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Background To study the concentrations of monocyte chemoattractant protein-1 (MCP-1), hepatocyte growth factor (HGF), and insulin-like growth factor-1 (IGF-1) in peritoneal fluid (PF) and serum, and to evaluate their expressions by PF and peripheral blood mononuclear cells (PFMCs and PBMCs, respectively), and ectopic and eutopic endometrial stromal cells of patients with endometriosis (EESCs and EuESCs, respectively) compared with controls. Methods The concentrations of mentioned cytokines in serum and PF, as well as their expression in PBMCs, PFMCs, EuESCs and EESCs from endometriosis patients and controls were assessed. Results The levels of MCP-1, HGF, and IGF-1 in serum and PF in women with endometriosis were significantly higher than the controls (P P MCP-1 and IGF-1 in the PFMCs, PBMCs and EESCs also showed an increased level compared to controls (P –P P P P P Conclusions The higher concentrations of mentioned cytokines in serum and PF and their higher expression by PFMCs and EESCs in endometriosis patients may contribute to the development of endometriosis.

Mesenchymal stromal cells (MSCs) have potent immunomodulatory abilities to regulate most of the immune cells. Not only the tissue origin of MSCs can affect their functions, but also their microenvironment can strongly influence their biology, particularly through toll-like receptors (TLR)/ligands interaction. In the present study, we compared MSCs derived from two different sources, i.e. human olfactory ecto-mesenchymal stem cells (OE-MSCs) and adipose tissue (AT-MSCs), in terms of their immunosuppressive effects before and after TLR3 and TLR4 stimulation through low-level and short-term TLR-priming protocol. After isolation and characterization of OE-MSCs and AT-MSCs, flow cytometry analyses were used to assess the expression of TLR3, TLR4 by MSCs. Secretion and expression levels of immune-related genes were analyzed using ELISA and RT-qPCR techniques. Based on the results, the proliferation potential of OE-MSCs was significantly higher than that of AT-MSCs. The gene expression and also protein levels of both TLR3 and TLR4 were significantly higher in OE-MSCs, compared to AT-MSCs. Among the examined cytokines and chemokines, OE-MSCs exhibited significantly higher levels of CCL5, IL-8, and TGF-β production, in comparison with AT-MSCs. However, IL-6 secretion by AT-MSCs was considerably more than that by OE-MSCs. OE-MSCs were only affected by the TLR4 ligand, and IL-8 and IL-6 production levels increased after LPS treatment. However, only IL-8 significantly increased after adding LPS or Poly (I:C) to the AT-MSC media. According to the obtained data, OE-MSCs exhibited a higher proliferative potential and greater expression levels of TLR3 and TLR4 genes, compared to AT-MSCs. However, unlike AT-MSCs, the expression of TLR3 by OE-MSCs was nonfunctional. Finally, based on our findings, OE-MSCs have a stronger secretion of immunosuppressive cytokines both before and after LPS or PIC treatment, compared to AT-MSCs.

Background: Epithelial-to-Mesenchymal Transition (EMT) is necessary for metastasis. Zinc- finger domain-containing transcription factors, especially Snail1, bind to E-box motifs and play a crucial role in the induction and regulation of EMT. Objective: We hypothesized if C-terminal region of Snail1 (CSnail1) may competitively bind to E-box and block cancer metastasis. Methods: The CSnail1 gene coding sequence was inserted into the pIRES2-EGFP vector. Following transfection of A549 cells with the designed construct, EMT was induced with TGF-β1 and the expression of essential EMT markers was evaluated by real-time PCR and immunoblotting. We also monitored cell migration. Results: CSnail1 inhibited TGF-β1-induced N-cadherin and vimentin mRNA expression and increased β-catenin expression in transfected TGF-β1-treated A549 cells. A similar finding was obtained in western blotting. CSnail1 also blocked the migration of transfected cells in the scratch test. Conclusions: Transfection of A549 cells with CSnail1 alters the expression of essential EMT markers and consequently suppresses tumor cell migration. These findings confirm the capability of CSnail1 in EMT blocking and in parallel to current patents could be applied as a novel strategy in the prevention of metastasis.

Variable antigen-binding domain of camelid single chain antibodies (VHH, Nanobody) and its conjugates are considered among very promising candidates in tumor diagnosis and treatment because of its small size, stability, and favorable bio-distribution. CD22 is a receptor that is expressed on most B cells and modulates their function. Targeting CD22 in B cell malignancies and disorders by monoclonal antibodies has shown promising results in vitro and in clinical trials. In this study, we investigate the impact of an anti-human CD22 VHH binding to CD22 on B cells and its internalization following attachment. Our findings demonstrate the proliferation inhibiting of these cells with no effect on apoptosis, in addition to the rapid internalization of the VHH.

Human platelet antigens (HPAs) are membranous glycoproteins considered as alloantigens due to their polymorphisms. HPA-incompatibility in multiple pregnancies or blood transfusion can induce the development of alloantibodies leading to thrombocytopenia. The frequency of HPAs varies among populations, so that deep knowledge of HPA frequencies will help us to reduce those incompatibilities. Herein, we studied the allele and genotype frequencies of HPA1-6, HPA9, and HPA15 among the Iranians with intra- and inter-populations analyses on 36 worldwide populations with diverse ethnicities. The analysis shows that the HPA2 and HPA5 have the greatest differences in genotype distribution between the Iranians and other nations, although similar to other populations, the sole allele found in HPA4, 6, and 9 is “a”. Despite other HPAs, the most frequent allele in HPA15 is “b”, which is also abundant in HPA3. Hierarchical clustering indicates the highest degree of global similarity in HPA genotype frequency among Iranian, Argentinian, Brazilian, and German Turkish populations. Our findings can be applied to decrease the risk of alloimmunizations and platelet disorders, especially in neonates.

