Iranian Journal of Allergy, Asthma and Immunology Tehran University of Medical Sciences en-US Iranian Journal of Allergy, Asthma and Immunology 1735-1502 Evaluation of Interleukin-21, 23 and 27 mRNA Expression and Protein Level in Liver Transplant Patients <p>Cytokines have prominent roles in activating of different T cells and shifting the immune response, in this study the role of three cytokines (<em>IL-21</em>, IL-23 and <em>IL-27</em>) is investigated in the liver transplant rejection. Three EDTA-treated blood samples were collected from each liver transplanted patient in 1<sup>st</sup>, 4<sup>th</sup> and 7<sup>th</sup> day of post-transplantation. The expression level of the mentioned cytokines was determined using real-time PCR for all samples. Also, the serum levels of cytokines were determined using ELISA tests. In acute rejection (AR) group (51 patients), mRNA expression pattern of <em>IL-21</em>and <em>IL-23</em> showed a steady increase, but this pattern was converse for <em>IL-27</em>. Our results in non-acute rejection (non-AR) group (54 patients) showed an elevation in day 4 and then a decrease in day 7 for <em>IL-21</em> and <em>IL-23</em> genes. This pattern was converse again for <em>IL-27</em> gene. In comparison between the two groups, in all 3 sampling times the mean of mRNA expression level of <em>IL-21</em> and <em>IL-23</em>, showed an increase in AR group which this increase was significant for <em>IL-21</em> in the 3<sup>rd</sup> (<em>p</em>=0.007) and for <em>IL-23</em> in 2<sup>nd</sup> (<em>p</em>=0.048) and 3<sup>rd</sup> (<em>p</em>=0.049) sampling time, but the pattern of mRNA expression for <em>IL-27</em> was contrary to the results of <em>IL-21</em> and <em>IL-23</em>. Furthermore, ELISA technique also, showed the serum level changes the same as cytokines. In this study <em>IL-21</em> and <em>IL-23</em> showed pro-inflammatory properties in the liver transplant rejected patients. Also, <em>IL-27</em> having different expression pattern, showed anti-inflammatory behavior which needs more considerations in future</p> <p>&nbsp;</p> Afsoon Afshari Ramin Yaghobi Negar Azarpira Bita Gerami Zadeh Seyed Ali Malek Hosseini Mojtaba Darbouy Mohammad Hossein Karimi ##submission.copyrightStatement## 2018-08-12 2018-08-12 17 4 298 307 Immunotherapeutic Effects of β-D Mannuronic Acid on IL-4, GATA3, IL-17 and RORC Gene Expression in the PBMC of Patients with Inflammatory Bowel Diseases <p class="Default">Inflammatory bowel diseases (IBD) are chronic relapsing immune-mediated disorders that result from an aberrant immunological response. IBD comprises of Crohn's disease (CD) and ulcerative colitis (UC). The precise aetiology of IBD has not been fully understood, however, recent studies support the hypothesis that patients with IBD have a dysregulated immune response to endogenous bacteria in the gastrointestinal tract (GIT). The increasing number of hospitalisation coupled with the high economic burden faced by IBD patients, calls for more concerted research efforts, to design a potent and credible treatment option for these strata of patients. This research was designed to test the efficacy and potency of β-D Mannuronic acid (M2000) in the treatment of IBD. Ten ml of blood was aseptically collected from 24 IBD patients and 24 normal controls. PBMC was isolated and stimulated with 1 µg/mL of LPS and incubated for 4 hours. The cells were later treated with 10 µg/mL or 50 µg/mL of Mannuronic acid and incubated for 24 hours at 37<sup>0</sup>C under 5% CO2 and 100% humidity. After the incubation, RNA was extracted from the cells, cDNA was synthesised, and the expression of the gene was evaluated using quantitative real-time PCR. The result indicated a significant down-regulation of RORC and IL-17 genes expression, while the expression of IL-4 and GATA3 genes were significantly up-regulated. These research findings have shown that M2000 a biocompatible agent, that has an immunotherapeutic, immunomodulatory and immunosuppressive effects on the PBMC of IBD patients.