Publications by authors named "Abbas Rezaei"

73 Publications

Progesterone-induced blocking factor (PIBF) influences the expression of membrane progesterone receptors (mPRs) on peripheral CD4 T lymphocyte cells in normal fertile females.

Hormones (Athens) 2021 Apr 29. Epub 2021 Apr 29.

Cellular and Molecular Research Center, Birjand University of Medical Sciences, Birjand, Iran.

Purpose: Progesterone-induced blocking factor (PIBF) is a protein secreted by lymphocytes exposed to progesterone (P4). P4 and PIBF have immunomodulatory effects on peripheral CD4 T cells during normal pregnancy. Membrane progesterone receptors (mPRs) may correlate with the immunomodulatory properties of P4 on T cells. Variation in expression of mPRs may influence P4 regulatory performance during pregnancy. On the other hand, PIBF increases in pregnant normal women compared to women who have experienced abortion. The present study aimed to determine whether PIBF, in addition to having a direct influence on the immune system, can affect P4 performance through its effect on mPR expression. Such novel research findings demonstrate the importance of PIBF in the maintenance of pregnancy.

Methods: Isolated peripheral blood mononuclear cells (PBMCs) from 30 healthy women were stimulated with the mitogen phytohemagglutinin (PHA). Cells were either exposed to various concentrations of PIBF or had no exposure at all in a culture medium at 37 °C for 3 days. The mean fluorescence intensity (MFI) of mPRα and mPRβ was evaluated using polyclonal and monoclonal antibodies on CD4 T cells.

Results: PIBF was able to significantly increase mPR expression on the surface of peripheral CD4 T cells (p ≤ 0.05).

Conclusion: This study characterized the effects of PIBF on mPR expression on peripheral CD4 T cells of healthy fertile women. Thus, a decrease in PIBF concentration during abnormal pregnancy can modulate mPR expression and regulatory performance of P4 on T cells. Future research into this issue is likely to open up a new understanding of the etiology of abortion.
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http://dx.doi.org/10.1007/s42000-021-00291-5DOI Listing
April 2021

Comparison of high-flow oxygenation with noninvasive ventilation in COPD exacerbation: A crossover clinical trial.

Clin Respir J 2021 Apr 22;15(4):420-429. Epub 2020 Dec 22.

Chronic Respiratory Diseases Research Center (CRDRC), National Research Institute of Tuberculosis and Lung Diseases (NRITLD), Shahid Beheshti University of Medical Sciences, Tehran, Iran.

Objectives: To compare the therapeutic effects of high-flow-oxygen-Therapy (HFT) and noninvasive-ventilation (NIV) for stabilizing chronic obstructive pulmonary disease during exacerbation.

Methods: In this randomized clinical trial at Masih-Daneshvari hospital, between July 2019 and Oct 2019, 30 exacerbated-COPD-patient with PaCO 64.58 ± 11.61 mm Hg, Respiratory Rate 24.43 ± 2.75, and PH 7.31 ± 0.02 were divided into two groups, N = 15. By a simple randomized allocation, patients receive either NIV or HFT for 1 hour, and following a washout period of 30 minutes, they switched to the other treatment option. Arterial Blood Gas Parameters, as well as Respiratory Rate (RR), Dyspnea Score, Heart Rate (HR), and Oxygen Saturation (SO ), were compared before and after the intervention and between groups.

Results: Baseline patient characteristics were similar in the two groups. Pre and post-analysis revealed that in both groups, all improved significantly. After the first period, there was no difference in all parameters between groups except for SO which was significantly higher in HFT (%92.1 ± 1) than that of NIV (%89 ± 1), P = .001. Likewise, following the washout period, patients in HFT and NIV had a dyspnea score of 1.93 ± 0.7 and 2.73 ± 0.9, respectively, P = .01. No carryover-effect and was observed but the period effect was significant for some outcomes. A significant improvement in SO and HR was observed by HFT according to treatment effect by combining two periods' results. During the study, no side effects were reported.

Conclusion: In this short-term study HFT appears feasible for patients with COPD exacerbation to reduce dyspnea score and improve respiratory distress.
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http://dx.doi.org/10.1111/crj.13315DOI Listing
April 2021

Thyroid peroxidase in human endometrium and placenta: a potential target for anti-TPO antibodies.

Clin Exp Med 2021 Feb 26;21(1):79-88. Epub 2020 Sep 26.

Reproductive Immunology Research Center, Avicenna Research Institute, ACECR, Tehran, Iran.

Autoimmune thyroid disease is the most common endocrine disorder during pregnancy. Thyroid autoantibodies (TAs) have been suggested to serve a role in implantation failure and spontaneous abortion. Until now, there are no data on the potential interaction of TAs with human reproductive organs. Here, we set out for the first time to test this hypothesis by studying the expression of thyroid peroxidase (TPO) at gene and protein level in human reproductive organs. Endometrial samples were taken from normal women, and placenta tissues were collected after full-term caesarian section. Expression of TPO messenger RNA (mRNA) was investigated by qRT-PCR. In addition, polyclonal anti-TPO antibodies were produced and the expression of TPO protein in mentioned tissues was evaluated by immunohistochemistry and Western blot analysis. The reactivity of anti-TPO antibody in human embryos was evaluated by immunofluorescent staining. For the first time, our study showed that TPO is expressed at gene and protein levels in endometrium and placenta. TPO expression was mainly localized to glandular and luminal epithelial cells in the endometrium. In placenta, the syncytiotrophoblasts and invasive trophoblast cells were the main cell types that expressed TPO protein. Specific band of approximately 110 kDa was observed in all endometrial and placental tissues by Western blot analysis. However, no expression of TPO protein was observed in human embryo. TPO expression in endometrium and placenta may explain higher frequency of abortion and infertility in patients with thyroid autoimmunity.
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http://dx.doi.org/10.1007/s10238-020-00663-yDOI Listing
February 2021

Producing Soluble Human Programmed Cell Death Protein-1: A Natural Supporter for CD4+T Cell Cytotoxicity and Tumor Cells Apoptosis.