Introduction Immune imbalance at the maternal-fetal interface plays a fundamental role in the pathogenesis of unexplained recurrent spontaneous abortion (URSA). Human amniotic epithelial cells (hAECs) possess pregnancy-friendly immunomodulatory effects. Here, we investigated how function of naive CD4+ T cells from URSA patients is affected by hAECs. Methods Phenotypic characteristics of hAECs were determined by flow cytometry and their effect on proliferation of allogeneic peripheral blood mononuclear cells (PBMCs) was evaluated by a BrdU cell proliferation assay. Naive CD4+ T cells were isolated from 25 URSA patients and 5 healthy women and co-cultured with hAECs. Immunomodulatory effects of hAECs on cytokines profile, proliferation of stimulated CD4+ T cells and induction of regulatory T cells (Tregs) were assessed by ELISA and flow cytometry, respectively. Functional competency of Tregs was evaluated in an allogeneic mixed lymphocyte reaction (MLR) system. Results hAECs did not elicit allogeneic proliferative responses of PBMCs, inhibited proliferation of naive CD4+ T cells, induced production of Th2 and suppressed production of Th1 and Th17 cytokines. hAECs showed the ability to induce differentiation of Tregs and production of transforming growth factor-beta1 (TGF-β1) and interleukin-10 (IL-10). This ability was found to be superior in control subjects compared to URSA patients. Indeed, Tregs generated in the presence of hAECs expressed higher levels of CTLA-4 compared to Tregs generated in their absence and restrained the proliferation of autologus PBMCs in MLR system. Conclusion Based on these findings, hAECs can be considered as one potential candidate in immunotherapy of patients with URSA.

Background Coronary artery disease (CAD) is characterized by narrowing/ blockade of coronary arteries that is mainly caused by atherosclerotic plaques. Considering the involvement of platelet abnormalities, such as defective aggregation and adhesion, in the cardiovascular-related disorders, genetic variations in human platelet alloantigens (HPA) have been implicated in the CAD susceptibility. Herein, we intended to determine the association of HPA-1 to -6, -9, and -15 biallelic polymorphisms with CAD in an Iranian population. Methods In this retrospective case–control study, 200 CAD subjects and 100 matched healthy individuals were enrolled. DNA samples were isolated from peripheral blood samples and genotyping of HPA polymorphisms was accomplished using polymerase chain reaction-sequence-specific primers. Results The alleles and genotypes of studied HPA polymorphisms were equally distributed among cases and controls and therefore no statistically significant differences were detected. Univariate analysis identified no association of combined haplotypes with CAD risk. However, multivariate analysis showed a positive association of the‌ HPA1b/2a/3b haplotype with CAD after adjustment for some covariates (including BMI, TG, LDL, FBS and blood pressure) that conferred a CAD susceptibility haplotype (P = 0.015; OR = 2.792; 95% CI 1.45–8.59). Conclusions Although alleles, genotypes, and haplotypes of HPA polymorphisms were not associated with CAD risk, HPA1b/2a/3b haplotype was found to be a dependent disease risk haplotype in Iranian population after correcting for confounding factors.

Unexplained recurrent spontaneous abortion (URSA) has been assumed to be caused by a defect in maternal immunological tolerance to the fetus. Human amniotic epithelial cells (hAECs) have stem cell-like features and the ability to modulate the innate and adoptive immune responses. This study aimed to investigate whether hAECs have immunomodulatory effects on naive CD4+ T cells from URSA patients. hAECs were obtained from 15 healthy pregnant women and phenotypic profile of hAECs was determined by flow cytometry. Naive CD4+ T cells were isolated from 25 URSA patients using an immunomagnetic separation method. Naive T cells were stimulated with anti-CD3/anti-CD28 antibodies and co-cultured with different numbers of hAECs for 3 and 6 days. Immunomodulatory effect of hAECs on activation of stimulated T cell was assessed by flow cytometry and Enzyme-linked immunoasorbent assay (ELISA). The hAECs effect on pro-inflammatory cytokines production of activated T cells was also measured by ELISA. Our results indicated that hAECs significantly inhibited the activation of naive T cells in a dose-dependent manner (p 0.0001-0.05). They significantly reduced the production of transforming growth factor-beta1 (TGF-β1) of stimulated CD4+T cells (p 0.0001-0.05). Moreover, hAECs had potent immunomodulatory effects on the production of interferon-gamma (IFN-γ) and interleukin-17A (IL-17A) of activated T cells (p 0.0001-0.01). These findings suggest that hAECs may be a suitable cell source to modulate abnormal immune responses in women with URSA.

Background:The metabolism of cholesteryl esters (CEs) is under the control of a gene network in macrophages. Several genes such asATF3andEGR2are related to cholesterol metabolism.Methods:In this study, theATF3andEGR2gene expression levels were evaluated in differentiated macrophages of subjects undergoing coronary artery angiography [controls (70% stenosis)] after treatment with small dense low density lipoprotein (sdLDL) particles. Monocytes were isolated using a RosetteSep Kit and were differentiated into macrophages using the M-CSF factor. A modified heparin-MgSO4-PEG method was used for the sdLDL preparation. TheATF3andEGR2gene expression levels were measured by the real-time quantitative polymerase chain reaction (RT-qPCR) technique.Results:In contrast with the control group (p=0.4), theATF3expression level reduced significantly in the differentiated macrophages from all patients [single vessel disease (SVD), fold change 17 times, p=0.02; two vessel disease (2VD), fold change 1.5 times, p=0.05; three vessel disease (3VD), fold change 3.5 times, p=0.04]. Also, theEGR2expression level reduced significantly in all groups (p0.8).Conclusions:We propose that the failure ofATF3gene expression improves the CE synthesis after sdLDL influx. Furthermore, the reducedEGR2gene expression level in the sdLDL-treated groups may be a negative factor in cholesterol homeostasis.