&nbsp;</p> Hussaini Alhassan Mohammed Ali Akbar Saboor-Yaraghi Homayoun Vahedi Ghordratollah Panahi Gholamreza Hemmasi Mir Saeed Yekaninejad Abbas Mirshafiey ##submission.copyrightStatement## 2018-08-12 2018-08-12 17 4 308 317 Mir-22-3p Enhances the Chemosensitivity of Gastrointestinal Stromal Tumor Cell Lines to Cisplatin through PTEN/PI3K/Akt Pathway <p class="a">Mir-22-3p is associated with many important biological processes, including neuroprotection, tumorigenesis, and various other tumor progressions. Our study aimed to investigate the roles of Mir-22-3p in chemosensitivity of gastrointestinal stromal tumor (GIST-T1) cells to cisplatin and explore its underlying mechanisms. Mir-22-3p high-expressing cell line was established by transfecting GIST-T1 cell line cells with Mir-22-3p mimic. After treatment with cisplatin (10 μM), Cell counting kits-8 (CCK-8) method was used to detect the cell viability. Flow cytometry was applied to measure the degree of cell apoptosis. Scratch wound healing test was used to detect the migration ability of cells. The protein and mRNA levels of the phosphatase and tensin homolog deleted on chromosome ten (PTEN)/phosphatidylinositol 3 kinase (PI3K)/protein kinase B (Akt) pathway-related factors were analyzed by Western blot and qRT-PCR. The mRNA level of Mir-22-3p was increased in transfected GIST-T1 cells compared with that in control cells. The survival rate and Bcl-2/Bax ratio of GIST-T1 cells treated with both Mir-22-3p analogue and cisplatin were significantly decreased, while the apoptosis rate and protein level of caspase-3 were significantly increased (<em>p</em>&lt;0.05). In addition, the mRNA and protein levels of PTENwere significantly increased in cells treated with both Mir-22-3p analogue and cisplatin (<em>p</em>&lt;0.05), while the expression levels of PI3K and Akt were significantly decreased (<em>p</em>&lt;0.05). Mir-22-3p overexpression can increase the chemosensitivity of cisplatin in human gastrointestinal stromal tumor cells by PTEN/PI3K/Akt pathway.  </p> Yugang Xu Ming Cheng Lei Mi Yunping Qiu Wenli Hao Leping Li ##submission.copyrightStatement## 2018-08-12 2018-08-12 17 4 318 325 Quantum Dot-labeled Tags Improve Minimal Detection Limit of CA125 in Ovarian Cancer Cells and Tissues <p>In recent years, a lot of attention has been paid to quantum dot (QD) nanoparticles as fluorescent sensors for sensitive and accurate detection of cancer biomarkers. Here, using a homemade specific monoclonal antibody against CA125 and QD525- or FITC-labeled probes, expression of this marker in an ovarian cancer cell line and cancer tissues were traced and optical properties of fluorophores were compared qualitatively and quantitatively. Our results clearly showed that besides lower background and exceptionally higher photobleaching resistance, QD525 exhibited higher fluorescent intensity for both ovarian cancer cell and tissues at different exposure times (<em>p</em>&lt;0.0001) and excitation filter sets (<em>p</em>&lt;0.0001) exemplified by significantly higher staining index (<em>p</em>&lt;0.016). More importantly, the FITC-labeled probe detected antigen-antibody complex at minimum concentration of 0.3 mg/mL of anti-CA125, while reactivity limit decreased to 0.078 mg/mL of anti-CA125 when QD525-labeled probe was applied showing four times higher reactivity level of QD525 probe compared to the same probe labeled with FITC. Based on our results, it seems that QDs are inimitable tags for sensitive detection and localization of ovarian cancer micrometastasis and molecular demarcation of cancer tissues in surgical practice, which subsequently figure out accurate therapeutic approaches. </p> Sorour Shojaeian Abdolamir Allameh Mahmood Jeddi-Tehrani Roya Ghods Jaleh Shojaeian Akram-Sadat Tabatabaei-Panah Amir-Hassan Zarnani ##submission.copyrightStatement## 2018-08-12 2018-08-12 17 4 326 335 Sex Hormones and Prolactin Levels and Their Association with Anti Cardiolipin Antibody in Patients with Systemic Lupus Erythematosus <p>Pathogenesis of systemic lupus erythematosus (SLE) is complex and multi-factorial. Among various suggested mechanisms for the disease, the hormonal theory has been considered as one of the most important mechanisms. Recently, the association of sex hormones with manifestations of antiphospholipid antibody syndrome (APLS) has been hypothesized. The aim of present