Monoclonal Antibody for Reducing Memory and Learning Problems in Schizophrenia 2 2 Schizophrenia is a chronic debilitating psychiatric illness that accounts for a significant portion of the burden caused by mental illnesses worldwide. Primary negative symptoms of schizophrenia are not secondary to extrapyramidal, depressive or positive symptoms 1,2. Negative symptoms are the core features of the illness which are associated with long-term functional disability and poor outcome 1,3. These symptoms include deficits in social and emotional functioning, blunted affect and lack of spontaneity. There is a growing body of evidence for the role of inflammation and immune system dysregulation in psychiatric disorders 4. Although the precise pathophysiology of schizophrenia is not completely known, a number of recent studies support the probable pathologic role of immunologic dysfunction in this disorder. Assessing serum cytokine levels such as interleukin 1 (IL-1), IL-2, IL-6, and chemokine CCL11 in schizophrenic patients demonstrates profound alterations compared to healthy matched controls 4. Furthermore, increased cyclooxygenase-2 (COX-2) expression as well as prostaglandin E2 production in schizophrenia, are among other postulated etiologies supported by recent studies 4. On the other hand, it has been shown that immune response imbalance is associated with decreased activity of indoleamine 2, 3-dioxygenase enzyme which subsequently leads to accumulation of kynurenic acid, an endogenous antagonist of glutamate N-methyl-D-aspartate (NMDA) receptor. Compared with anit-inflammatory agents like celecoxib and NAC, monoclonal antibodies also have more potent anti-inflammatory properties. Indeed, COX-2 inhibitors and N-acetylcysteine have moderate efficacy in treatment of schizophrenia and autism 1,2,5. British scientists have begun testing a radically new approach to treating schizophrenia based on emerging evidence that it could be a disease of the immune system. Evidence for prenatal and premorbid immune risk factors for the development of schizophrenia in the offspring is highlighted 6,7. Then key evidence for immune dysfunction in patients with schizophrenia is considered. A collaboration between the Medical Research Council (MRC) and King’s College London, is based on emerging evidence that schizophrenia may be an immune disease. The drug, natalizumab, works by targeting microglia, a type of immune cell residing in the brain which are thought to be overactive in people at risk of developing schizophrenia 6,7. 125 125 Shahin Akhondzadeh Psychiatric Research Center, Roozbeh Hospital, Tehran University of Medical Sciences Psychiatric Research Center, Roozbeh Hospital, Tehran University of Medical Sciences Iran Editorial 315.pdf Akhondzadeh S, Tabatabaee M, Amini H, Ahmadi Abhari SA, Abbasi SH, Behnam B. Celecoxib as adjunctive therapy in schizophrenia: a double-blind, randomized and placebo-controlled trial. Schizophr Res 2007;90(1-3):179-185.##Farokhnia M, Azarkolah A, Adinehfar F, Khodaie-Ardakani MR, Hosseini SM, Yekehtaz H, et al. N-acetylcysteine as an adjunct to risperidone for treatment of negative symptoms in patients with chronic schizophrenia: a randomized, double-blind, placebo-controlled study. Clin Neuropharmacol 2013;36(6):185-192.##Akhondzadeh S. Hippocampal synaptic plasticity and cognition. J Clin Pharm Ther 1999;24(4):241-248.##Müller N. Inflammation in schizophrenia: Pathogenetic aspects and therapeutic considerations. Schizophr Bull 2018 Apr 10. [Epub ahead of print].##Asadabadi M, Mohammadi MR, Ghanizadeh A, Modabbernia A, Ashrafi M, Hassanzadeh E, et al. Celecoxib as adjunctive treatment to risperidone in children with autistic disorder: a randomized, double-blind, placebo-controlled trial. Psychopharmacology (Berl) 2013;225(1):51-59.##Miller BJ, Buckley PF. The case for adjunctive monoclonal antibody immunotherapy in schizophrenia. Psychiatr Clin North Am 2016;39(2):187-198.##Miller BJ, Dias JK, Lemos HP, Buckley PF. An open-label, pilot trial of adjunctive tocilizumab in schizophrenia. J Clin Psychiatry 2016;77(2):275-276.##

Apoptosis of Adipose-Derived Stem Cells Induced by Liposomal Soybean Phosphatidylcholine Extract 2 2 Background: Recently, Phosphatidylcholine (PC) has been used as an off-label treatment for lipolysis injection, which is associated with inflammatory reaction due to sodium deoxycholate, an emulsifier, so that inflammation as side effect occurs in those patients. Liposome formulation from soybean lipid was thought to be a better and safer alternative. This study aimed to analyze the mechanism of Liposomal Soybean Phosphatidylcholine (LSPC) extract from Indonesian soybeans (containing 26% PC) to induce Adipose-derived Stem Cells (ASCs) death in vitro.  Methods: Liposomes were prepared using thin film hydration method followed by a stepwise extrusion process to produce a small amount of 41.0-71.3 nm. Liposomal soybean phosphatidylcholine extract (LSPCE), liposomal purified PC (LPCC), and solution of PC+SD were used for comparison. Annexin V fluorescein Isothiocyanate/ Propidium Iodide (FITC/PI) double staining by flow cytometry and also measurement of caspase-3 activity using ELISA were used to quantify the rate of apoptosis. ASCs viability was measured using MTT assay after induction with liposomes. Morphological changes were shown using a phase-contrast, inverted microscope and Transmission-Electron Microscope (TEM).  Results: The flow cytometry results showed that cells treated with both LSPCE and LPCC showed increase in early apoptosis beginning at 6 hr after incubation, which was confirmed by caspase 3 measurement. MTT assay showed that both LSPCE and LPCC could decrease viability of cells. Cells treated with LSPCE and LPCC showed some rounded cells, which was an early sign of cell death. Cells treated with SD showed extensive membrane damage with necrosis features using TEM.  