Killer cell immunoglobulin like receptors (KIRs) have a principal role in regulating the effector functions of NK cells, particularly in viral infections. The major ligands for KIRs are human leukocyte antigen (HLA) class I molecules. The aim of this study is to investigate the possible association of KIR genes, their known HLA ligands and compound KIR–HLA genotypes with hepatitis B virus (HBV) infection. Our study group consisted of 202 Iranian HBV-infected patients (52 spontaneously recovered, 50 asymptomatic carriers, 50 chronic sufferers and 50with liver cirrhosis) and 100 ethnic-matched healthy control subjects. KIR and HLA genotyping was performed by a polymerase chain reaction–sequence-specific primer (PCR–SSP). The frequencies of the KIR2DL5A, KIR2DS1, and KIR3DS1 genes were significantly elevated in recovered individuals when compared with both control and patient groups. Also, KIR2DL5, and KIR3DP1 full were escalated in recovered individuals in comparison with patient groups. In addition, HLA-Bw4 ligand and HLA-A Bw4 were highly frequent in recovered individuals compared with healthy controls. Furthermore, the KIR3DS1 + HLA-Bw4, KIR3DS1 + HLA-Bw4 Iso80 , and KIR3DS1 + HLA-A Bw4 genotypes were significantly more common in recovered individuals than both healthy control and patient groups. Interestingly, AA genotype had less frequency and Bx had higher frequency in recovered individuals compared with both healthy control and patient groups. Our findings suggest a potential impact of the NK cells’ activating phenotype that leads to the HBV clearance in infected individuals.

Background: Islet transplantation could be an ideal alternative treatment to insulin therapy for type 1 diabetes Mellitus (T1DM). This clinical and experimental field requires a model that covers problems such as requiring a large number of functional and viable islets, the optimal transplantation site, and the prevention of islet dispersion. Hence, the methods of choice for isolation of functional islets and transplantation are crucial. Methods: The present study has introduced an experimental model that overcomes some critical issues in islet transplantation, including in situ pancreas perfusion by digestive enzymes through common bile duct. In comparison with conventional methods, we inflated the pancreas in Petri dishes with only 1 ml collagenase type XI solution, which was followed by hand-picking isolation or Ficoll gradient separation to purify the islets. Then we used a hydrogel composite in which the islets were embedded and transplanted into the peritoneal cavity of the streptozotocin-induced diabetic C57BL/6 mice. Results: As compared to the yield of the classical methods, in our modified technique, the mean yield of isolation was about 130-200 viable islets/mouse pancreas. In vitro glucose-mediated insulin secretion assay indicated an appropriate response in isolated islets. In addition, data from in vivo experiments revealed that the allograft remarkably maintained blood glucose levels under 400 mg/dl and hydrogel composite prevents the passage of immune cells. Conclusion: In the model presented here, the rapid islet isolation technique and the application of biomimetic hydrogel wrapping of islets could facilitate islet transplantation procedures.

Our study aimed to assess the influence of common methylprednisolone therapy on innate inflammatory factors in potential brain-dead organ donors (BDDs). The study groups consisted of 50 potential BDDs who received 15 mg/kg/d methylprednisolone and 25 live organ donors (LDs) as control group. Innate immunity gene expression profiling was performed by RT-PCR array. Soluble serum cytokines and chemokines, complement components, heat shock protein 70 (HSP70) and high mobility group box-1 (HMGB1) were measured by ELISA. Surface expression of TLR2 and TLR4 were determined using flow cytometry. Gene expression profiling revealed up-regulation of TLRs 1, 2, 4, 5, 6, 7 and 8, MYD88, NF-κB, NF-κB1A, IRAK1, STAT3, JAK2, TNF-α, IL-1β, CD86 and CD14 in the BDD group. Remarkably, the serum levels of C-reactive protein and HSP70 were considerably higher in the BDD group. In addition, serum amounts of IL-1β, IL-6, TNF-α, HMGB1, HSP70, C3a and C5a, but not IL-8, sCD86 or monocyte chemoattractant protein-1, were significantly increased in the BDD group. Significant differences were observed in flow cytometry analysis of TLR2 and TLR4 between the two groups. In summary, common methylprednisolone therapy in BDDs did not adequately reduce systemic inflammation, which could be due to inadequate doses or inefficient impact on other inflammatory-inducing pathways, for example oxidative stress or production of damage-associated molecules.

Toll-like receptors (TLRs) are inevitable elements for immunity development and antibody production. TLRs are in close interaction with Bruton's tyrosine kinase which has been found mutated and malfunctioned in the prototype antibody deficiency disease named X-linked agammaglobulinemia (XLA). TLRs' ability was evaluated to induce transcription of TLR-negative regulators, including suppressor of cytokine signaling 1 (SOCS1), interleukin-1 receptor-associated kinase 3 (IRAK-M), tumor necrosis factor alpha-induced protein 3 (TNFAIP3, A20), and Ring finger protein 216 (RNF216), and Tumor necrosis factor-α (TNF-α) and Interferon-α (IFN-α) production via Lipopolysaccharides (LPS) and CpG-A oligodeoxynucleotides (CpG-A ODN). Measured by TaqMan real-time polymerase chain reaction (PCR), meaningfully increased transcripts of SOCS1 and RNF216 were found in XLA peripheral blood mononuclear cells (PBMCs). Also, TLR inductions of XLA have led to similar downregulations in the regulator's transcription which was different from that in healthy donors. Cytokine measurement by enzyme-linked immunosorbent assay (ELISA) revealed a significant lower TNF-α production both before and after LPS. By selected molecules in this study, TLRs' potential defectiveness range expands TLRs expression, downstream signaling, and cytokine production. The results show new potential elements that could play a part in TLRs defect and pathogenesis of agammaglobulinemia as well.