Iran J Biotechnol 2019 Dec 1;17(4):e2104. Epub 2019 Dec 1.

Immunology Department, Faculty of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran.

Background: Programmed cell death protein-1 (PD-1)/PD-L1 pathway is one of the immune checkpoint pathways involved in the regulation of the immune responses and the suppression of anti-tumor defense. PD-1/PD-L1 blocking antibodies improve immune responses such as cytotoxic activity of CD8/CD4T cells and increase mortality of tumor cells as well; however, their use is accompanied by adverse side effects.

Objectives: We aimed to produce a native blocker of human PD-1/PD-L1, for developing T cells cytotoxicity and tumor cells apoptosis.

Materials And Methods: We designed and cloned soluble human PD-1-GFP-pcDNA3.1/hygro construct in strain TOP10 cells and then transfected this construct into the HEK cells. The concentration of the secreted shPD-1 in the supernatant was measured and the supernatant was used for blocking PD-L1 on the MDA-MB-231 cells. The cytotoxicity of CD8/CD4T cells and the apoptosis of MDA-MB-231 cells, under the influence of shPD-1 in the co-culture of T cells with the MDA-MB-231 cells, were evaluated using flow cytometry technique.

Results: The GFP expression in the transfected cells illustrated the successful designing, transfection, and production of shPD-1. Soluble human PD-1 concentration in the supernatant of the transfected HEK cells was significantly higher than the untransfected cells. In addition, shPD-1 significantly blocked PD-L1 on the MDA- MB-231 cells, improved the cytotoxicity of CD4T cells, and increased the apoptosis of MDA-MB-231 cells.

Conclusion: Overall, increased CD4T cell cytotoxicity and tumor cells apoptosis under the influence of shPD-1, confirmed the effectiveness of shPD-1 as a natural blocker of PD-L1and as an augmenter of the anti-tumorimmune responses.
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http://dx.doi.org/10.30498/IJB.2019.85180DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7357696PMC
December 2019

Decreased Toll-like Receptor (TLR) 2 and 4 Expression in Spermatozoa in Couples with Unexplained Recurrent Spontaneous Abortion (URSA).

Iran J Allergy Asthma Immunol 2019 Nov 5;18(6):701-706. Epub 2019 Nov 5.

Department of Immunology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran.

Studies have shown that toll-like receptors (TLRs) play some important roles in reproductive processes such as ovulation, spermatogenesis, sperm capacitation, fertilization, and pregnancy to the best of our knowledge, no study has evaluated the expression and role of these molecules and their impairment in spermatozoa; accompanied by pregnancy complications such as recurrent spontaneous abortion (RSA). Therefore, this study investigates the alteration of toll-like receptor 2 (TLR2) and toll-like receptor 4 (TLR4) expression in spermatozoa in men whose spouse have unexplained RSA. Fifteen fertile couples and fifteen couples with unexplained recurrent spontaneous abortion (URSA) were included in this study. The level of TLR2 and TLR4 expression in untreated and lipopolysaccharide (LPS) or PAM3CYS in treated spermatozoa were examined by flow cytometry. The results showed reduced expression of TLR4 in untreated spermatozoa and decreased LPS or PAM3CYS levels in treated spermatozoa in the URSA group compared to the control group. No significant differences were found in TLR2 expression of untreated spermatozoa in RSA and control groups. After the treatment of spermatozoa with LPS, the TLR2 expression was decreased in both groups. After the treatment of spermatozoa with PAM3CYS, the level of TLR2 expression was significantly increased in the URSA group; while no significant differences were shown in the control group in comparison to untreated spermatozoa. We have concluded that decreased TLR4 expression and a differently increased TLR2 expression in response to ligand treatment in spermatozoa is associated with URSA.
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http://dx.doi.org/10.18502/ijaai.v18i6.2183DOI Listing
November 2019

Optimization of Expansion and Activation of Human Natural Killer Cells against Breast Cancer Cell Line.

Avicenna J Med Biotechnol 2020 Jan-Mar;12(1):17-23

Department of Immunology, Faculty of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran.

Background: Regarding to the increase of cancer deaths in recent years and disability of common therapies to eradicate cancers, as well as expansion of Natural Killer (NK) cell therapy, it seems so vital to find new useful therapies against cancers. Breast cancer is the second main cause of cancer death among women. As it is impossible for a majority of patients to receive NK cell therapy, an attempt was made to establish a low-cost and efficient method for expanding and activating NK cells against breast cancer cell line (MCF7).

Methods: NK cells were isolated from Peripheral Blood Mononuclear Cells (PBMCs) applying either MACS based NK cell enrichment kit or antibodies and complement as cytotoxic method. Then, the NK cells were cultured in Stem Cell Growth Medium (SCGM) with feeder layer (irradiated PBMCs) along with PHA or OKT3. IL-2, IL-15 and IL-21 were used to expand NK cells and finally their cytotoxic activity was investigated by flow cytometry.

Results: Highly pure NK cells were obtained and no significant difference between the two isolation methods was found. Using IL-2 plus IL-15, the number of NK cells increased up to100 fold after 16 days. No significant effect was observed after IL-21 treatment.

Conclusion: Our data indicated that cytotoxicity method can be considered a low-cost alternative for NK cell isolation kits. It seems that culturing NK cells for 14 days in either PHA or OKT3 supplemented SCGM medium would be more effective than culturing for 16 days in the presence of IL-21.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7035457PMC
March 2020

Can the Decreased Expression of Human Leukocyte Antigen Class Ⅰ and Ⅱ by Spermatozoa Lead to Recurrent Spontaneous Abortion?

Iran J Pathol 2020 ;15(1):19-22

Department of Immunology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran.