Conclusion: The results above demonstrated that LSPCE induced apoptosis of ASCs. 126 133 Reza Purwoko érpour Medical-Spa Skin and Mesotherapy Centre érpour Medical-Spa Skin and Mesotherapy Centre Indonesia Iis Rosliana érpour Medical-Spa Skin and Mesotherapy Centre érpour Medical-Spa Skin and Mesotherapy Centre Indonesia Siti Sobariah érpour Medical-Spa Skin and Mesotherapy Centre érpour Medical-Spa Skin and Mesotherapy Centre Indonesia Nabila Hermana érpour Medical-Spa Skin and Mesotherapy Centre érpour Medical-Spa Skin and Mesotherapy Centre Indonesia Silvani Permatasari érpour Medical-Spa Skin and Mesotherapy Centre érpour Medical-Spa Skin and Mesotherapy Centre Indonesia Dewi Wulandari Department of Pharmacy, Faculty of Medicine, Universitas Indonesia Department of Pharmacy, Faculty of Medicine, Universitas Indonesia Indonesia Puji Sari Department of Biology, Faculty of Medicine, Universitas Indonesia Department of Biology, Faculty of Medicine, Universitas Indonesia Indonesia Ernie Purwaningsih Department of Pharmacy, Faculty of Medicine, Universitas Indonesia Department of Pharmacy, Faculty of Medicine, Universitas Indonesia Indonesia L Chaidir Agro Industrial Technology Development Biomedical Laboratory, Serpong Agro Industrial Technology Development Biomedical Laboratory, Serpong South Tangerang Hans-Joachim Freisleben German Indonesian Medical Association German Indonesian Medical Association Indonesia Jeanne Pawitan Department of Histology, Faculty of Medicine, Universitas Indonesia Department of Histology, Faculty of Medicine, Universitas Indonesia Indonesia Kusmarinah Bramono Department of Dermatovenerology, Faculty of Medicine, University of Indonesia Department of Dermatovenerology, Faculty of Medicine, University of Indonesia Indonesia Adipose-derived stem cellsApoptosisLiposomesPhosphatidylcholines 316.pdf Karl GH. Efficacy of injections of phosphatidylcholine into fat deposits a non-surgical alternative to liposuction in body-contouring. Indian J Plast Surg 2005;38(2):119-122.##Noh Y, Heo CY. The effect of phosphatidylcholine and deoxycholate compound injections to the localized adipose tissue: an experimental study with a murine model. Arch Plast Surg 2012;39(5):452-456.##Thomas M, D'Silva JA, Borole AJ. Injection lipolysis with a cocktail of phosphatidylcholine and deoxycholate: an Indian experience. Plast Reconstr Surg Glob Open 2016;4(9):e861.##Hexsel DM, Serra M, de Oliveira Dal’Forno T, Zechmeister do Prado D. Cosmetic uses of injectable phosphatidylcholine on the face. Otolaryngol Clin North Am 2005;38(5):1119-1129.##Palmer M, Curran J, Bowler P. Clinical experience and safety using phosphatodylcholine injections for the localized reduction of subcutaneous fat: a multicentre, retrospective UK study. J Cosmet Dermatol 2006;5(3):218-226.##Rotunda AM, Suzuki H, Moy RL, Kolodney MS. Detergent effects of sodium deoxycholate are a major feature of an injectable phosphatidylcholine formulation used for localized fat dissolution. Dermatol Surg 2004;30(7):1001-1008.##Rzany B, Griffiths T, Walker P, Lippert S, McDiarmid J, Havlickova B. Reduction of unwanted submental fat with ATX-101 (deoxycholic acid), an adipocytolytic injectable treatment: results from a phase III, randomized, placebo-controlled study. Br J Dermatol 2014;170(2):445-453.##Yagima Odo ME, Cucé LC, Odo LM, Natrielli A. Action of sodium deoxycholate on subcutaneous human tissue: local and systemic effects. Dermatol Surg 2007;33(2): 178-188; discussion 188-9.##Bechara FG, Mannherz HG, Jacob M, Mazur AJ, Sand M, Altmeyer P, et al. Induction of fat cell necrosis in human fat tissue after treatment with phosphatidylcholine and deoxycholate. J Eur Acad Dermatol Venereol 2012;26(2):180-185.##Klein SM, Schreml S, Nerlich M, Prantl L. In vitro studies investigating the effect of subcutaneous phosphatidylcholine injections in the 3t3-l1 adipocyte model: lipolysis or lipid dissolution? Plast Reconstr Surg 2009;124(2):419-427.##Li H, Lee JH, Kim SY, Yun HY, Baek KJ, Kwon NS, et al. Phosphatidylcholine induces apoptosis of 3T3-L1 adipocytes. J Biomed Sci 2011;18:91.##Purwoko RY, Rosliana I, Purwaningsih EH, Chaidir, Freisleben HJ, Pawitan A. Liposome formulation of purified soybean phosphatidylcholine extract from argomulyo variety soy to replace the toxicity of injectable phosphatidylcholine solution containing sodium deoxycholate. Int J Pharm Tech Res 2016;9(2):166-175.##Pawitan JA, Liem IK, Suryani D, Busami A, Purwoko RY. Simple lipoaspirate washing using a coffee filter. Asian Biomed 2013;7(3):333-338.##Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini F, Krause D, et al. Minimal criteria for defining multipotent mesenchymal stromal cells: The international society for cellular therapy position statement. Cytotherapy 2006;8(4):315-317.##Kepp O, Galluzzi L, Lipinski M, Yuan J, Kroemer G. Cell death assays for drug discovery. Nat Rev Drug Discov 2011;10(3):221-237.##Galluzzi L, Joza N, Tasdemir E, Maiuri MC, Hengartner M, Abrams JM, et al. No death without life: vital functions of apoptotic effectors. Cell Death Differ 2008;15(7):1113-1123.##Galluzzi L, Aaronson SA, Abrams J, Alnemri ES, Andrews DW, Baehrecke EH, et al. Guidelines for the use and interpretation of assays for monitoring cell death in higher eukaryotes. Cell Death Differ 2009;16(8):1093-1107.##Sundquist T, Moravec R, Niles A, O’Brien M, Riss T. Timing your apoptosis assays. Cell Notes 2016;16:18-21.##El Kamshouhsy A, Abel Maksoud R, El Mahdy N. Evaluation of the efficacy of injection lipolysis using phosphatidylcholine/deoxycholate versus deoxycholate alone in treatment of localized fat deposits. J Clin Exp Dermatol 2012;3(2):1-9.##Zhang W, He H, Feng Y, Da S. Separation and puriﬁcation of phosphatidylcholine and phosphatidylethanolamine from soybean degummed oil residues by using solvent extraction and column chromatography. J Chromatogr B Analyt Technol Biomed Life Sci 2003;798(2):323-331.##Slee EA, Adrain C, Martin SJ. Executioner caspase-3, -6, and -7 perform distinct, non-redundant roles during the demolition phase of apoptosis. J Biol Chem 2001;276(10):7320-7326.##Yang JY, Della-Fera MA, Rayalam S, Ambati S, Baile CA. Enhanced pro-apoptotic and anti-adipogenic effects of genistein plus guggulsterone in 3T3-L1 adipocytes. Biofactors 2007;30(3):159-169.##Zhang Y, Yu W, Jiang X, Lv K, Sun S, Zhang F. Analysis of the cytotoxicity of differentially sized titanium dioxide nanoparticles in murine MC3T3-E1 preosteoblasts. J Mater Sci Mater Med 2011;22(8):1933-1945.##