Testicular ischemia is the main consequence of testicular torsion, in both clinical and experimental aspects. Preservation and auto-transplantation of spermatogonial stem cells (SSCs) could be a new treatment for infertility in testicular ischemia following testicular torsion. To apply the idea in this study, animals were randomly divided into four groups of control, sham, with torsion, and with torsion followed by transplantation (TT). Isolated SSCs from neonatal mice were cultured and identified by flow cytometry (C-KIT−, INTEGRIN β1 +) and RT-PCR (Reverse transcription polymerase chain reaction) for specific spermatogonial cell markers (Oct4, Gfrα-1, Plzf, Vasa, Itgα 6 , and Itgβ 1 ). SSCs were transplanted upon a 2-h testicular torsion in the TT group. Cultured cells were transplanted into ischemia reperfusion testicle 2 weeks post-testicular torsion. Eight weeks after SSCs transplantation, the SSCs-transplanted testes and epididymis were removed for sperm analysis, weight and histopathological evaluation, and pre- and post-meiotic gene expression assessment by qRT-PCR. Our findings indicated that all evaluated parameters (epididymal sperm profile, Johnsen score, Plzf, Gfrα-1, Scp-1, Tekt-1 expressions, and histopathological profile) were significantly decreased following testicular torsion (group 3) when compared to the control group (p ≤ 0.05). However, all abovementioned parameters showed a significant increase/improvement in torsion-transplantation group compared to torsion group. However, these parameters in the TT group were significantly lower in the sham and control groups (p ≤ 0.05). SSCs transplantation could up-regulate the expression of pre- and post-meiotic genes in testicular ischemia, which resulted in improvement of both testicular function and structure after testicular torsion.

Autoimmune hepatitis (AIH) is an uncommon autoimmune liver disease of unknown etiology. The aim of this study was to determine the frequency of HLA-DRB1 alleles in Iranian patients with AIH and investigate the association between HLA alleles and the different types of the disease. Fifty-four AIH patients and 100 age- and sex-matched healthy controls were subjected to low resolution HLA-DRB typing performed by polymerase chain reaction-sequence-specific primers (PCR-SSP) technique. The results revealed higher frequencies of HLA-DRB1(∗)03, and DRB1(∗)13 alleles in patients with AIH compared to controls. However, DRB1(∗)11 was less frequent in AIH patients. In type I AIH patients HLA-DRB1(∗)03, HLA-DRB1(∗)04, HLA-DRB1(∗)08, and HLA-DRB1(∗)13 were the most frequent alleles. While in type II, the most frequent alleles were HLA-DRB1(∗)07 and HLA-DRB1(∗)13. The seronegative patients showed more frequency of HLA-DRB1(∗)03 and HLA-DRB3. In contrary, the frequency of HLA-DRB1(∗)11, HLA-DRB1(∗)15 and HLA-DRB5 in type 1 was less than healthy individuals. These findings indicate the role of HLA-DRB haplotypes in AIH susceptibility and protection, in the Iranian population.

Objectives Angiogenesis targeting is an attractive approach for cancer treatment. Delta-like ligand 4 (DLL4) plays a pivotal role in neovascular development and its inhibitors have recently entered clinical trials for solid tumors. The aim of this study was to evaluate the possibilities of using anti-DLL4 antibody fragment as an angiogenesis maturation inhibitor. Materials and methods In this study, a DLL4-specific Nanobody, named 3Nb3, was selected and assessed by western blotting and internalization assays. Functional assessments included MTT, apoptosis, and chicken chorioallantoic membrane (CAM) assays. Results Based on the results, 3Nb3 specifically binds to DLL4 and internalizes into MKN cell. Furthermore, 3Nb3 significantly inhibited the proliferation of cells and also neovascularization in the CAM. Conclusions These data demonstrated the potential of Nanobody for application in targeting DLL4. Our findings may provide a basis for the development of novel therapeutic techniques to inhibit growth and neovascularization of tumors.

Common variable immunodeficiency (CVID), a clinically symptomatic primary immunodeficiency disease (PID), is characterized by hypogammaglobulinemia leading to recurrent infections and various complications. Recently, some defects in the signaling of TLRs have been identified in CVID patients which led us to investigate the expression of TLR4 and 9 negative regulatory molecules and their upregulation status following their activation. Using TaqMan real-time PCR, SOCS1, TNFAIP3, RFN216, and IRAK-M transcripts among peripheral blood mononuclear cells (PBMCs) were measured with/without TLR4 and 9 activations. TLR4 and 9 were activated by lipopolysaccharide (LPS) and unmethylated CpG-oligodeoxynucleotide (CpG-ODN), respectively. Production of IFN-α and TNF-α cytokines, as a part of the functional response of mentioned TLRs, was also measured using ELISA. Deficient transcripts of IRAK-M and TNFAIP3 in unstimulated PBMCs and lower production of TNF-α and IFN-α after treatments were observed. Upregulation of RFN216 and TNFAIP3 after TLR9 activation was abnormal compared to healthy individuals. Significant correlations were found between abnormal IRAK-M and TNFAIP3 transcripts, and lymphadenopathy and inflammatory scenarios in patients, respectively. It seems that the transcriptional status of some negative regulatory molecules is disturbed in CVID patients, and this could be caused by the underlying pathogenesis of CVID and could involve complications like autoimmunity and inflammatory responses.

Immune imbalance at the maternal-fetal interface plays a fundamental role in the pathogenesis of unexplained recurrent spontaneous abortion (URSA). Human amniotic epithelial cells (hAECs) possess pregnancy-friendly immunomodulatory effects. Here, we investigated how function of naive CD4Phenotypic characteristics of hAECs were determined by flow cytometry and their effect on proliferation of allogeneic peripheral blood mononuclear cells (PBMCs) was evaluated by a BrdU cell proliferation assay. Naive CD4hAECs did not elicit allogeneic proliferative responses of PBMCs, inhibited proliferation of naive CD4Based on these findings, hAECs can be considered as one potential candidate in immunotherapy of patients with URSA.