Background & Objective: Unexplained recurrent spontaneous abortion (URSA) is defined as an unknown cause of occurrence of three or more clinically detectable pregnancy losses before 20 weeks of gestation, but it occurs presumably as a result of the immune system dysfunctions. We supposed that the disruption of semen or spermatozoa might be responsible for the dysfunction of the immune system in women with URSA. Semen and spermatozoa (as antigens) induce female reproductive tract (FRT) immunity. This stimulated immunity is necessary for pregnancy occurrence. The disruption of semen or spermatozoa can be a result of altering a variety of surface molecules on spermatozoa, especially polymorphic human leukocyte antigen (HLA) molecules or antigens. Despite the importance of HLA antigens in reproduction, to the best of our knowledge, no one has studied the relation of HLA expression between spermatozoa and URSA. Therefore, this paper aims to assess this relation.

Methods: Semen samples were collected from 15 URSA couples and 20 normal couples. After purification of normal spermatozoa, the HLA class I and II expressions were evaluated by flow cytometry methods.

Results: Results showed that the expression of both HLA class I and II by spermatozoa, in URSA couples, was significantly less than the control couples.

Conclusion: The decreased expression of polymorphic HLA class Ⅰ and Ⅱ by spermatozoa can be related to URSA occurrence.
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http://dx.doi.org/10.30699/IJP.2019.102943.2026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6995681PMC
January 2020

NOX1 Regulates Collective and Planktonic Cell Migration: Insights From Patients With Pediatric-Onset IBD and NOX1 Deficiency.

Inflamm Bowel Dis 2020 07;26(8):1166-1176

Dr. von Hauner Children's Hospital, Department of Pediatrics, University Hospital, Ludwig-Maximilians-Universität München, Munich, Germany.

Background: Genetic defects of pediatric-onset inflammatory bowel disease (IBD) provide critical insights into molecular factors controlling intestinal homeostasis. NOX1 has been recently recognized as a major source of reactive oxygen species (ROS) in human colonic epithelial cells. Here we assessed the functional consequences of human NOX1 deficiency with respect to wound healing and epithelial migration by studying pediatric IBD patients presenting with a stop-gain mutation in NOX1.

Methods: Functional characterization of the NOX1 variant included ROS generation, wound healing, 2-dimensional collective chemotactic migration, single-cell planktonic migration in heterologous cell lines, and RNA scope and immunohistochemistry of paraffin-embedded patient tissue samples.

Results: Using exome sequencing, we identified a stop-gain mutation in NOX1 (c.160C>T, p.54R>*) in patients with pediatric-onset IBD. Our studies confirmed that loss-of-function of NOX1 causes abrogated ROS activity, but they also provided novel mechanistic insights into human NOX1 deficiency. Cells that were NOX1-mutant showed impaired wound healing and attenuated 2-dimensional collective chemotactic migration. High-resolution microscopy of the migrating cell edge revealed a reduced density of filopodial protrusions with altered focal adhesions in NOX1-deficient cells, accompanied by reduced phosphorylation of p190A. Assessment of single-cell planktonic migration toward an epidermal growth factor gradient showed that NOX1 deficiency is associated with altered migration dynamics with loss of directionality and altered cell-cell interactions.

Conclusions: Our studies on pediatric-onset IBD patients with a rare sequence variant in NOX1 highlight that human NOX1 is involved in regulating wound healing by altering epithelial cytoskeletal dynamics at the leading edge and directing cell migration.
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http://dx.doi.org/10.1093/ibd/izaa017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7365810PMC
July 2020

Gene expression analysis of membrane progesterone receptors in women with recurrent spontaneous abortion: a case control study.

BMC Res Notes 2019 Dec 4;12(1):790. Epub 2019 Dec 4.

Department of Immunology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran.

Objective: Recurrent spontaneous abortion (RSA) is a condition which is defined as three consecutive pregnancy losses prior to 20 weeks from the last menstrual period. Progesterone is a steroid hormone that has an essential role in the implantation and maintenance of pregnancy. The progesterone signaling is performed by nuclear progesterone receptors (NPRs) and membrane progesterone receptors (mPR). The aim of this study was to analyze gene expression of mPR-α, mPR-β and NPR in the endometrium of patients with a history of RSA compared to normal fertile women.

Results: In this study, endometrial samples were obtained from 10 women with a history of RSA and 10 fertile women during days 10-14 of menstrual cycle. Relative expression of mPR-α, mPR-β and NPR genes were studied by a quantitative real time polymerase chain reaction (qRT-PCR) and compared between the two groups. The mean relative expression of mPR-β gene was significantly lower in the case group compared to the fertile women (p < 0.05). However, the gene expression of mPR-α and NPR showed no significant difference between two groups. The findings suggest a reduction of endometrial gene expression of mPR-β in RSA patients may play an important role in pathogenesis of RSA.
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http://dx.doi.org/10.1186/s13104-019-4787-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6894300PMC
December 2019

IL-12Rβ1 deficiency corresponding to concurrency of two diseases, mendelian susceptibility to mycobacterial disease and Crohn's disease.

J Clin Tuberc Other Mycobact Dis 2019 Dec 20;17:100123. Epub 2019 Sep 20.

Aquired Immunodeficiency Research Center, Isfahan University of Medical Sciences, Isfahan, Iran.

Background: The interleukin-12 receptor β1 (IL-12Rβ1) deficiency is a primary immunodeficiency (PID), affecting the immunological pathway of interleukin 12/interferon- γ (IL12/IFN-γ) axis and interleukin 23 receptor (IL23R). Defect in this pathway is mainly affecting the cellular immunity-related disorders. IL-12Rβ1 is a receptor chain of both the IL-12 and the IL-23 receptors and thus, deficiency of IL-12Rβ1 abolishes both IL-12 and IL-23 signaling.

Material And Methods: In this study, we performed whole exon sequencing and confirmatory Sanger sequencing in . Evaluation of the IL12/IFN-γ axis was performed by assessment of patients' whole blood cell to IL12/IFN-γ responding. Total and surface IL-12Rβ1expression was evaluated, in peripheral blood mononuclear cells (PBMCs) and T cell- derived PBMCs, and Th17 count was assessed.