LACK Gene’s Immune Response Induced by Cocktail DNA Vaccine with IL-12 Gene Against Cutaneous Leishmaniasis in BALB/c Mice 2 2 Background: Leishmaniasis is caused by parasitic protozoa of the genus Leishmania which is an obligate intracellular parasite in the infected host. Individuals who have been recovered from clinical leishmaniasis develop strong immunity against reinfection. DNA vaccines are the new type of vaccines that induce expression of protein eukaryotic cells. DNA vaccines can be stimulated by the cellular and humoral immune responses using one or several genes.  Methods: A DNA vaccine containing plasmids encoding the pcLACK+pcTSA genes of Leishmania major (L. major) (MHRO/IR/75/ER) in the vicinity of IL-12 gene expression was made and then its protective efficacy in comparison with single-gene of LACK was evaluated. Also, BALB/c mice were immunized intramuscularly three times. The humoral and cellular immune responses were evaluated after immunization with pcLACK, pcLACK+pcTSA+pCAGGS-IL12, and then challenged with L. major.   Results: Humoral response and IFN-γ values were significantly higher than control groups after immunization with pcLACK, pcLACK+pcTSA+pCAGGS-IL12 and challenge with L. major (p≤0.05). IL-4 values were increased in the control groups in such a way that they were remarkably higher than the pcLACK, pcLACK+pcTSA+ pCAGGS-IL12 groups (p≤0.05) after immunization and challenge with L. major. Conclusion: The survival time of the immunized mice with pcLACK, pcLACK+pcTSA+ pCAGGS-IL12 groups was higher than the control groups. Then, DNA vaccine of pcLACK appeared to be likely able to induce more protection against infection with L. major in mice. Therefore, cocktail DNA is effective to enhance specific immunity. 134 140 Oghlniaz Jorjani Laboratory Science Research Center, Golestan University of Medical Sciences Laboratory Science Research Center, Golestan University of Medical Sciences Iran Fatemeh Ghaffarifar Department of Parasitology, Faculty of Medical Sciences, Tarbiat Modares University Department of Parasitology, Faculty of Medical Sciences, Tarbiat Modares University Iran Zohreh Sharifi Research Center of Iranian Blood Transfusion Organizations Research Center of Iranian Blood Transfusion Organizations Iran Abdolhossein Dalimi Department of Parasitology, Faculty of Medical Sciences, Tarbiat Modares University Department of Parasitology, Faculty of Medical Sciences, Tarbiat Modares University Iran Hajar Ziaei-Hezarjaribi Department of Parasitology, Faculty of Medicine, Mazandaran University of Medical Sciences Department of Parasitology, Faculty of Medicine, Mazandaran University of Medical Sciences Iran Benyamin Talebi Private Veterinary Physician Private Veterinary Physician Iran Cutaneous leishmaniasisDNA vaccineImmune response 317.pdf Webb JR, Campos-Neto A, Ovendale PJ, Martin TI, Stromberg EJ, Badaro R, et al. Human and murine immune responses to a novel Leishmania major recombinant protein encoded by members of a multicopy gene family. Infect Immun 1998;66(7):3279-3289.##World Health Organization-TDR. The TDR fifteenth program report. Research Progress 1999-2000. New and Improved Tools; 2003. www.who.int/tdr/research/progress 9900/tools/vdr.htm.##Alvar J, Cañavate C, Gutiérrez-Solar B, Jiménez M, Laguna F, López-Vélez R, et al. Leishmania and human immunodeficiency virus coinfection: the first 10 years. Clin Microbiol Rev 197;10(2):298-319.##Desjeux P, Alvar J. Leishmania/HIV co-infections: epidemiology in Europe. Ann Trop Med Parasitol 2003;97 Suppl 1:3-15.##Dumonteil E, McMahon-Pratt D, Price V. Report on the fourth TDR/IDRI meeting on second-generation vaccines against leishmaniasis, Universidad Autonoma de Yucatan, Yucatan, Mexico. Document TDR/PDR/LEISH/VAC/01.1.##Sacks D, Noben-Trauth N. The immunology of susceptibility and resistance to Leishmania major in mice. Nat Rev Immunol 2002;2(11):845-858.##Gurunathan S, Wu CY, Freidag BL, Seder RA. DNA vaccines: a key for inducing long-term cellular immunity. Curr Opin Immunol 2000;12(4):442-447.##Tang DC, DeVit M, Johnston SA. Genetic immunization is a simple method for eliciting an immune response. Nature 1992;356(6365):152-154.##Méndez S, Gurunathan S, Kamhawi S, Belkaid Y, Moga MA, Skeiky YA, et al. The potency and durability of DNA-and protein-based vaccines against Leishmania major evaluated using low-dose, interadermal challenge. J Immunol 2001;166(8):5122-5128.##Gurunathan S, Saks DL, Brown DR, Reiner SL, Chavest H, Glaichenhaus N, et al. Vaccination with DNA encoding the immunodominant LACK parasite antigen confers protective immunity to mice infected with Leishmania major. J Exper Med 1997;186(7):1137-1147.##Gonzalez-Aseguinolaza G, Taladriz S, Marquet A, Larraga V. Molecular cloning, cell localization and binding affinity to DNA replication proteins of the p36/LACK protective antigen from Leishmania infantum. Eur J Biochem 1999;259(3):909-916.##Pérez-Jiménez E, Kochan G, Gherardi MM, Esteban M. MVA-LACK as a safe and efficient vector for vaccination against leishmaniasis. Microbes Infect 2006;8(3):810-822.