study was to assess the serum levels of anticardiolipin antibody (ACA), sex hormones and prolactin in SLE female patients and their association with the disease. This study comprised 40 SLE female patients and 41 healthy age-matched female subjects. For all patients and controls, the serum levels of ACA (IgG and IgM), estradiol, testosterone, progesterone, dehydroepiandrosterone sulfate (DHEA-S) and prolactin were measured by ELISA method. Our study revealed that serum levels of testosterone, DHEA-S and progesterone were significantly lower in SLE patients than control (<em>p</em>&lt;0.001). However, serum levels of estradiol and prolactin were significantly higher in SLE patients compared to controls (<em>p</em>&lt;0.001). There was a significant difference between mild and moderate severity patients group for ACA positivity (95% CI 13.67-41.3; <em>p</em>=0.03). Also, SLE patients with positive ACA showed significantly lower (<em>p</em>&lt;0.001) serum levels of testosterone, DHEA-S and progesterone and significantly higher (<em>p</em>&lt;0.001) estradiol and prolactin serum levels compared to negative ACA patients. The results of our study indicated that expression and metabolism of sex hormones and prolactin are different in female SLE patients compared to healthy subjects. It seems, change in serum levels of these hormones is related to higher SLE disease activity, increased thrombotic risks and increased renal involvement. </p> Dariyush Raeisi Mohammad Erfan zare Atefeh Nasir Hamidraza Sherkatolabbasieh Shiva Shafeizadeh ##submission.copyrightStatement## 2018-08-12 2018-08-12 17 4 336 345 Characterization of CD4+ and CD8+ T Cell Subsets and Interferon Regulatory Factor 4 (IRF4) in MS Patients Treated with Fingolimod (FTY-720): A Follow-up Study <p>Fingolimod is a novel immunomodulatory drug used in patients with relapsing multiple sclerosis (MS) which reversibly inhibits egress of lymphocytes from lymph nodes. In this longitudinal study, the frequency of Interferon- gamma (IFN-γ)+, IL4+, IL17+ and IL10+ CD4+ and CD8+ T cell subsets were measured in Fingolimod treated patients before and after 12 months’(12M) therapy using flow cytometry and compared to those of naive, Betaferon treated MS patients and healthy individuals. Additionally, the level of transcription factor IRF4 and IL-6, IL-23, TGF-β1 cytokines, required for differentiation of IL-17+ T cells, were assessed by RT-PCR and ELISA, respectively. In Fingolimod treated MS patients, we observed a significant decrease in the percentage of IFN-γ+/IL17+ CD4+ and CD8+ T cell subsets. In contrast, Fingolimod increased IL10+ CD4+ T cells. We also showed that IFN-γ+IL17+ co-producing CD8+ T cells were reduced in patients under fingolimod therapy. furthermore, Fingolimod could reduce the expression level of IRF4 in patients while IL6 was increased in the supernatant of cultured peripheral blood mononuclear cells. Our data showed that Fingolimod treatment alters CD4+ and CD8+ T cell subsets and reduces expression of IRF-4, which affects the proportion of pathogenic memory T cells in peripheral blood.</p> Bahare Laribi Mohammad Ali Sahraian Mehdi Shekarabi Rahimeh Emamnejad Mohsen Marzban Shokufeh Sadaghiani Maryam Izad ##submission.copyrightStatement## 2018-08-12 2018-08-12 17 4 346 360 Collagen II-primed Foxp3 Transduced T Cells Ameliorate Collagen-induced Arthritis in Rats: The Effect of Antigenic Priming on T Regulatory Cell Function <p>Regulatory T cells (Tregs) play a major role in the prevention of autoimmune diseases. Transfer of Foxp3 gene into conventional T cells converts their phenotype to regulatory T cells. Therefore, the question arises as to whether adoptively transferred in vitro differentiated Treg cells specific for a locally expressed antigen might have better inhibitory effects on the progression of the disease as compared with antigen-nonspecific T reg cells. Herein, we investigated the therapeutic potential of primed and unprimed retrovirus mediated Foxp3-overexpression T cells following intravenously injected of these cells into affected rats with collagen-induced arthritis (CIA), an animal model of rheumatoid arthritis. Our analyses demonstrate that systemic administration