Results: In the present study, we described a c.1791 + 2T > G mutation at a splicing site position in using whole exome sequencing, and confirmed with targeted Sanger sequencing in a 26- year-old patient with Mendelian susceptibility to mycobacterial disease (MSMD) and Crohn's disease (CD). Complete lack of IL-12Rβ1 protein expression was detected in patient's PBMCs, compared to the healthy control. Furthermore, no IL-12Rβ1 protein was expressed on the cell surface. Interestingly, IL-12Rβ1-mutant cells showed an impaired response to IL12, and stimulation, confirming that the mutation is causative in this patient.

Conclusion: A 3'splicing site mutation in , can be corresponding to the abolished expression of in patients' cells, and associated with an impaired IL12-mediated signaling, which may lead not only to MSMD, but also to inflammatory bowel disease (IBD).
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http://dx.doi.org/10.1016/j.jctube.2019.100123DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6879969PMC
December 2019

The expression of human leukocyte antigen by human ejaculated spermatozoa.

Mol Genet Genomic Med 2019 12 18;7(12):e1005. Epub 2019 Oct 18.

School of Geosciences, University of Aberdeen, Aberdeen, Scotland, UK.

Background: After coitus and insemination, an inflammatory response is evident in the female reproductive tract (FRT). Semen contains a variety of immune-activating components that have a major role in the induction of an immune response in the FRT. One of the most important is (human leukocyte antigen) HLA molecules which are present in soluble form in seminal plasma and in membrane form on the surface of cells (such as epithelial and leukocytes) existing in semen. Nevertheless, there is considerable debate over the expression of HLA antigens by human spermatozoa. Considering the critical role of HLA molecules in reproduction and the induction of an immune response, it is very important to clearly define HLA expression by spermatozoa and the role of these molecules in sperm morphology, motility, and strength to fertilize an egg. Therefore, the objective of this study was to determine HLA expression by ejaculated spermatozoa. The results of this study will facilitate the design of future studies.

Method: Semen samples were collected from 50 healthy men with normal semen status by masturbation after 2-3 days of sexual abstinence. After purification of normal spermatozoa, HLA class I & II expression was evaluated by quantitative real-time PCR and flow cytometry methods.

Results: The results showed the expression of both HLA class I & class II by spermatozoa. The results also showed that the expression of HLA class Ⅱ was significantly more than HLA class Ⅰ.

Conclusion: Spermatozoa express both HLA class I & class II molecules.
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http://dx.doi.org/10.1002/mgg3.1005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6900355PMC
December 2019

Roles of GM-CSF in the Pathogenesis of Autoimmune Diseases: An Update.

Front Immunol 2019 4;10:1265. Epub 2019 Jun 4.

Department of Immunology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) was first described as a growth factor that induces the differentiation and proliferation of myeloid progenitors in the bone marrow. GM-CSF also has an important cytokine effect in chronic inflammatory diseases by stimulating the activation and migration of myeloid cells to inflammation sites, promoting survival of target cells and stimulating the renewal of effector granulocytes and macrophages. Because of these pro-cellular effects, an imbalance in GM-CSF production/signaling may lead to harmful inflammatory conditions. In this context, GM-CSF has a pathogenic role in autoimmune diseases that are dependent on cellular immune responses such as multiple sclerosis (MS) and rheumatoid arthritis (RA). Conversely, a protective role has also been described in other autoimmune diseases where humoral responses are detrimental such as myasthenia gravis (MG), Hashimoto's thyroiditis (HT), inflammatory bowel disease (IBD), and systemic lupus erythematosus (SLE). In this review, we aimed for a comprehensive analysis of literature data on the multiple roles of GM-CSF in autoimmue diseases and possible therapeutic strategies that target GM-CSF production.
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http://dx.doi.org/10.3389/fimmu.2019.01265DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6593264PMC
October 2020

Development of α4 integrin DNA aptamer as a potential therapeutic tool for multiple sclerosis.

J Cell Biochem 2019 09 20;120(9):16264-16272. Epub 2019 May 20.

Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Islamic Republic of Iran.

One of the most important molecules for multiple sclerosis pathogenesis is α4 integrin, which is responsible for autoreactive leukocytes migration into the brain. The monoclonal antibody, natalizumab, was introduced to market for blocking the extravasation of autoreactive leukocytes via inhibition of α4 integrin. However, the disadvantages of antibodies provided a suitable background for other agents to be replaced with antibodies. Considering the profound advantages of aptamers over antibodies, aptamer isolation against α4 integrin was intended in the current study. The α4 integrin-specific aptamers were selected using cell-systematic evolution of ligands by exponential enrichment (SELEX) method with human embryonic kidney (HEK)-293T overexpressing α4 integrin and HEK-293T as target and control cells, respectively. Evaluation of selected aptamer was performed through flow cytometric analysis. The selected clones were then sequenced and analyzed for any possible secondary structure and affinity. The results of this study led to isolation of 13 different single-stranded DNA clones in 11 rounds of selection which were categorized to three clusters based on common structural motifs and the equilibrium dissociation constant (K ) of the most stable structure was calculated. The evaluation of SELEX progress showed growth in aptamer affinity with increasing of the number of cycles. Taken together, the findings of this study demonstrated the isolation of α4-specific single-stranded DNA aptamers with suitable affinity for ligand, which can further be replaced with natalizumab.
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http://dx.doi.org/10.1002/jcb.28907DOI Listing
September 2019

Vitamin D3 induces the expression of membrane progesterone receptors (mPRs) on naive CD4 T lymphocyte cells in women of reproductive age.

Int Immunopharmacol 2019 Jul 5;72:55-61. Epub 2019 Apr 5.

School of Medicine, Isfahan University of Medical Sciences, Department of Immunology, Isfahan, Iran; Acquired Immunodeficiency Research Center, Isfahan University of Medical Sciences, Isfahan, Iran. Electronic address:

Objective: Vitamin D3 and progesterone (P4) both belong to steroid hormones. These hormones have effects on the function of each other in different ways. The immunomodulatory activity of vitamin D3 and P4 and their role in inducing maternal tolerance for fetus have been shown in various studies. The purpose of this study was to evaluate the effect of vitamin D3 on the expression of membrane progesterone receptors (mPRs) on CD4 T cells.