##Monnerat S, Martinez-Calvillo S, Worthey E, Myler PJ, Stuart KD, Fasel N. Genomic organization and gene expression in a chromosomal region of Leishmania major. Mol Biochem Parasitol 2004;134(2):233-243.##Campos-Neto A, Porrozzi R, Greeson K, Coler RN, Webb JR, Seiky YA, et al. Protection against cutaneous Leishmaniasis induced by recombinant antigens in murine and nonhumans primate models of the human disease. Infect Immun 2001;69(6):4103-4108.##Campos-Neto A, Webb JR, Greeson K, Coler RN, Skeiky YA, Reed SG. Vaccination with plasmid DNA encoding TSA/LmSTI1 Leishmanial fusion proteins confers protection against Leishmania major Infection in Susceptible BALB/c mice. Infect Immun 2002;70(6):2828-2836.##Stäger S, Smith DF, Kaye PM. Immunization with a recombinant stage-regulated surface protein from Leishmania donovani induces protection against visceral leishmaniasis. J Immunol 2000;165(12):7064-7071.##Park AY, Hondowicz BD, Scott P. IL-12 is required to maintain a Th1 response during Leishmania major infection. J Immunol 2000;165(2):896-902.##Ghaffarifar F, Jorjani O, Sharifi Z, Dalimi A, Hassan ZM, Tabatabaie F, et al. Enhancement of immune response induced by DNA vaccine cocktail expressing complete LACK and TSA genes against Leishmania major. APMIS 2013;121(4):290-298.##Ahmed SB, Touihri L, Chtourou Y, Dellagi K, Bahloul C. DNA based vaccination with a cocktail of plasmids encoding immunodominant Leishmania (Leishmania) major antigens confers full protection in BALB/c mice. Vaccine 2009;27(1):99-106.##Méndez S, Belkaid Y, Seder RA, Sacks D. Optimization of DNA vaccination against cutaneous leishmaniasis. Vaccine 2002;20(31-32):3702-3708.##Jorjani O, Ghaffarifar F, Sharifi Z, Dalimi A. Cloning and expression of recombinant plasmid containing LACK gene of Leishmania major (MHRO/IR/75/ER) in CHO cells. ISESCO J Sci Technol 2012;8(13):37-43.##Crowther JR, editor. Methods in molecular biology. 1st ed. Vol. 42, ELISA theory and practice. New Jersey: Humana Press; 1995. 216 p.##Ashford RW, Bates PA. Leishmaniasis in the old world. In: Collier L, Balows A, Sussman M, editors. Topley & Wilson’s Microbiology and Microbial Infections. New York: Oxford University Press; 1998. p. 215-4.##Zadeh-Vakili A, Taheri T, Taslimi Y, Doustdari F, Salmanian AH, Rafati S. Immunization with the hybrid protein vaccine, consisting of leishmania major cysteine proteinases type I (CPB) and type II (CPA), partially protects against leishmaniasis. Vaccine 2004;22(15-16):1930-1940.##Handman E. Leishmaniasis: current status of vaccine development. Clin Microbiol Rev 2001;14(2):229-243.##Sakai T, Hisaeda H, Nakano Y, Ishikawa H, Maekawa Y, Ishii K, et al. Gene gun-mediated delivery of an interleukin-12 expression plasmid protects against infections with the intracellular protozoan parasites Leishmania major and Trypanosoma cruzi in mice. Immunology 2000;99(4):615-624.##Hezarjaribi HZ, Ghaffarifar F, Dalimi A, Sharifi Z. Evaluation of protective effect of IL-22 and IL-12 on cutaneous leishmaniasis in BALB/c mice. Asian Pac J Trop Med 2014;7:940-945.##Hezarjaribi HZ, Ghaffarifar F, Dalimi A, Sharifi Z, Jorjani O. Effect of IL-22 on DNA vaccine encoding LACK gene of Leishmania major in BALB/c mice. Exp Parasitol 2013;134(3):341-348.##Ramos I, Alonso A, Marcen JM, Peris A, Castillo JA, Colmenares M, et al. Heterologous prime-boost vaccination with a non-replicative vaccinia recombinant vector expressing LACK confers protection against canine visceral leishmaniasis with a predominant Th1-specific immune response. Vaccine 2008;26(3):333-344.##Sánchez-Sampedro L, Gómez CE, Mejías-Pérez E, Sorzano CO, Esteban M. High quality long-term CD4+ and CD8+ effector memory populations stimulated by DNA-LACK/MVA-LACK regimen in Leishmania major BALB/c model of infection. PLoS One 2012;7(6):e38859.##

Soluble Expression of Humanized Anti-CD20 Single Chain Antibody in Escherichia coli by Cytoplasmic Chaperones Co-expression 2 2 Background: CD20 is an important cell surface receptor that is used for target therapy of B cell lymphoma and some related blood diseases due to vital function of CD20. In previous studies, a Rituximab based humanized single chain variable fragment (scFv) antibody showed good reactivity against B cell related cancer cells. But this recombinant protein produced Inclusion Bodies (IBs) in Escherichia coli (E. coli) cytoplasm. The aim of this study was to investigate the effect of coexpression with cytoplasmic chaperones on expression and solubility of humanized anti-CD20 scFv in E. coli. Methods: For this purpose, the fragment coding for anti-CD20 huscFv subcloned into the pET22b (+) and transformed into the E. coli BL21 (DE3) was evaluated. In order to inhibit the production of IBs, the effects of co-expression with cytoplasmic chaperones GroEL, DnaK, GroES, Tig, DnaJ and GrpE were investigated.  Result: Coexpression with cytoplasmic chaperones led to increased soluble expression of anti-CD20 recombinant protein. Among investigated chaperones, pKJE7 chaperone plasmid containing DnaJ, GrpE, DnaK chaperone genes had significant effects with an expression yield of 325 µg/ml soluble anti-CD20 scFv.  