CD22 is a B-cell-specific trans-membrane glycoprotein, which is found on the surface of the most B cells and modulates their function, survival, and apoptosis. Recently, targeting this cell surface biomarker in B-cell malignancies and disorders has attracted a lot of attention. The variable domain of camelid single-chain antibodies (VHH, nanobody) is a form of antibodies with novel properties including small size (15-17 kDa), thermal and chemical stability, high affinity and homology to human antibody sequences. In this study, a novel anti-CD22-specific VHH (Nb) has been developed and characterized by the screening of an immunized phage display library and its binding to CD22

Antibody-based targeting of angiogenesis is a key approach for cancer treatment. Delta-like ligand 4 (DLL4) plays a pivotal role in tumor neovascular development and angiogenesis during tumor progression. It forecasts the prognosis of human malignancies and blocking its signaling can help to inhibit neovascularization and tumor metastasis. Nanobodies are the smallest antigen-binding domains of heavy chain antibodies in camelidae. The aim of this study was to develop a Nanobody against DLL4 and apply binding and functional approaches to target it. In this work, a Nanobody library against human recombinant DLL4 was developed. After panning, the periplasmic-extract (PE) of individual colonies were screened through ELISA. The interactions between Nanobody and DLL4 were assessed using immunohistochemistry and FACS. The functional assessment was carried out via tube formation assay. We selected a Nanobody (3Nb3) with a high binding signal to DLL4, associated with a binding affinity of 3.6 nM. It was demonstrated that 3Nb3 binds to native DLL4 on the surface of MKN cells and gastric carcinoma tissue, and also inhibits the maturation of capillary-like structures in HUVECs. The results were indicative of the potential of Nanobody for DLL4 identification and can broaden the scope for development of cancer diagnosis and treatment techniques.

Objective Human amniotic epithelial cells (hAECs) which are isolated from the amniotic membrane have stem cell-like properties and immunomodulatory effects. Several protocols have been proposed for isolation of hAECs, nevertheless, there is no report concerning isolation of highly viable hAECs, with desirable yield, and without significant purity reduction. In the current study, a detailed protocol with some modification of previous ones is presented in which the amendments led to isolation of hAECs with high purity, yield and viability. Moreover, isolated hAECs were subjected to immuno-phenotyping and their physiological status was assessed using a proliferation assay. Results The average yield of obtained hAECs using the new modified method was 190 × 106 cells with a mean viability of 87%, with less than 1% contamination with mesenchymal stem cells (MSCs). The isolated cells were > 95% positive for the epithelial cell markers. The lowest initial plating efficiency of the cells was 80%. Freshly isolated hAECs had the ability to proliferate for 5–6 passages in a standard culture medium.

Background Allogeneic islet transplantation could be an ideal alternative therapy for Type 1 Diabetes Mellitus (T1DM). Adipose Tissue-derived Mesenchymal Stem Cells (AT-MSCs) characterized by immunomodulatory and protective effects may have the potential to improve the outcome of this highly immunogenic transplant. Methods Syngenic AT-MSCs along with allograft islets embedded in hydrogelic composite and transplanted intraperitoneally in Streptozotocin (STZ) induced diabetic C57BL/6 mice. Results In vitro experiments of co-imbedded islets and AT-MSCs in a hydrogel revealed AT-MSCs are able to significantly increase insulin secretion. During a 32 days of post-transplant period, blood glucose monitoring showed a decrease from over 400 mg/dl to less than 150 mg/dl and at the end of 32 days, mice have been dissected and assessed. Graft histopathology demonstrated that hydrogel makes an artificial immune isolation site and AT-MSCs contribute greatly to the reduction of the immune cells infiltration. Analyses of mononuclear cells isolated from Mesenteric Lymph Nodes (MLNs) and spleen showed that AT-MSCs co-transplanted with allograft decreased pro-inflammatory cytokines and increased regulatory cytokines (for both MLNs and spleen) and regulatory T cells (Treg) population (only for MLNs). In addition, real time-PCR assays revealed that transcript levels of IDO, iNOS, and PDX1, significantly increased in allograft islets in the presence of AT-MSCs. Conclusions according to results, this investigation indicates that AT-MSCs can be regarded as promising complementary candidates for engineered-cell therapy using hydrogel composites in islet transplantation.

Unexplained recurrent spontaneous abortion (RSA) might be caused by the mother's immunological rejection of the fetus. In this cross-sectional study, the percentage of T helper 17 (Th17), T regulatory (Treg) cells and their cytokines as the main players of immunomodulation in peripheral blood lymphocytes during the luteal phase of 20 women with unexplained RSA were compared with 20 normal non-pregnant women. The percentage of Treg cells in the former was significantly lower compared with controls. The percentage of Th17 cells in the former was higher than controls. Expression of IL-23, IL-17, IL-6 cytokines in the former was significantly higher than controls, but the higher expression of IL-21 was not significant. The gene expression of TGF-β and FoxP3 in the former was lower than controls. Significant positive correlations were found between the percentage of Th17 cells with IL-23, IL-6 and IL-17 and between expression of IL-23 and IL-6 and IL-17. IL-6 gene expression showed a significant positive correlation with IL-17. Therefore, imbalance of Th17-Treg cells and the consequent changes in cytokine expression might be implicated in the pathogenesis of unexplained RSA and may provide new insight into the immunoregulatory events at the maternal-fetal interface.