of collagen II primed Foxp3-transduced T cells could markedly ameliorate CIA inflammatory responses at clinical (<em>p</em>&lt;0.0014) and pathological exchanges including cellular infiltration (<em>p</em>=0.002), bone erosion (<em>p</em>=0.0013) and synovial hyperplasia (<em>p</em>=0.002). In contrast, collagen II unprimed Foxp3-transduced T cells like as collagen II primed or unprimed GFP-transduced T cells did not reveal any beneficial effects on arthritis features as compared with untreated group (<em>p</em>&gt;0.05). Therefore, we believe that collagen II primed Foxp3-transduced T cells are interacting locally and systemically with immune cells which reveled with decreasing of T cells infiltration into joints along with specific CII IgG production. Considering the results described here, it appears that the using patients' T cells which previously exposed to specific antigens may have more effective therapeutic advantage in the production of induced regulatory T cells in the treatment of arthritis.</p> Mahdi Zavvar Mohsen Abdolmaleki Hamid Farajifard Farshid Noorbakhsh kayhan Azadmanesh Mohammad Vojgani Mohammad Hossein Nikcnam ##submission.copyrightStatement## 2018-08-12 2018-08-12 17 4 361 371 Nicotine Modulates the Release of Inflammatory Cytokines and Expression of TLR2, TLR4 of Cord Blood Mononuclear Cells <p>The underlying mechanisms of how nicotine affects cord umbilical cells remain largely elusive. Nicotine rapidly crosses the blood-brain barrier (10 to 20 s) and binds to nicotinic acetylcholine receptors (nAChRs). Nicotine considered as a major compound found in cigarette smoke and the mechanism of nicotine action in immune response is not well understood. Cigarette smoke well known by activation of toll like receptors (TLRs) especially TLR4 and 9 which stimulates the immune response by induction of releases of cytokines mainly CXCL-8 which in turn triggers lungs reactions specially induction of neutrophils recruitments. In this study we isolated human umbilical mononuclear cells (UCBMC) from umbilical cord blood and exposed to the nicotine for detection any cytokines and TLRs modulation. We have found that nicotine (at concentration 0.01µM) induced release of TNF-a and IL-6 but not CXCL-8 production. Besides we have shown that nicotine did not effect on TLR4 surface expression however up-regulated the TLR2 surface expression. Moreover expression of CD11a and CXCR4 after nicotine incubation was upregulated as demonstrated by flow cytometry analysis, These data indicated that nicotine by stimulation of inflammatory cytokines induces immune response. The present study provides evidence that nicotine selectively regulates the release of cytokines and expression of TLRs. Further studies are needed to exploring details of its effects and signaling.</p> Laya Takbiri Osgoei Kazem Parivar Marzieh Ebrahimi Esmaeil Mortaz ##submission.copyrightStatement## 2018-08-12 2018-08-12 17 4 372 378 Evaluation of mRNA Expression Levels of TNFα, TNFR1 and IL1β in Lung Tissue 20 Years after Sulfur-mustard Exposure <p>Despite many years having passed since exposure to sulfur mustard (SM) gas, there are many exposed subjects who are still suffering from delayed pulmonary complications. The levels of pro-inflammatory cytokines in the lung of these subjects have not been investigated in delay phase. In this study, we evaluated mRNA expression of pro-inflammatory  cytokines (tumor necrosis factor alpha (TNFα), tumor necrosis factor receptor type 1 (TNFR1), and interleukin 1 beta (IL-1β)  ) in lung biopsy of SM-exposed subjects and compared them with control (non-exposed) subjects. We used formalin-fixed, paraffin-embedded (FFPE) tissue for this purpose. Lung FFPE blocks of SM-exposed subjects (30 samples) and a control group (30 samples) were collected from archival pathology department. The total mRNA of FFPE tissues were extracted and the mRNA expression of pro-inflammatory  cytokines were determined by quantitative Real Time PCR (RT-qPCR). The obtained results from two groups were compared to each other and non-parametric statistical analyses were carried out on them. Our studies showed that the mRNA expression of TNFα, TNFR1 and IL-1β  in lung tissue of SM injured and control people have no significant difference (p-value= 0.159, 0.832 and 0.314 respectivly). TNFR1 showed direct correlation with TNFα (r=0.867, <em>p</em>=0.002) and IL-1β (r= 0.65, <em>p</em>=0.006). The evaluation of mRNA expression in pro-inflammatory cytokines in lung of SM-exposed subjects after 20 years showed that these mediators are similar to those of non-exposed group and there was no acute inflammation in lung of these patients.