Conclusion: The result of this study demonstrated remarkable effect of pKJE7 chaperone on enhancement of soluble expression of anti-CD20 huscFv antibody in E. coli. 141 146 Mohammadreza Yousefi Department of Biotechnology, Higher Education Institute of Rab-RashidDrug Applied Research Center, Tabriz University of Medical Sciences Department of Biotechnology, Higher Education Institute of Rab-RashidDrug Applied Research Center, Tabriz University of Medical Sciences IranIran Safar Farajnia Drug Applied Research Center, Tabriz University of Medical Sciences Drug Applied Research Center, Tabriz University of Medical Sciences Iran Ahad Mokhtarzadeh Department of Biotechnology, Higher Education Institute of Rab-RashidFaculty of Medicine, Gonabad University of Medical Sciences Department of Biotechnology, Higher Education Institute of Rab-RashidFaculty of Medicine, Gonabad University of Medical Sciences IranIran Bahman Akbari Department of Medical Biotechnology, School of Medicine, Kermanshah University of Medical Sciences Department of Medical Biotechnology, School of Medicine, Kermanshah University of Medical Sciences Iran Shiva Ahdi Khosroshahi Biotechnology Research Center, Tabriz University of Medical Sciences Biotechnology Research Center, Tabriz University of Medical Sciences Iran Mina Mamipour Department of Biotechnology, Higher Education Institute of Rab-Rashid Department of Biotechnology, Higher Education Institute of Rab-Rashid Iran Hassan Dariushnejad Immunology Research Center, Tabriz University of Medical Sciences Immunology Research Center, Tabriz University of Medical Sciences Iran Vahideh Ahmadzadeh Biotechnology Research Center, Tabriz University of Medical Sciences Biotechnology Research Center, Tabriz University of Medical Sciences Iran Molecular chaperonesNon Hodgkin lymphomaSingle chain antibody 318.pdf Jazirehi AR, Bonavida B. Cellular and molecular signal transduction pathways modulated by rituximab (rituxan, anti-CD20 mAb) in non-Hodgkin's lymphoma: implications in chemosensitization and therapeutic intervention. Oncogene 2005;24(13):2121-2143.##Brüggemann M, Winter G, Waldmann H, Neuberger M. The immunogenicity of chimeric antibodies. J Exp Med 1989;170(6):2153-2157.##Janas E, Priest R, Wilde JI, White JH, Malhotra R. Rituxan (anti‐CD20 antibody)‐induced translocation of CD20 into lipid rafts is crucial for calcium influx and apoptosis. Clin Exp Immunol 2005;139(3):439-446.##Pucca MB, Bertolini TB, Barbosa JE, Galina SVR, Porto GS. Therapeutic monoclonal antibodies: scFv patents as a marker of a new class of potential biopharmaceuticals. Braz J Pharm Sci 2011;47(1):31-38.##Wang R, Xiang S, Feng Y, Srinivas S, Zhang Y, Lin M, et al. Engineering production of functional scFv antibody in E. coli by co-expressing the molecule chaperone Skp. Front Cell Infect Microbiol 2013;3:72.##Choi JH, Lee SY. Secretory and extracellular production of recombinant proteins using Escherichia coli. Appl Microbiol Biotechnol 2004;64(5):625-635.##Sahdev S, Khattar SK, Saini KS. Production of active eukaryotic proteins through bacterial expression systems: a review of the existing biotechnology strategies. Mol Cell Biochem 2008;307(1-2):249-264.##Terpe K. Overview of bacterial expression systems for heterologous protein production: from molecular and biochemical fundamentals to commercial systems. Appl Microbiol Biotechnol 2006;72(2):211-222.##Veisi K, Farajnia S, Zarghami N, Khoram Khorshid HR, Samadi N, Ahdi Khosroshahi S, Zarei Jaliani H. Chaperone-assisted soluble expression of a humanized anti-EGFR ScFv antibody in E. Coli. Adv Pharm Bull 2015;5(Suppl 1):621-627.##Welch WJ, Brown CR. Influence of molecular and chemical chaperones on protein folding. Cell Stress Chaperones 1996;1(2):109-115.##Becker J, Craig EA. Heat‐shock proteins as molecular chaperones. Eur J Biochem 1994;219(1‐2):11-23.##Buchner J. Supervising the fold: functional principles of molecular chaperones. FASEB J 1996;10(1):10-19.##Grall N, Livny J, Waldor M, Barel M, Charbit A, Meibom KL. Pivotal role of the Francisella tularensis heat-shock sigma factor RpoH. Microbiology 2009;155(Pt 8):2560-2572.##Nishihara K, Kanemori M, Yanagi H, Yura T. Overexpression of trigger factor prevents aggregation of recombinant proteins in Escherichia coli. Appl Environ Microbiol 2000;66(3):884-889.##Ahmadzadeh V, Farajnia S, Hosseinpour Feizi MA, Khavarinejad RA. Design, expression and characterization of a single chain anti-CD20 antibody; a germline humanized antibody derived from Rituximab. Protein Expr Purif 2014;102:45-51.##Smith MR. Rituximab (monoclonal anti-CD20 antibody): mechanisms of action and resistance. Oncogene 2003;22(47):7359-7368.##Weisser NE, Hall JC. Applications of single-chain variable fragment antibodies in therapeutics and diagnostics. Biotechnol Adv 2009;27(4):502-520.##Sørensen HP, Mortensen KK. Soluble expression of recombinant proteins in the cytoplasm of Escherichia coli. Microb Cell Fact 2005;4(1):1.##Nishihara K, Kanemori M, Kitagawa M, Yanagi H, Yura T. Chaperone coexpression plasmids: differential and synergistic roles of DnaK-DnaJ-GrpE and GroEL-GroES in assisting folding of an allergen of Japanese cedar pollen, Cryj2, in Escherichia coli. Appl Environ Microbiol 1998;64(5):1694-1699.##Lee KH, Kim HS, Jeong HS, Lee YS. Chaperon in GroESL mediates the protein folding of human liver mitochondrial aldehyde dehydrogenase in Escherichia coli. Biochem Biophys Res Commun 2002;298(2):216-224.##Hu X, O’Hara L, White S, Magner E, Kane M, Wall JG. Optimisation of production of a domoic acid-binding scFv antibody fragment in Escherichia coli using molecular chaperones and functional immobilisation on a mesoporous silicate support. Protein Expr Purif 2007;52(1):194-201.##Shuo-shuo C, Xue-zheng L, Ji-hong S. Effects of co-expression of molecular chaperones on heterologous soluble expression of the cold-active lipase Lip-948. Protein Expr Purif 2011;77(2):166-172.##Maeng BH, Nam DH, Kim YH. Coexpression of molecular chaperones to enhance functional expression of anti-BNP scFv in the cytoplasm of Escherichia coli for the detection of B-type natriuretic peptide. World J Microbiol Biotechnol 2011;27(6):1391-1398##Heo MA, Kim SH, Kim SY, Kim YJ, Chung J, Oh MK, et al. Functional expression of single-chain variable fragment antibody against c-Met in the cytoplasm of Escherichia coli. Protein Expr Purif 2006;47(1):203-209.##Sonoda H, Kumada Y, Katsuda T, Yamaji H. Functional expression of single-chain Fv antibody in the cytoplasm of Escherichia coli by thioredoxin fusion and co-expression of molecular chaperones. Protein Expr Purif 2010;70(2):248-253.##Sonoda H, Kumada Y, Katsuda T, Yamaji H. Cytoplasmic production of soluble and functional single-chain Fv-Fc fusion protein in Escherichia coli. Biochem Eng J 2011;53(3):253-259.##