Vitamin D has various roles in many biological actions such as calcium homeostasis, cell proliferation, and cell differentiation to many target tissues. These effects are mediated by the active form of vitamin D, 1,25(OH)2D3, which binds to a cytoplasmic protein called vitamin D receptor (VDR). VDR gene has four common single nucleotide polymorphisms (SNPs) that are defined by the presence of restriction sites for FokI (F/f), TaqI (T/t), BsmI (B/b), and ApaI (A/a). The association of VDR gene polymorphisms with several diseases has been investigated. In most studies, VDR genotyping was performed by polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) assays, which are cumbersome and time consuming, and their results are sometimes difficult to interpret. We modified previously reported primers for VDR genotyping and set up a single amplification-refractory mutation system (ARMS)-PCR method for simultaneous genotyping of four common VDR polymorphisms. In this study, 218 DNA samples were analyzed for VDR genetic variants by this ARMS-PCR technique; 136 of them were re-genotyped by PCR-RFLP assays to compare genotyping results. We obtained allelic frequencies of 69 vs. 31 % for F/f, 34 vs. 66 % for B/b, 70 vs. 30 % for T/t, and 52 vs. 48 % for A/a in this sample of the Iranian population. In addition, comparisons of the results of these two methods showed good uniformity in VDR genotypes; although, in some samples, ambiguity in restriction patterns was present. As ARMS-PCR is more rapid, economic, and user friendly than PCR-RFLP, its substitution would be welcomed in disease association and pharmacogenetic studies of VDR variants.

Etiological factors for coronary artery disease (CAD) involve a wide range of gene and environmental interactions. One of the systems being implicated in the pathophysiology of CAD is the renin-angiotensin system (RAS). However, the genetic polymorphisms of this system have not been widely studied in Iranian patients diagnosed with CAD. The aim of this study was to assess the relationship between six gene polymorphisms of RAS components and CAD in a sample of Iranian population. A total of 374 participants were enrolled in a case/control study. The presence of CAD was determined by coronary angiography. Genotyping of six RAS gene polymorphisms was performed using a modified PCR-RFLP method. Our results revealed, for the first time, a significant independent association of angiotensin-converting enzyme (ACE) A-240T polymorphism and incidence of CAD among Iranian women (P=0.005, OR=20.4, 95% CI=2.49-41.2). There has also been a significant difference in genotype distribution of ACE A-240T (P=0.008) and angiotensin II receptor type 2 C3123A polymorphism (P=0.032) in Iranian female participants. In conclusion, TT genotype of ACE A-240T seems to be a genetic risk factor for CAD in Iranian women.

Genetic factors contribute substantially to the likelihood of developing major depressive disorder (MDD). The importance of renin-angiotensin system (RAS) elements in cognition and behaviour and their involvement in aetiology and treatment of depression imply that RAS gene polymorphism(s) associated with RAS overactivity might also be associated with depression. In the present study, genotype and allele frequencies of six common polymorphisms of genes encoding for RAS components were determined in DNAs extracted from venous blood of 191 depressed and 104 healthy individuals using polymerase chain reaction (PCR) and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and serum angiotensin-converting enzyme (ACE) activity was assayed using a high-performance liquid chromatography (HPLC) method. The results showed, for the first time, that GG genotype of ACE A2350G was significantly associated with MDD among Iranian participants (P=0.001; odds ratio (OR)=6.2; 95% confidence interval (CI)=2.1-18.3). Significant higher serum ACE activity (P=0.0001) as well as higher diastolic blood pressure (P=0.036) were observed in depressed patients compared to the healthy control group. Depressed patients carrying GG genotype of the A2350G polymorphism had a significantly higher serum ACE activity (P=0.02) than individuals with either AA or AG genotype. In conclusion, this study supports the hypothesis of RAS overactivity in depression in that the genotype associated with higher serum ACE activity in an Iranian population was also associated with MDD.

Systemic lupus erythematosus (SLE) is a multi-factor autoimmune disorder with diverse clinical manifestations and unclear pathogenesis. Genetic components play important roles in the incidence and development of SLE. Among these, APRIL as a cytokine has roles in the stimulation and antibody production in B cells. APRIL was hypothesized to be associated with SLE. The aim of this study was to assess the involvement of the APRIL gene in SLE susceptibility in Iranian patients. A single-nucleotide polymorphism (SNP) for rs11552708 of APRIL gene was analyzed by real-time PCR in 60 SLE Iranian children and 64 healthy controls. DNA samples of patients and healthy controls were extracted from peripheral blood leukocytes by phenol-chloroform. Serum samples obtained from 45 children with SLE and 45 healthy controls were assayed by enzyme-linked immunosorbent assay (ELISA). The G/G genotype (odds ratio (OR) 0.67, 95% confidence interval (CI) 0.22–2.07; P = 0.68) and G allele (OR 0.81, 95% CI 0.25–2.56; P = 0.89) frequencies of polymorphism at codon 67 (67G) do not differ significantly in the SLE patients compared with those in the healthy controls. The serum APRIL levels in the SLE patients (mean ± SD = 29.27 ng/ml ± 20.77, range from 0 to 55.33 ng/ml) were significantly higher than those in the healthy controls (P = 0.02). Our results demonstrated that rs11552708 of the APRIL gene is not associated with SLE susceptibility in Iranian children. Likewise, these findings suggest that APRIL antagonist could be a potential therapeutic target to control SLE in children.

article Objectives: Major depressive disorder (MDD) is an increasingly recognized risk factor of coronary artery disease (CAD). The aim of this study was to assess the relationship between renin-angiotensin system (RAS) genetic polymorphisms and CAD in a sample of depressed Iranian patients. Design and methods: A total of 191 patients with a history of unipolar depression were enrolled in a case/control study. The presence of MDD was reconfirmed at study entry using DSM-IV criteria and CAD was diagnosed by coronary angiography. Genotyping of six RAS genes polymorphisms was performed by a modified PCR-RFLP method. Results: DD genotype of ACE I/D was independently associated with the incidence of CAD in depressed patients (P=0.011, OR=9.41, 95% CI: 1.68-17.81). Moreover, serum creatinine (P=0.033, OR=11.91, 95%CI: 7.23-15.62) was an independent predictor of CAD among depressed individuals. Conclusion: ACE I/D polymorphism may play a major role in the development of CAD amongst Iranian depressed patients.