</p> Marzieh Eghtedardoost Zuhair Mohammad Hassan Tooba Ghazanfari Alireza Sadeghipour Mostafa Ghanei ##submission.copyrightStatement## 2018-08-12 2018-08-12 17 4 379 387 Frequency and Pattern of IgE-mediated Sensitization to Aero and Food Allergens in Ahvaz, Province of Khuzestan in Southwestern Iran <p>Efficient diagnosis of allergy and proper treatment need identification of the causative allergens eliciting clinical symptoms. The present study was performed to identify the most common aero- and food allergens and determine the pattern of sensitization among people of Ahvaz (southwestern Iran), one of the most polluted cities worldwide. Based on the physical examination and medical records, patients were referred to the Allergy laboratory for “in vitro” IgE determination. Specific and total IgE was determined by the ImmunoCAP system (Thermo Fisher-Phadia, Uppsala, Sweden). A total of 666 consecutive patients (51.1% female) were tested for 202 different allergens. The majority of requests (57%) belonged to food allergens. Sensitization to at least one allergen was found in 47.6% of patients. In a selected group of allergens for which specific IgE had been tested in at least 100 patients, the most common sensitizing aeroallergens were Russian thistle, grass pollen, and willow; while wheat, honey, and shrimp were the most frequent food allergens, respectively. Sensitization profiles based on measurement of specific IgE indicated that Russian thistle, grasses, and wheat were the most prevalent allergens in people with allergic symptoms living in Ahvaz.</p> Mohammad Shahrooei Abbas Fayezi Raheleh Shokouhi Shoormasti Faramarz Zakavi Leila Golpasand Hagh Johan Van Eldere Xavier Bossuyt Erna Van Hoeyveld ##submission.copyrightStatement## 2018-08-12 2018-08-12 17 4 393 397 Pro and Anti-inflammatory Cytokine Production in CD4+ T Lymphocytes in Children with Asthma and Allergic Rhinitis Exposed to the Monocyte Locomotion Inhibitory Factor (MLIF) <p><em>Entamoeba histolytica</em> produces, in axenic culture, the monocytes locomotion inhibitory factor (MLIF), a oligopeptide with selective anti-inflammatory properties. We evaluated the effect of MLIF on the expression of pro- and anti-inflammatory cytokines in CD4+ T lymphocytes from children with asthma and allergic rhinitis. Twelve children with severe asthma, 12 children with allergic rhinitis and 6 healthy controls were recruited for this study between May and December 2016. CD4+ T cells were cultured for 24 h at 37°C, 5% CO<sub>2</sub> in the presence of MLIF, 1-phorbol 12-myristate 13-acetate (PMA), MLIF+PMA or RPMI. Interleukin-10 (IL-10), IL-4, interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α) expression levels were measured in the supernatants of T-cell cultures using the enzyme-linked immunosorbent assay (ELISA). Pro- and anti-inflammatory cytokines were inhibited by MLIF (IFN-γ <em>p</em>=0.0036, TNF-α <em>p</em>&lt;0.001, IL-4 <em>p</em>=0.0082) in asthmatic patients, however IFN-γ was not significantly inhibited (NS) in patients with allergic rhinitis when compared to the RPMI group. In CD4+ T cells treated with PMA+MLIF, the expression levels of IFN-γ, TNF-α and IL-4 were strongly inhibited (<em>p</em>&lt;0.001, <em>p</em>&lt;0.001 and <em>p</em>&lt;0.0094), compared to PMA treatment alone, for both, rhinitis and asthma. IL-10 expression was not affected by MLIF in neither of the two diseases. We conclude that MLIF alters the pro/anti-inflammatory balance and induces inhibition of IL-4, IFN-γ and TNF-α, but does not affect IL-10.</p> Sara Rojas-Dotor Adriana González-Hernández Francisco Humberto León-Aguilar Víctor Juárez-Téllez Patricia Gómez de León Cruces ##submission.copyrightStatement## 2018-08-12 2018-08-12 17 4 388 392