A Feasibility Study to Evaluate Bacillus subtilis as a Host for Producing Recombinant Human Parathyroid Hormone 2 2 Background: Biosynthetic teriparatide (1-34) (TPD) is a N-terminally truncated version of human parathyroid hormone (hPTH). The recombinant form of this polypeptide has been expressed in Escherichia coli (E. coli) and approved as the first anabolic treatment of osteoporosis in the EU and the USA. Feasibility of expression and secretion of a tag- fused form of TPD into Bacillus subtilis (B. subtilis) was examined due to several advantages of B. subtilis over E. coli in production of recombinant proteins with pharmacological activities. Methods: A codon optimized gene containing TPD open reading frame carrying enterokinase site in its upstream was fully synthesized. According to our cloning scheme, this synthetic polynucleotide was used as a template for PCR amplification using engineered primers in such a way that a polyhistidin tag was added in frame to the upstream of the amplicon as well as two restriction sites at its ends. The resulted amplicon, a cassette containing His-tag, enterokinase site and TPD, from 5’ to 3’, was cloned into pTZ57R/T vector and subjected to sequencing.The cassette was then subcloned into pHT43 shuttle vector and transformed into B. subtilis. Expression of target protein was analyzed by SDS-PAGE and western blotting upon induction by IPTG. Results: The accuracy of construction of pHT43-TPD was confirmed by sequencing and restriction map analyses. SDS-PAGE and western blotting results showed that the recombinant fusion form of hPTH was successfully expressed and secreted into cytoplasm and extracellular medium. Conclusion: TPD may be successfully expressed and secreted in B. subtilis; however, optimization of expression conditions is required for enhancing target protein yield. 147 151 Mahdi Karimi Department of Molecular Genetics, Research Centre for Biosciences and Biotechnology, Malek-Ashtar University of Technology Department of Molecular Genetics, Research Centre for Biosciences and Biotechnology, Malek-Ashtar University of Technology Iran Farida Behzadian Department of Molecular Genetics, Research Centre for Biosciences and Biotechnology, Malek-Ashtar University of Technology Department of Molecular Genetics, Research Centre for Biosciences and Biotechnology, Malek-Ashtar University of Technology Iran Hamideh Rouhaninejad Department of Molecular Genetics, Research Centre for Biosciences and Biotechnology, Malek-Ashtar University of Technology Department of Molecular Genetics, Research Centre for Biosciences and Biotechnology, Malek-Ashtar University of Technology Iran Sanaz Yari Department of Molecular Genetics, Research Centre for Biosciences and Biotechnology, Malek-Ashtar University of Technology Department of Molecular Genetics, Research Centre for Biosciences and Biotechnology, Malek-Ashtar University of Technology Iran Bacillus subtilishPTHTeriparatide 319.pdf Migliore A, Broccoli S, Massafra U, Bizzi E, Frediani B. Mixed-treatment comparison of anabolic (teriparatide and PTH 1-84) therapies in women with severe osteoporosis. Curr Med Res Opin 2012;28(3):467-473.##Gentleman E, Fredholm YC, Jell G, Lotfibakhshaiesh N, O'Donnell MD, Hill RG, et al. The effects of strontium-substituted bioactive glasses on osteoblasts and osteoclasts in vitro. Biomaterials 2010;31(14):3949-3956.##Hodsman AB, Bauer DC, Dempster DW, Dian L, Hanley DA, Harris ST, et al. Parathyroid hormone and teriparatide for the treatment of osteoporosis: a review of the evidence and suggested guidelines for its use. Endoc Rev 2005;26(5):688-703.##D'Amelio P, Tamone C, Sassi F, D'Amico L, Roato I, Patanè S, et al. Teriparatide increases the maturation of circulating osteoblast precursors. Osteoporos Int 2012;23(4):1245-1253.##Chintamaneni S, Finzel K, Gruber BL. Successful treatment of sternal fracture nonunion with teriparatide. Osteoporos Int 2010;21(6):1059-1063.##Ponnapakkam T, Katikaneni R, Sakon J, Stratford R, Gensure RC. Treating osteoporosis by targeting parathyr-oid hormone to bone. Drug Discov Today 2014;19(3):204-208.##Zhang X, Zhang R, Bao T, Rao Z, Yang T, Xu M, et al. The rebalanced pathway significantly enhances acetoin production by disruption of acetoin reductase gene and moderate-expression of a new water-forming NADH oxidase in Bacillus subtilis. Metab Eng 2014;23:34-41.##Chen J, Zhao L, Fu G, Zhou W, Sun Y, Zheng P, et al. A novel strategy for protein production using non-classical secretion pathway in Bacillus subtilis. Microb Cell Fact 2016;15:69.##Westers L, Westers H, Quax WJ. Bacillus subtilis as cell factory for pharmaceutical proteins: a biotechnological approach to optimize the host organism. Biochim Biophys Acta 2004;1694(1-3):299-310.##Xue GP, Johnson JS, Dalrymple BP. High osmolarity improves the electro-transformation efficiency of the gram-positive bacteria Bacillus subtilis and Bacillus licheniformis. J Microbiol Methods 1999;34(3):183-191.##Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: a laboratory manual. 3rd ed. USA: Cold Spring Harbor; 2001. 1546 p.##van Staa TP, Dennison EM, Leufkens HG, Cooper C, et al. Epidemiology of fractures in England and Wales. Bone 2001;29(6):517-522.##Gullberg B, Johnell O, Kanis J. World-wide projections for hip fracture. Osteoporos Int 1997;7(5):407-413.