Materials And Methods: Naive CD4 T cells were isolated from peripheral blood of 38 healthy women of childbearing age. After stimulating by anti-CD3 and anti-CD28 monoclonal antibodies (mAb), these cells were exposed to either various concentrations of vitamin D3 or no exposure at all in a culture medium at 37 °C for 3 days. In the final stage, the mean fluorescence intensity (MFI) of mPRα and mPRβ were evaluated using polyclonal and monoclonal antibodies and several gating strategies on CD4 T cells.

Results: Vitamin D3 significantly increased the expression of mPR α and mPR β on the surface of CD4 T cells (p ≤ 0.05).

Conclusion: The present study demonstrated the potential effect of vitamin D3 on increasing the expression of P4 receptors on CD4 T cells. This study shows a new aspect of correlation between vitamin D3 and P4 that may influence P4 performance. Therefore, our findings suggest that the appropriate level of this vitamin may affect the optimum P4 immunomodulatory activity during pregnancy.
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http://dx.doi.org/10.1016/j.intimp.2019.03.053DOI Listing
July 2019

Immunomodulatory effects of human amniotic epithelial cells on naive CD4 T cells from women with unexplained recurrent spontaneous abortion.

Placenta 2018 11 18;71:31-40. Epub 2018 Jun 18.

Department of Immunology, Faculty of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran. Electronic address:

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.
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http://dx.doi.org/10.1016/j.placenta.2018.06.008DOI Listing
November 2018

MicroRNA-92a Drives Th1 Responses in the Experimental Autoimmune Encephalomyelitis.

Inflammation 2019 Feb;42(1):235-245

Department of Immunology, Faculty of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran.

Dysregulation of microRNAs (miRNAs) has been linked to the progress of a number of autoimmune diseases including multiple sclerosis (MS), and its animal model, experimental autoimmune encephalomyelitis (EAE). IFN-γ-producing Th1 cells are major players in MS/EAE pathogenesis. It is known that differentiation of T cells towards the Th1 phenotype is influenced by various factors including miRNAs. The miR-92a shows substantial upregulation in MS; however, little is known about its role in the development of autoimmune and inflammatory responses. Herein, we investigated the role of miR-92a in the pathogenesis of MS, focusing on its potential effects on differentiation of Th1 cells. The expression levels of miR-92a were assessed in the spinal cord tissues and splenocytes from mice with EAE using real-time RT-PCR. Next, using transfection with miR-92a mimic sequences, the potential involvement of miR-92a in Th1 polarization was investigated by flow cytometric analysis. Moreover, the expression levels of miR-92a targets were explored in spinal cord tissues of EAE mice. miR-92a expression was enhanced in mouse spinal cord samples at the peak of EAE disease. Overexpression of miR-92a in splenocytes led to increased differentiation of Th1 cells compared with cells transfected with negative control sequences. Enhanced miR-92a expression was accompanied by reduced expression TSC1 or DUSP10, predicted miR-92a targets, in EAE spinal cords. Our data point to a potential role for miR-92a in neuroinflammatory responses in EAE. Our results indicate that miR-92a might affect Th1 differentiation, likely due to downregulation of TSC1 and DUSP10.
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http://dx.doi.org/10.1007/s10753-018-0887-3DOI Listing
February 2019

High Frequency of Tc22 and Th22 Cells in Myasthenia Gravis Patients and Their Significant Reduction after Thymectomy.

Neuroimmunomodulation 2018 2;25(2):80-88. Epub 2018 Aug 2.

Department of Immunology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran.

Objectives: Myasthenia gravis (MG) is an autoimmune disease accompanied by a thymic pathology and in most patients thymectomy (TE) is used as the therapeutic approach. Both B and T cells play an important role in MG pathogenesis.

Methods: Twelve pre- and post-TE MG patients and 12 healthy controls (HCs) were enrolled. The mean percentages of Th22 and Tc22 cells were evaluated in MG patients (before and 6 months after TE) and HCs.

Results: The mean percentage of Tc22 cells in pre-TE patients was significantly higher than in HCs (p < 0.05), and after TE Tc22 cells significantly decreased compared to pre-TE (p < 0.05). The frequency of Th22 cells in pre-TE MG patients was not significantly different from HCs, but after TE Th22 cells were significantly decreased compared to pre-TE (p < 0.05).

Conclusion: Our findings suggest a possible role of Th22 and Tc22 in MG pathogenesis.
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http://dx.doi.org/10.1159/000490855DOI Listing
January 2019

Stimulation of Camel Polyclonal Antibody against Human T cell Immunoglobulin and Mucin 3.

Iran J Biotechnol 2017 27;15(3):166-171. Epub 2017 Sep 27.

Immunology Department, Faculty of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran.

T cell Immunoglobulin, Mucin (TIM)-3, is a type I transmembrane glycoprotein belonging to TIM family. This receptor expresses on T helper type 1 (Th1) cells that binds to galectin-9 (Gal9); inducing an inhibitory signal. As a result, apoptosis of Th1 cells occurs and cytotoxicity of CD8 T cells becomes evident in vitro. Therefore, this immunomodulatory molecule may be used as a novel target for clinical purposes. The production of camel polyclonal antibodies against TIM-3-expressing cell line was the purpose of this study. In this study, we aimed to use HEK 293 cells expressing human TIM-3 to obtain camel polyclonal antibody against TIM-3 by immunization. A pre-synthesized human TIM-3cDNA was inserted into pcDNA3.1 plasmid and the new construct was transfected in HEK cell. TIM-3 expression was confirmed by qRT-PCR and flow cytometry. A camel (6 months old) was immunized with the lysate prepared from rTIM-3 expressing HEK cells 4 times. The anti-TIM-3 antibody level was evaluated using ELISA method. TIM-3 was successfully cloned in HEK cells with 88% success rate. High level of anti-TIM-3 antibody was detected in the serum of the camel immunized with the recombinant cell lysate, after final injection. Our rhTIM-3 cell display system can be useful for future diagnostic or therapeutic approaches.
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http://dx.doi.org/10.15171/ijb.1427DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5811063PMC
September 2017

Real-Time PCR: an Appropriate Approach to Confirm ssDNA Generation from PCR Product in SELEX Process.

