.56, 3.12, 6.25, 12.5, and 50% v/v) added to the culture media at the time of spore inoculation. Aflatoxin production in fungal mycelia grown for 96 h in culture media containing 50% neem leaf and seed extracts was inhibited by approximately 90 and approximately 65% respectively. Under similar conditions, culture media amended with 1.56% of leaf or seed extract caused approximately 23 and approximately 7% inhibition respectively. Mycelial samples exposed to selected concentrations of the plant extract (1.56 or 50% v/v) collected and processed for morphological studies. Semi-thin longitudinal and cross sections prepared from control (untreated) and treated mycelia (1.56% v/v) revealed that alterations are limited to the vacuolation of the mycelial cytoplasm. Nevertheless, exposure to high concentration i.e., 50% v/v of the extract resulted in vacuolation of the mycelial cytoplasm and vesicle deformation causing attenuation of cell wall at variable intervals. Herniation of the cytoplasmic contents that was protruding from the mycelium was associated with deformation of the mycelium. Some mycelia showed a cleft between the cell wall and cytoplasm. Association of aflatoxin production with morphological changes suggest that probably integrity of the cell barriers particularly cell wall is critical in regulation of aflatoxin production and excretion.

Liver failure, in an acute or chronic form, is a growing health problem ranking as one of the leading causes of death worldwide. Inborn errors of metabolism characterized by defects in hepatic enzymes or other proteins with metabolic functions, such as receptors or transporters accompanied with environmental factors involve etiology and presentation of liver failure. Currently, the only established long-term successful treatment for these conditions is Orthotropic Liver Transplantation (OLT) with long-waiting lists of patients to be transplanted. In recent years, cellular therapy using human hepatocytes is being evaluated worldwide as an alternative to organ transplantation in patients with liver failure. Besides, scientists are trying to modify stem cells and their progenitor cells to improve their efficacy for treatment and repair of damaged tissue. The cell-based therapies have been a particularly active area of investigation in recent years. Experimental studies showed that transplantation of MSC-derived hepatocytes as well as MSC to a mice model of liver failure can effectively contribute to liver regeneration and rescue animals from liver failure Clinical trials with mesenchymal stem cells (MSC) using autologous bone marrow cell fusion (ABMI) therapy in patient suffering with cirrhosis show improvement of liver function. Hepatocyte transplantation method has been performed for indications such as, acute liver failure (ALF), end-stage liver disease, and inborn errors of metabolism. The cell based therapy for liver diseases comprises of two stages, firstly, the cell isolation and preparation, secondly, the transplantation stage. The protocols used for cell preparation and infusion from peripheral vein have been successfully performed in animal models as well as clinical trials. In recent years using tissue engineering protocols, 3D scaffolds of different composition have been prepared which support MSC proliferation and differentiation into active hepatocytes. The biocompatible nanofibrousbiomatrix scaffolds that support and enhance stem cell proliferation and differentiation are useful for transplantation and development of bioartificial liver system. Very recently scientists focused on decellularization of the liver organ and cell seeding of whole liver which is implicated for transplantation. The advantages and disadvantages of these protocols will be discussed in this meeting. Keyword: Acute liver failure, Cell therapy, Stem cells.

Aflatoxin B1(AFB1)-glutathione(GSH) conjugation is the major pathway for the detoxification of aflatoxin metabolites. This reaction is catalysed by GSH S-transferase (GST) and play a major role in modulation of AFB1 adduct formation to nuclear DNA. Changes recorded in hepatic GST activity during development of rats can alter the balance between AFB1-GSH conjugation and AFB1-DNA adduct formation. Measurment of cytosolic GST using 1-chloro-2,4-dinitrobenzene (CDNB) as the substrate showed that the enzyme activity is initially lower in weanling tissues as compared to that of adults. But nevertheless hepatic and renal cytosolic GST activity is increased significantly in growing rats pretreated with AFB1. Kinetic studies of AFB1-GSH conjugate formation in kidneys and livers of the two-age groups of rats treated with a single i.p. dose of AFB1 (400 microg/kg b.w.) revealed that at the end of 24 h of AFB1 administration the rate of the conjugate formation in kidneys of immature rats was approximately twice of that measured in adults. Age-related differences in the GST activity as well as AFB1-GSH conjugation was more pronounced in kidneys. The conjugate formation in kidneys of growing rats during 6-24 h following AFB1 administration shows that urinary excretion of aflatoxin metabolites is relatively rapid in growing rats. The major portion of the AFB1-GSH is formed in liver but contribution of the renal tissue to the formation of detoxification metabolites can not be ruled out. These data demonstrate that aflatoxin metabolites are eliminated more efficiently from kidneys of a growing rat. AFB1-induced GST induction in renal tissues of growing animals during 24 h of the carcinogen administration could be considered as an important mechanism for GSH conjugate formation and aflatoxin detoxification. Therefore GST induction in response to hepatotoxic drugs can confer resistance to young animals being exposed for the first time to such drugs. It is also worthmentioning that the GST activity measured before AFB1 administration does not reflect the rate of AFB1 detoxification via GSH conjugation.