##Hamedifar H, Salamat F, Saffarion M, Ghiasi M, Hosseini A, Lahiji H, et al. A novel approach for high level expression of soluble recombinant human parathyroid hormone (rhPTH 1-34) in Escherichia coli. Avicenna J Med Biotechnol 2013;5(3):193-201.##Durban MA, Silbersack J, Schweder T, Schauer F, Bornscheuer UT. High level expression of a recombinant phospholipase C from Bacillus cereus in Bacillus subtilis. Appl Microbiol Biotechnol 2007;74(3):634-639.##Birch JR, Onakunle Y. Biopharmaceutical proteins: opportunities and challenges. Methods Mol Biol 2005:1-16.##Mongkolthanaruk W. Classification of Bacillus beneficial substances related to plants, humans and animals. J Microbiol Biotechnol 2012;22(12):1597-1604.##Luan C, Zhang HW, Song DG, Xie YG, Feng J, Wang YZ. Expressing antimicrobial peptide cathelicidin-BF in Bacillus subtilis using SUMO technology. Appl Microbiol Biotechnol 2014;98(8):3651-3658.##Shields DC, Sharp PM. Synonymous codon usage in Bacillus subtilis reflects both translational selection and mutational biases. Nucleic Acids Res 1987;15(19):8023-8040.##Jeong SJ, Kwon GH, Chun J, Kim JS, Park CS, Kwon DY, et al. Cloning of fibrinolytic enzyme gene from Bacillus subtilis isolated from Cheonggukjung and its expression in protease-deﬁcient Bacillus subtilis. J Microbiol Biotechnol 2007;17(6):1018-1023.##

The Distinct Role of Small Heat Shock Protein 20 on HCV NS3 Expression in HEK-293T Cell Line 2 2 Background: Hepatitis C (HCV) is known as a serious blood-borne disease that infects millions of people globally. NS3 is a conserved non-structural sequence of hepatitis C virus which has a major role in activating specific CTL responses. As known, there is no effective vaccine against HCV infection, thus it is required to design a specific regimen of vaccination. Recently, the strong immunological properties of Heat shock proteins (Hsps) led to their use as immunomodulators and an antigen carrier for subunit vaccine candidates. In the current study, the role of Hsp20 was evaluated as a HCV NS3 gene carrier in mammalian cell line. Methods: At first, the recombinant plasmids of pEGFP-Hsp20, pEGFP-NS3, and pEGFP-Hsp20-NS3 were constructed and their accuracy was confirmed by digestion and sequencing. Then, all recombinant plasmids were transfected into HEK293T cells by Lipofectamine and TurboFect gene delivery systems. Finally, the expression of proteins was assessed by fluorescent microscopy, western blotting, and flow cytometry.  Results: In western blotting, the 47, 59, and 79 kDa bands were detected for pEGFP-Hsp20, pEGFP-NS3, and pEGFP-Hsp20-NS3, respectively. The percentage of NS3-Hsp20-GFP protein expression was ~67% by TurboFect and ~50% by Lipofectamine indicating high potency of TurboFect delivery system. Furthermore, the expression of Hsp20 (~83%) was higher than NS3 (~58%) in the cells transfected by TurboFect using flow cytometry analysis. This result was confirmed in the expression of Hsp20-NS3 fusion (~67%) in which Hsp20 increased the delivery of HCV NS3 in vitro. The same data were obtained by Lipofectamine transfection reagent.  Conclusion: Briefly, our data confirmed the role of Hsp20 as a suitable antigen carrier for DNA vaccine design. 152 157 Marzieh Basirnejad Department of Molecular and Cellular Sciences, Faculty of Advanced Sciences & Technology, Pharmaceutical Sciences Branch, Islamic Azad University Department of Molecular and Cellular Sciences, Faculty of Advanced Sciences & Technology, Pharmaceutical Sciences Branch, Islamic Azad University Iran Azam Bolhassani Department of Hepatitis and AIDS, Pasteur Institute of Iran Department of Hepatitis and AIDS, Pasteur Institute of Iran Iran Seyed Mehdi Sadat Department of Hepatitis and AIDS, Pasteur Institute of Iran Department of Hepatitis and AIDS, Pasteur Institute of Iran Iran Hepatitis C virusSmall heat-shock proteinsVaccines 320.pdf Chevaliez S, Pawlotsky JM, editors. HCV genome and life cycle. Norfolk (UK): Horizon Bioscience; 2006. 451 p. (Tan SL, editor. Hepatitis C Viruses: Genomes and Molecular Biology).##Larson AM. Diagnosis and management of acute liver failure. Curr Opin Gastroenterol 2010;26(3):214-221.##Thimme R, Oldach D, Chang KM, Steiger C, Ray SC, Chisari FV. Determinants of viral clearance and persistence during acute hepatitis C virus infection. J Exp Med 2001;194(10):1395-1406.##Sasaki S, Takeshita F, Xin KQ, Ishii N, Okuda K. Adjuvant formulations and delivery systems for DNA vaccines. Methods 2003;31(3):243-254.##Petrovsky N, Aguilar JC. Vaccine adjuvants: current state and future trends. Immunol Cell Biol 2004;82(5):488-496.##Qazi KR, Qazi MR, Julián E, Singh M, Abedi-Valugerdi M, Fernández C. Exposure to mycobacteria primes the immune system for evolutionarily diverse heat shock proteins. Infect Immun 2005;73(11):7687-7696.##Suzue K, Young RA. Heat shock proteins as immunological carriers and vaccines. EXS 1996;77:451-465.##van Noort JM, Bsibsi M, Nacken P, Gerritsen WH, Amor S. The link between small heat shock proteins and the immune system. Int J Biochem Cell Biol 2012;44(10):1670-1679.##Kampinga HH, Garrido C. HSPBs: small proteins with big implications in human disease. Int J Biochem Cell Biol 2012;44(10):1706-1710.##Basha E, O’Neill H, Vierling E. Small heat shock proteins and α-crystallins: dynamic proteins with flexible functions. Trends Biochem Sci 2012;37(3):106-117.##Bakthisaran R, Tangirala R, Rao ChM. Small heat shock proteins: Role in cellular functions and pathology. Biochim Biophys Acta 2015;1854(4):291-319.##Lindquist S. The heat-shock response. Annu Rev Biochem 1986;55(1):1151-1191.##Taylor RP, Benjamin IJ. Small heat shock proteins: a new classification scheme in mammals. J Mol Cell Cardiol 2005;38(3):433-444.##Kim YE, Hipp MS, Bracher A, Hayer-Hartl M, Hartl FU. Molecular chaperone functions in protein folding and proteostasis. Annu Rev Biochem 2013;82:323-355.