Iran J Biotechnol 2017 19;15(2):143-148. Epub 2017 Aug 19.

Department of Immunology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, 313, Iran.

Aptamers are single stranded DNA (ssDNA) or RNA molecules. The potential of aptamers for binding to the different targets has made them be widely used as the preferred diagnostic and therapeutic tools. DNA aptamers present several advantages over the RNA oligonucleotides due to their higher stability, easier selection, and production. Selection of DNA aptamers which is facilitated through a systematic evolution of ligand by exponential enrichment (SELEX) method is much dependent on the successful conversion of double stranded DNA (dsDNA) to ssDNA. There are different methods available for ssDNA generation. While visualization of ssDNA is limited to the gelbased method, the method is not applicable in the initial rounds of SELEX due to more than 1015 different sequences. This study was designed to evaluate the effi ciency of another technique for confi rming the ssDNA generation in comparison to the polyacrylamide electrophoresis (PAGE) analysis. Real-time PCR was employed in the present study for PCR amplifi cation of the initial library that was followed by enzymatic digestion of the dsDNA. Subsequently melting curve analysis was carried out to evaluate ssDNA generation from dsDNA. Moreover, PAGE analysis was performed and the results were compared with the melt curve analysis. The melt curves, revealed dsDNA conversion to the ssDNA based on a significant reduction of Tm from 73.8 to 41.5 °C. Applying PAGE analysis, it was not effectively feasible to show ssDNA generation from the corresponding initial dsDNA library, while, it was effi cient enough to confirm ssDNA generation in accordance with the increasing the number of SELEX rounds. The present study has proven the applicability of the real-time PCR as a suitable confirmatory technique for validating ssDNA generation in the DNA aptamer selection process for the initial library preparation.
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http://dx.doi.org/10.15171/ijb.1550DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5811056PMC
August 2017

The Increase in Protein and Plasmid Yields of with Optimized Concentration of Ampicillin as Selection Marker.

Iran J Biotechnol 2017 19;15(2):128-134. Epub 2017 Aug 19.

Department of Immunology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, 81746-73461 Iran.

is still the common host for ing and heterologous protein expression. Various strategies have been employed to increase protein expression in , but, it seems that external factors such as selection marker concentration can drastically affect the yield of protein and plasmid. Alterations of protein expression and plasmid yields of in different concentrations of ampicillin, as selection marker, will be determined. In order to improve heterologous expression, the system will be redesigned and optimized. The expression cassette of codon optimized EGFP for was synthesized in pUC57. The pUC57-GFP was transformed into . The expression of GFP was verified by SDS-PAGE and flow cytometry after induction by IPTG (0.5 mM) and incubation with 0, 100, 200 and 300 μg.mL ampicillin. Plasmid copy numbers of samples were determined by Real-Time PCR on AMP gene using regression line of diluted standard curve. GFP expressing clones formed fair green colonies on LB agar supplemented with 0.5 mM IPTG and showed fluorescence in FL1 filter of flow cytometry and an extra protein band on SDS-PAGE gel. The fluorescent intensity of GFP in 0, 100, 200 and 300 μg.mL ampicillin in medium were 549.83, 549.78, 1443.52, 684.87, and plasmid copy numbers were 6.07×10 , 3.21×10 , 2.32×10 , 8.11×10 , respectively. The plasmid yields were 55 ng.μL, 69 ng.μL, 164 ng.μL and 41 ng.μL, respectively. Protein and plasmid yields of are variable in different concentrations of ampicillin and need to be optimized in newly designed expression systems. Protein and plasmid yield in the optimized concentration (200 μg.mL) was significantly (p < 0.01) higher than other doses.
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http://dx.doi.org/10.15171/ijb.1467DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5811054PMC
August 2017

Human amniotic epithelial cells inhibit activation and pro-inflammatory cytokines production of naive CD4+ T cells from women with unexplained recurrent spontaneous abortion.

Reprod Biol 2018 Jun 3;18(2):182-188. Epub 2018 May 3.

Department of Immunology, School of Medicine, Iran University of Medical Sciences, Shahid Hemmat Highway, Tehran, 14496, Iran. Electronic address:

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.
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http://dx.doi.org/10.1016/j.repbio.2018.04.002DOI Listing
June 2018

Gene-knocked out chimeric antigen receptor (CAR) T cells: Tuning up for the next generation cancer immunotherapy.

Cancer Lett 2018 06 12;423:95-104. Epub 2018 Mar 12.

Department of Medical Immunology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran. Electronic address:

Recently clinical trials utilizing genetically engineered T cells expressing a chimeric antigen receptor (CAR) that is half monoclonal antibody and half T-cell receptor have demonstrated remarkable response in patients with advanced cancers like relapsed or refractory acute lymphoblastic leukemia (ALL) and lymphoma. Moreover, emerging chimeric genome editing tools such as zinc-finger nucleases (ZNFs), transcription activator-like effector nucleases (TALENs) and clustered regulatory interspaced short palindromic repeat (CRISPR)/Cas composed of sequence-specific DNA binding module(s) linked to a non-specific DNA cleavage domain have made possible to dramatically expand the ability to manipulate cells aim to treat and/or study a wide range of diseases including cancer. Here, we will discuss how joint application of these two chimeras will help us to manipulate CAR T cells aiming to enhance the efficacy of CAR T cell therapy in preclinical and clinical settings.
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http://dx.doi.org/10.1016/j.canlet.2018.03.010DOI Listing
June 2018

Increased Proportion of Tc17 and Th17 Cells and Their Significant Reduction after Thymectomy May Be Related to Disease Progression in Myasthenia Gravis.