Butylated hydroxytoluene (BHT) is known to inhibit tumor formation due to several chemical carcinogens including aflatoxin B1 (AFB1). Pre-treatment of laboratory animals with high doses of BHT (0.75% for 15 days in diet) is associated with pathological effects. The mechanism of action of BHT against AFB1 carcinogenesis is by induction of liver glutathione (GSH) S-transferases. As a result, the formation of AFB1-DNA binding is effectively inhibited. In the present study, effects of low and high doses of dietary BHT on in vitro activation and inactivation of AFB1 was examined. BHT feeding to rats at a dose of 0.75% for 15 days caused a significant increase in the GSH S-transferase activity. Addition of liver cytosolic fractions prepared from the rats pre-treated with high dose BHT to the cell free system caused a 48% inhibition in AFB1-DNA binding. In contrast, low dose BHT feeding (0.06% for 6 months) had little influence on GSH S-transferase activity. This was corroborated when addition of liver cytosol from low dose BHT-treated rats failed to inhibit microsome-mediated AFB1-DNA binding as compared to that of control. It is concluded that the permitted dose of BHT, added to processed food as preservative, plays no role in the biotransformation of AFB1.

Objective: The aim of this study was to determine the oxidative stress; serum level of Total Antioxidant Capacity (TAC)and Malondialdehyde (MDA) level and the depression score among the depressed rotational shift workers in Shahid Tondgouyan Refinery in Tehran (Iran). Methods: A cross-sectional study was conducted among all the 189 shift workers in Shahid Tondgouyan oil refinery who were eligible to participate in the study. They did not take any antidepressants for two months or any supplements for two weeks prior to the study entry. Written consent was obtained from the participants. 21- Item Beck Depression Inventory was used to measure the depression level. Furthermore, body weight, height and systolic and diastolic blood pressure were collected from all the participants. The levels of Total Antioxidant Capacity (TAC) and Malondialdehyde (MDA) were measured by 8 ml fasting blood sample. MDA was determined by thiobarbituric acid reaction. Serum total antioxidants were measured using the spectrophotometric BTS. In the ABTS test, 2,2’-azinobis (3-ethylbenzthiazoline-6-acid) (ABTS) is converted into its radical cation (ABTS•+) by addition of sodium persulphate. This blue-green radical cation absorbs light at 734 nm. ABTS•+ is reactive towards most antioxidants. Descriptive statistics, ANOVA, ANCOVA and regression tests and correlation were used to analyze the data using SPSS software version 16. Results: The age of the participants ranged from 21 to 52 years. The mean age of the participants was 30.58 year (±6.97yr). Of all the participants, 28% (n= 53) had no depression symptoms (depression score between 0 and 9), 65.1% (n=123) were categorized as having mild depression (depression score between 10 and 18) and 6.9% (n=13) were categorized as having moderate depression (depression score between 19 and 29). The participants’ BMI ranged from 15.9 to 34.3; the mean BMI of the participants was 24.82 kg/m2 (+ 3.81 kg/m2). The mean of the serum TAC level was 2.51 (± 0.56) mg/dl, and the mean serum MDA level was 3.67(± 1.08) μmol/l. There was a significant difference in the mean TAC concentration between the non-depressed group and the group with mild depression (p=0.029). Conclusion: Depression was associated with reduced mean TAC concentration and an increase in MDA level. There was a linear relationship between the depression score and shift work experience among the rotational shift workers, which showed a high level of stress and depression among the shift-workers.

Background: It has been reported that the activity of glutathione S-transferase (GST) is over-expressed in plasma and esophagus biopsies in Iranian patients suffering from esophageal squamous cell carcinoma (SCC). The aim of this study was to find out the frequency of GST-P genotypes in these patients. Moreover, the association of GST-P genotypes with p53 protein accumulation in esophageal epithelium was investigated. Materials and Methods: DNA isolated from paraffin-embedded tissue biopsies from patients suffering from esophageal SCC (n = 56) were collected. polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) using Alw261 enzyme was applied to determine GST-P genotypes (Ile 105 Val). All the samples were also subjected to immunohistochemistry (IHC) for p53. Results: The frequency of GST-P genotypes in Iranian esophagus SCC patients for Ile/Ile, Ile/Val and Val/Val was 73.2, 21.5 and 5.3%. There was no association between GST-P polymorphism and p53 accumulation in esophageal epithelial cells. Conclusions: The frequency of GST-P polymorphism was not associated with p53 protein accumulation in esophagus epithelium. The frequency of polymorphic variants of GST-P, Ile/Ile, Ile/Val and Val/Val in SCC patients may suggest that Ile to Val substitution in GST-P gene dose not represent susceptibility to SCC in high-risk Iranian population.