Most of renin-angiotensin system (RAS) gene polymorphisms have not yet been studied in the Iranian population. In the present study, the frequencies of common polymorphisms in the RAS genes, including angiotensin-converting enzyme (ACE) insertion/deletion (I/D) and three single-nucleotide polymorphisms (SNPs), i.e., A-240T, T-93C and A2350G, angiotensinogen M235T, angiotensin II receptor type 1 A1166C and angiotensin II receptor type 2 C3123A variants were determined in DNAs extracted from venous blood of 104 healthy Iranian volunteers. Genotyping was performed by PCR-RFLP method. Serum ACE activity was also assayed using reverse phase HPLC. Combined polymorphisms of TT (A-240T) and GG (A2350G) was significantly associated with decreased serum ACE activity (P=0.042) and decreased diastolic blood pressure (P=0.040). The angiotensin II receptor type 1 A1166C polymorphism (CC genotype) showed a significant association with declined diastolic blood pressure (P=0.028). Serum ACE activity was significantly higher in men compared to women (P=0.033). ACE activity also showed a direct association with diastolic blood pressure (P0.001). No association was obtained among each single polymorphism with body mass index (BMI), fasting blood sugar (FBS), lipid profile and ACE activity. In conclusion, combined polymorphisms of A-240T and A2350G seem to affect serum ACE level as well as diastolic blood pressure in our study population. However, it also might be hypothesized that they are in strong linkage disequilibrium with other functional mutations not studied yet. Our findings revealed that gene interactions can play an important role in various biological conditions.

Contribution of killer cell immunoglobulin-like receptors (KIR) and their human leucocyte antigen (HLA) class I ligands in the pathogenesis of autoimmune diseases has been shown in several studies. In this study, the possible association of KIR genes, their known HLA ligands and compound KIR/HLA genotypes with ankylosing spondylitis (AS) was assessed. Combined KIR/HLA ligand genotyping was performed by a polymerase chain reaction-sequence-specific primers assay in 35 Iranian patients with AS, and genotypes were compared to those in 200 healthy individuals. The frequencies of telomeric cluster genes KIR2DL5A, KIR2DS1 and KIR3DS1 were significantly increased in AS patient group (P(c) = 0.0082, P(c) = 0.0195 and P(c) = 0.0328, respectively). Conversely, HLA-Bw4 ligand (the presence of one or more -B Bw4(Ile80) , -B Bw4(Thr80) and -A Bw4 epitopes) (P(c) = 0.0004) and HLA-B Bw4(Ile80) (P(c) = 0.053) were less frequent in these patients. Meanwhile, compound KIR/HLA genotype analyses revealed lower frequency of KIR3DL1+HLA-B Bw4(Ile80) (P(c) = 0.0343) and higher frequency of KIR2DS1+HLA-C2 (P(c) = 0.0308) combinations in patients with AS than in controls. In addition, the genotypes iKIR+HLA > aKIR+HLA (P(c) = .0308) and iKIR+HLA > aKIR (P(c) = 0.0258) were statistically less common, and genotypes iKIR+HLA = aKIR+HLA (P(c) = 0.0081) and iKIR+HLA < aKIR (P(c) = 0.077) were more common in patient group. Our findings suggest a role for excessive or inappropriate NK cell activation through 'KIR/HLA' system in AS disease.

Summary Natural killer (NK) cells eliminate infected and transformed cells while still are self-tolerant. Interactions of the independently segregating Killer cell immunoglobulin-like receptors (KIR) and human leucocyte antigens (HLA) loci play a critical role in NK cell regulation. Different compound KIR-HLA genotypes can impart different thresholds of activation to the NK-cell repertoire and such genotypic variation has been found to confer altered risk in a number of human diseases including viral infections, autoimmune disorders, reproduction abnormalities and cancers. In this study, we presented a novel combined KIR-HLA polymerase chain reaction–sequence-specific primers genotyping assay for simultaneous determination of KIR genes and their three major HLA class I ligand groups (C1, C2, and Bw4). Moreover, known inhibitory and activating KIR + HLA (iKIR + HLA: 2DL2/3 + C1, 2DL1 + C2, 3DL1 + Bw4; and aKIR + HLA: 2DS2 + C1, 2DS1 + C2, 3DS1 + Bw4) combinations as well as co-inheritance of aKIR genes and iKIR + HLA pairs were analysed in a total of 200 unrelated healthy Iranian individuals. All tested subjects had at least one of the three iKIR + HLA pairs and the frequencies of various inhibitory combinations in the study group were: 31.5%, three iKIR + HLA pairs, 53.5%, two iKIR + HLA pairs, and 15%, 0ne iKIR + HLA pair. Furthermore, we revealed that majority of Iranians (69%) carry compound genotypes with greater number of inhibitory pairings than activating combinations (iKIR + HLA > aKIR + HLA). Conversely, iKIR + HLA

Killer-cell immunoglobulin-like receptors (KIR) are a family of inhibitory and activating receptors that are expressed mainly by natural killer cells. The KIR gene family is highly polymorphic, and its genomic diversity is achieved through differences in gene content as well as allelic polymorphism. The number of KIR loci has been reported to be various among individuals and therefore resulting in different KIR haplotypes. This study represents the first report on the distribution of 17 presently defined KIR genes and pseudogenes in the Iranian population. In our study, 200 unrelated healthy individuals were KIR typed by a novel polymerase chain reaction-sequence-specific primers genotyping assay, and Iranian KIR genes distribution was compared with other ethnic groups. Over all, twenty-six different genotype profiles were found in our population and all KIR genes were observed. The most frequent non-framework KIR genes detected in our population were KIR2DL1 (96.5%), KIR3DL1 (91.5%), KIR2DS4 (91.5%) and the pseudogene KIR2DP1 (96.5%). The most commonly observed KIR genotype in Iranian population with a frequency of 27.5% consisted of KIR2DL1, KIR2DL3, KIR2DL4, KIR3DL1, KIR3DL2, KIR3DL3 and KIR2DS4 genes and the pseudogenes KIR2DP1 and KIR3DP1, which was compatible with a homozygote group-A haplotype. In addition, we found a new genotype (KIR2DL2, KIR2DL4, KIR2DL5, KIR3DL2, KIR3DL3, KIR2DS2, KIR2DS3, KIR2DS5, KIR3DS1 and KIR3DP1) in our samples. The results show that distribution of KIR genes in the Iranian population has common general features with the Caucasian populations studied before but still with unique, decreased or increased frequencies of several loci.