Neuroimmunomodulation 2017 8;24(4-5):264-270. Epub 2018 Feb 8.

Department of Immunology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran.

Objective: Myasthenia gravis (MG) is an autoimmune disease mediated by autoantibodies against the neuromuscular junction. The thymus has an important role in the pathogenesis of MG because most patients have thymic pathology, and thymectomy (TE) can reduce the severity of the disease.

Methods: In this study, the frequency of Th17 and Tc17 cells was studied in 12 MG patients (pre-TE and 6 months post-TE) and in 12 healthy controls (HC).

Results: The frequency of Tc17 cells in the pre-TE patients was significantly higher than in the HC (p < 0.05), and after TE, these cells had significantly decreased compared to before TE (p < 0.05). The frequency of Th17 cells in pre-TE patients was significantly higher than in the HC (p < 0.05), and after TE, these cells had significantly decreased compared to before TE (p < 0.05).

Conclusion: Our findings indicated a possible role of Tc17 and Th17 in MG pathogenesis.
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http://dx.doi.org/10.1159/000486037DOI Listing
January 2019

Down-regulation of inflammatory signaling pathways despite up-regulation of Toll-like receptors; the effects of corticosteroid therapy in brain-dead kidney donors, a double-blind, randomized, controlled trial.

Mol Immunol 2018 02 16;94:36-44. Epub 2017 Dec 16.

Immunology Research Center (IRC), Iran University of Medical Sciences, Tehran, Iran. Electronic address:

Background: The brain death of a potential organ donor induces a systemic inflammatory response, resulting in inferior organ quality and function. Our study aimed to evaluate the effects of methylprednisolone (MPN) therapy on pattern recognition receptor (PRR) signaling in potential brain-dead (BD) kidney donors.

Material And Methods: To evaluate the effects of MPN therapy on PRR signaling in BD kidney donors we performed a prospective randomized treatment-versus-control study. Fifty-one potential kidney donors were randomly divided into three groups: brain-dead donors (BDDs) who received 15 mg/kg/d of methylprednisolone (group T1, n = 17), BDDs who received 15 mg/kg/d of MPN at the time of filling consent for kidney donation and 100 mg/2 h until kidney harvest (group T2, n = 17), and normal donors as controls n = 17. Gene expression for Toll-like receptors (TLRs) 1-9 and their signaling pathway molecules including MYD88, TRIF, NF-KB1, IRAK, IRF3, and IRF7, as well as the inflammatory cytokines RANTES, IL-1β, TNF-α, IL-6, CXCL8, IL-18, IFN-α, and IFN-β was determined by PCR array. Due to the crucial role of TLRs 2 and 4 in pattern recognition, surface expression of these molecules was analyzed by flow cytometry. Plasma levels of inflammatory cytokines were measured by immunoassay. Finally, serum creatinine and cystatin C were measured in 100 kidney recipients one week and one, three, and six months after transplant.

Result: Polymerase chain reaction (PCR) array gene expression revealed greater expression of TLRs and signaling molecules in group T1 than in the controls. Surface expression of TLRs 2 and 4 were significantly greater in group T2 than in group T1 (P < .05). Plasma concentrations of inflammatory cytokines were significantly greater in group T1 than in controls (P < .05). The recipients that received kidneys from group T1 had significantly higher levels of creatinine and cystatin C than the recipients of kidneys from both group T1 and controls (P<0.05).

Conclusion: Administration of MPN to BDDs at specified periods until kidney harvest resulted in less systemic inflammation in the BDDs and improved renal function in kidney graft recipients compared with common MPN therapy.
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http://dx.doi.org/10.1016/j.molimm.2017.12.012DOI Listing
February 2018

Prophylactic DNA vaccine targeting Foxp3 regulatory T cells depletes myeloid-derived suppressor cells and improves anti-melanoma immune responses in a murine model.

Cancer Immunol Immunother 2018 03 9;67(3):367-379. Epub 2017 Nov 9.

Department of Immunology, Building No. 7, School of Medicine, Tehran University of Medical Sciences, Poursina Avenue, Tehran, 14155-6447, Iran.

Regulatory T cells (Treg) and myeloid-derived suppressor cells (MDSC) are the two important and interactive immunosuppressive components of the tumor microenvironment that hamper anti-tumor immune responses. Therefore, targeting these two populations together might be beneficial for overcoming immune suppression in the tumor microenvironment. We have recently shown that prophylactic Foxp3 DNA/recombinant protein vaccine (Foxp3 vaccine) promotes immunity against Treg in tumor-free conditions. In the present study, we investigated the immune modulatory effects of a prophylactic regimen of the redesigned Foxp3 vaccine in the B16F10 melanoma model. Our results indicate that Foxp3 vaccination continuously reduces Treg population in both the tumor site and the spleen. Surprisingly, Treg reduction was associated with a significant decrease in the frequency of MDSC, both in the spleen and in the tumor environment. Furthermore, Foxp3 vaccination resulted in a significant reduction of arginase-1(Arg-1)-induced nitric oxide synthase (iNOS), reactive oxygen species (ROS) and suppressed MDSC activity. Moreover, this concurrent depletion restored production of inflammatory cytokine IFN-γ and enhanced tumor-specific CTL response, which subsequently resulted in the reduction of tumor growth and the improved survival rate of vaccinated mice. In conclusion, our results revealed that Foxp3 vaccine promotes an immune response against tumor by targeting both Treg and MDSC, which could be exploited as a potential immunotherapy approach.
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http://dx.doi.org/10.1007/s00262-017-2088-6DOI Listing
March 2018

Method and key points for isolation of human amniotic epithelial cells with high yield, viability and purity.

BMC Res Notes 2017 Nov 2;10(1):552. Epub 2017 Nov 2.

Department of Immunology, Faculty of Medicine, Isfahan University of Medical Sciences, Hezar-Jereeb Ave, Isfahan, Iran.

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 × 10 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.
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http://dx.doi.org/10.1186/s13104-017-2880-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5669002PMC
November 2017

Role of Apoptosis in the Pathogenesis of Common Variable Immunodeficiency (CVID).