Intercellular adhesion molecule 1 (ICAM-1) is a cell surface glycoprotein member of the immunoglobulin superfamily and is actively involved in immune and inflammatory responses. We introduce a novel polymerase chain reaction-sequence-specific primers (PCR-SSP) method for rapid and simultaneous genotyping of ICAM-1 G241R and K469E polymorphisms. In a total of 184 DNA samples that have been previously analyzed for these polymorphisms using polymerase chain reaction-restriction fragment length polymorphism technique, re-genotyping of all samples with this new assay showed accurate and reproducible results. As PCR-SSP-based genotyping protocols are more convenient and cost-effective to do, it could therefore offer a valuable tool for assessment of ICAM-1 polymorphisms to which more confirmatory studies are needed.

1. The genetically polymorphic cytochrome P450 enzymes 2C9 (CYP2C9) and 2C19 (CYP2C19) are involved in the metabolism and elimination of a number of widely used drugs. The polymorphisms give rise to substantial interindividual and interethnic variability in drug excretion rates and final serum concentrations. For this reason, therapeutic responses and adverse drug reactions may vary from one person to another. In the present study we determined CYP2C9 and CYP2C19 genotypes in a random Iranian population to compare allele frequencies with previous findings in other ethnic groups. 2. Allelic variants of CYP2C9 (*1/*2/*3) and CYP2C19 (*1/*2/*3) were determined in 200 unrelated healthy Iranian volunteers by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assays. 3. Fifteen subjects (7.5%) were homozygous for the CYP2C9*2 allele, whereas 21 individuals (10.5%) were heterozygous for this allele and 164 subjects (82%) had the wild-type allele (CYP2C9*1). No CYP2C9*3 was detected in the population sampled. Six subjects (3%) were homozygous for CYP2C19*2, whereas 44 individuals (22%) were heterozygous for this allele. In the remaining subjects (75%), no CYP2C19*2 was found. In addition, no CYP2C19*3 was detected in the population sampled. 4. Based on our data, the frequency of the CYP2C9*2 allelic variant in Iranians is similar to that in other Caucasian populations; however, the frequency of the CYP2C9*3 allele differed significantly (P0.05). Conversely, there was no difference in the frequency of CYP2C19 allelic variants between the present study and other studies evaluating this allele in Caucasian populations (P0.05).

Background: The FAS and FAS-Ligand (FASL) system is an important apoptosis pathway in the liver. The FAS-mediated pathway functions by binding the FASL on the activated cytotoxic T lymphocytes and Natural Killer (NK) cells to the FAS receptor on infected hepatocytes. FAS and FASL polymorphisms, which are related to apoptosis, might influence the outcome of Hepatitis B Virus (HBV) infection. Objectives: Thus, the present study aimed to determine if FAS and FASL promoter polymorphisms are associated with the clinical outcome of HBV infection. Patients and Methods: DNA samples were obtained from the infected individuals including chronic carrier (n = 50), chronic hepatitis (n = 50), cirrhosis (n = 25), naturally recovered (n = 26) and compared with those of their matched healthy controls (n = 100). Genotyping for polymorphisms of FAS-670 A/G and -1377 G/A, and FASL -844 C/T was performed using polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) assays. Results: Multiple analyses for genetic association of FAS and FASL polymorphisms were not statistically different between HBV patients (n = 125) and healthy controls (n = 100). However, genotype and allele frequencies of FASL-844 C/T were significantly different between recovered individuals and patients with cirrhosis (P = 0.02 and P=0.01, respectively). Whereas, FAS-670A/G and -1377G/A polymorphisms were similarly distributed in these two groups (P = 0.8 and P = 0.47, respectively). Conclusions: The current study results showed that bearing -844T allele in FASL promoter region has a protective effect on cirrhosis and is involved in recovery from infection. In conclusion, it is proposed that HBV infection outcome might be influenced by FASL-844C/T polymorphism through alteration in apoptosis of hepatocytes.

To evaluate expression of glucocorticoid-induced tumor necrosis factor receptor (GITR), cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) and IL-10 in peripheral blood mononuclear cells (PBMCs) of 20 women with unexplained recurrent spontaneous abortion (URSA) compared to 20 normal non-pregnant women (NNP) during luteal phase in the window of implantation.Quantitative real-time PCR (qRT-PCR) was performed using the Taqman method for expression of GITR and SYBR Green method for expression of CTLA-4 and IL-10.Expression of CTLA-4 in the NNPs (median; interquartile range; 3; 1.8-10) was significantly higher than the URSAs (0.72; 0.26-3.81, p = 0.015). Expression of GITR in the NNPs (53; 10-139) was significantly higher than the URSAs (6; 3-27, p = 0.005). However, IL-10 expression in the URSAs was significantly higher than the NNPs, did not meet a significant value. A significant correlation was found between CTLA-4 and GITR expression in the study population (p = 0.0001).Expression of CTLA-4 and GITR were significantly down-regulated in the URSAs compared to NNPs at the window of implantation, which shows the essential role of Treg cells in creating an immunological privileged site for fetus as an allograft at the maternal-fetal interface by high expression levels of CTLA-4 and GITR during a normal pregnancy.