Endocr Metab Immune Disord Drug Targets 2017 Nov;17(4):332-340

bResearch Center for Immunodeficiencies, Pediatrics Center of Excellence, Children`s Medical Center, Tehran University of Medical Science, Tehran, Iran.

Background: Common variable immunodeficiency (CVID) is a heterogeneous immune deficiency characterized by hypogammaglobulinemia. Since B cell maturation and differentiation is defective in this disorder, we evaluated apoptosis in B cells of patients with CVID compared with healthy donors (HD).

Methods: Determination of peripheral blood B-cell subsets in CVID and HDs, was performed using flow cytometry. We compared total apoptosis, early apoptosis and late apoptosis/necrosis in unstimulated and stimulated B-cells of patients with CVID and HDs. We also assessed the expression of the anti-apoptotic molecule BCL2 mRNA levels in B-cells by real-time PCR in CVID patients compared with HDs.

Results: Total B-cell apoptosis was increased in both unstimulated and stimulated B-cells from CVID patients compared with HDs (p=0.02 and p=0.004). Early apoptosis in stimulated B-cells (p=0.04) and late apoptosis/necrosis of B-cells in both unstimulated and stimulated B-cells (p=0.04 and p=0.03, respectively) were significantly higher in CVID patients compared with HDs. There was a significant inverse correlation between the percentages of post germinal center B-cells in the peripheral blood of CVID patients compared with percentage of apoptotic B-cells. However, anti-apoptotic BCL2 expression was not significantly reduced in B-cells from CVID patients compared with HDs (p=0.16).

Conclusion: Increased apoptosis of B-cells may be a factor in abnormality of differentiated B-cell subsets and the impaired endogenous immunoglobulin production in CVID patients. Further studies of the expression of pro/anti-apoptotic mediators in B-cells of CVID patients may shed light on the mechanism behind this increased B-cell apoptosis, and present potential therapeutic interventions in the future.
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http://dx.doi.org/10.2174/1871530317666170919120245DOI Listing
November 2017

Innate inflammatory gene expression profiling in potential brain-dead donors: detailed investigation of the effect of common corticosteroid therapy.

Innate Immun 2017 07 15;23(5):440-448. Epub 2017 May 15.

1 Immunology Departatment, Faculty of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran.

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.
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http://dx.doi.org/10.1177/1753425917709508DOI Listing
July 2017

Preparation and characterization of a novel nanobody against T-cell immunoglobulin and mucin-3 (TIM-3).

Iran J Basic Med Sci 2016 Nov;19(11):1201-1208

Biotechnology Research Center, Biotechnology Department, Venom & Bio-therapeutics Molecules Lab, Pasteur Institute of Iran, Tehran, Iran.

Objectives: As T-cell immunoglobulin and mucin domain 3 (TIM-3) is an immune regulatory molecule; its blocking or stimulating could alter the pattern of immune response towards a desired condition. Based on the unique features of nanobodies, we aimed to construct an anti-TIM-3 nanobody as an appropriate tool for manipulating immune responses for future therapeutic purposes.

Materials And Methods: We immunized a camel with TIM-3 antigen and then, synthesized a VHH phage: mid library from its B cell's transcriptome using nested PCR. Library selection against TIM-3antigen was performed in three rounds of panning. Using phage-ELISA, the most reactive colonies were selected for sub-cloning in soluble protein expression vectors. The Nanobody was purified and confirmed with a nickel-nitrilotriacetic acid (Ni-NTA) column, SDS-PAGE and Western blotting. A flowcytometric analysis was performed to analyze the binding and biologic activities of theTIM-3 specific nanobody with TIM-3 expressing HL-60 and HEK cell lines.

Results: Specific 15kD band representing for nanobody was observed on the gel and confirmed with Western blotting. The nanobody showed significant specific immune-reactivity against TIM-3 with a relatively high binding affinity. The nanobody significantly suppressed the proliferation of TIM-3 expressing HL-60 cell line.

Conclusion: Finally, we successfully prepared a functional anti-humanTIM-3 specific nanobody with a high affinity and an anti-proliferative activity on an AML cell line in vitro.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5126221PMC
November 2016

Deciphering biological characteristics of tumorigenic subpopulations in human colorectal cancer reveals cellular plasticity.

J Res Med Sci 2016 1;21:64. Epub 2016 Aug 1.

Department of Immunology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran.

Background: It is supposed that human colorectal cancer consists of a phenotypically distinct population of tumorigenic cancer cells known as cancer stem cells (CSCs) which play a pivotal role in cancer progression, maintenance, metastasis, and the relapse. The aim of this effort was to investigate and compare biological characterizations of CD133 with CD133 cell subsets isolated from both primary and metastatic human colorectal tumors.

Materials And Methods: Using our optimized protocols, unfixed colorectal tumors were enzymatically and mechanically dissociated into single cells followed by evaluation of postdigestion viability. The obtained single cell suspensions were then subjected to cell sorting using magnetic beads according to CD133 marker. The resultant CD133 and CD133 cell subsets were cultured in specific cell culture medium followed by aldehyde dehydrogenases (ALDH) activity assessment and flow cytometric analyses.

Results: The results demonstrate that CD133 cells have smaller size and lower complexity of intracellular structure, sphere formation ability, and ALDH enzyme activity while CD133 cells isolated from primary colon cancer samples were not able to form a sphere and did not show ALDH enzyme activity. Intriguingly, CD133 cells isolated from metastatic colorectal cancer specimen were able to form a sphere and shown ALDH enzyme activity. The present study indicates that our results are in agreement with SC theory and possibility of the existence of cellular plasticity among cancer subpopulations should be portrayed.

Conclusion: We also conclude that this cellular plasticity is greatly affected by tumor microenvironment cues and the role of CSCs niche in cancer therapeutic strategies should be precisely considered.
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http://dx.doi.org/10.4103/1735-1995.187355DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5122187PMC
August 2016