##Velichko AK, Markova EN, Petrova NV, Razin SV, Kantidze OL. Mechanisms of heat shock response in mammals. Cell Mol Life Sci 2013;70(22):4229-4241.##Kappé G, Franck E, Verschuure P, Boelens WC, Leunissen JA, de Jong WW. The human genome encodes 10 alpha-crystallin-related small heat shock proteins: HspB1-10. Cell Stress Chaperones 2003;8(1):53-61.##Sun Y, MacRae TH. Small heat shock proteins: molecular structure and chaperone function. Cell Mol Life Sci 2005;62(21):2460-2476.##Bolhassani A, Rafati S. Heat-shock proteins as powerful weapons in vaccine development. Expert Rev Vaccines 2008;7(8):1185-1199.##Newport GR. Heat shock proteins as vaccine candidates. Semin Immunol 1991;3(1):17-24.##Diepolder HM, Zachoval R, Hoffmann RM, Wierenga EA, Santantonio T, Jung MC, et al. Possible mechanism involving T-lymphocyte response to non-structural protein 3 in viral clearance in acute hepatitis C virus infection. Lancet 1995;346(8981):1006.##Lazdina U, Hultgren C, Frelin L, Chen M, Lodin K, Weiland O, et al. Humoral and CD4(+) T helper (Th) cell responses to the hepatitis C virus non-structural 3 (NS3) protein: NS3 primes Th1-like responses more effectively as a DNA-based immunogen than as a recombinant protein. J Gen Virol 2001;82(6):1299-1308.##Missale G, Bertoni R, Lamonaca V, Valli A, Massari M, Mori C, et al. Different clinical behaviors of acute hepatitis C virus infection are associated with different vigor of the anti-viral cell-mediated immune response. J Clin Invest 1996;98(3):706-714.##Tsai S, Liaw Y, Chen MH, Huang CY, Kuo GC. Detection of type 2‐like T‐helper cells in hepatitis C virus infection: Implications for hepatitis C virus chronicity. Hepatology 1997;25(2):449-458.##Sällberg M, Zhang ZX, Chen M, Jin L, Birkett A, Peterson DL, et al. Immunogenicity and antigenicity of the ATPase/helicase domain of the hepatitis C virus non-structural 3 protein. J Gen Virol 1996;77(11):2721-2728.##Gurunathan S, Klinman DM, Seder RA. DNA vaccines: immunology, application, and optimization. Annu Rev Immunol 2000;18(1):927-974.##Naderi M, Saeedi A, Moradi A, Kleshadi M, Zolfaghari MR, Gorji A, et al. Interleukin-12 as a genetic adjuvant enhances hepatitis C virus NS3 DNA vaccine immunogenicity. Virol Sin 2013;28(3):167-173.##Jiao X, Wang RY, Qiu Q, Alter HJ, Shih JW. Enhanced hepatitis C virus NS3 specific Th1 immune responses induced by co-delivery of protein antigen and CpG with cationic liposomes. J Gen Virol 2004;85(Pt 6):1545-1553.##Qazi KR, Wikman M, Vasconcelos NM, Berzins K, Stahl S, Fernández C. Enhancement of DNA vaccine potency by linkage of Plasmodium falciparum malarial antigen gene fused with a fragment of HSP70 gene. Vaccine 2005;23(9):1114-1125.##Barrios C, Lussow AR, Van Embden J, Van Der Zee R, Rappuoli R, Costantino P, et al. Mycobacterial heat-shock proteins as carrier molecules. II: The use of the 70-kDa mycobacterial heat-shock protein as carrier for conjugated vaccinescan circumvent the need for adjuvants and Bacillus Calmette Guérin priming. Europ J Immunol 1992;22(6):1365-1372.##Ebrahimi SM, Tebianian M. Role of mycobacterial heat shock protein 70 (mHSP70) as genetic vaccine adjuvants. World Appl Sci J 2011;14(10):1569-1575.##McNulty S, Colaco CA, Blandford LE, Bailey CR, Baschieri S, Todryk S. Heat‐shock proteins as dendritic cell‐targeting vaccines-getting warmer. Immunology 2013;139(4):407-415.##Montalvo-Alvarez AM, Folgueira C, Carrión J, Monzote-Fidalgo L, Cañavate C, Requena JM. The Leishmania HSP20 is antigenic during natural infections, but, as DNA vaccine, it does not protect BALB/c mice against experimental L. amazonensis infection. J Biomed Biotechnol 2008;2008:695432.##Jaramillo Ortiz JM, Del Médico Zajac MP, Zanetti FA, Molinari MP, Gravisaco MJ, Calamante G, et al. Vaccine strategies against Babesia bovis based on prime-boost immunizations in mice with modified vaccinia Ankara vector and recombinant proteins. Vaccine 2014;32(36):4625-4632.##Brown WC, Ruef BJ, Norimine J, Kegerreis KA, Suarez CE, Conley PG, et al. A novel 20-kilodalton protein conserved in Babesia bovis and B. bigemina stimulates memory CD4(+) T lymphocyte responses in B. bovis-immune cattle. Mol Biochem Parasitol 2001;118(1):97-109.##Bepperling A, Alte F, Kriehuber T, Braun N, Weinkauf S, Groll M, et al. Alternative bacterial two-component small heat shock protein systems. Proc Natl Acad Sci USA 2012;109(50):20407-20412.##Park TG, Jeong JH, Kim SW. Current status of polymeric gene delivery systems. Adv Drug Deliv Rev 2006;58(4):467-486.##Lin C, Thomson JA, Rice CM. A central region in the hepatitis C virus NS4A protein allows formation of an active NS3-NS4A serine proteinase complex in vivo and in vitro. J Virol 1995;69(7):4373-4380.##He QQ, Cheng RX, Sun Y, Feng DY, Chen ZC, Zheng H. Hepatocyte transformation and tumor development induced by hepatitis C virus NS3 c-terminal deleted protein. World J Gastroenterol 2003;9(3):474-478.##Jiao X, Wang RY, Feng Z, Hu G, Alter HJ, W‐K Shih J. DNA immunization encoding the secreted nonstructural protein 3 (NS3) of hepatitis C virus and enhancing the Th1 type immune response. J Viral Hepat 2004;11(1):18-26.##Lang KA, Yan J, Draghia-Akli R, Khan A, Weiner DB. Strong HCV NS3-and NS4A-specific cellular immune responses induced in mice and Rhesus macaques by a novel HCV genotype 1a/1b consensus DNA vaccine. Vaccine 2008;26(49):6225-6231.##Behzadi MA, Alborzi A, Pouladfar G, Dianatpour M, Ziyaeyan M. Expression of NS3/NS4A proteins of Hepatitis C virus in Huh7 cells following engineering its eukaryotic expression vector. Jundishapur J Microbiol 2015;8(11):e27355.##Mehrlatifan S, Mirnurollahi SM, Motevalli F, Rahimi P, Soleymani S, Bolhassani A. The structural HCV genes delivered by MPG cell penetrating peptide are directed to enhance immune responses in mice model. Drug Deliv 2016;23(8):2852-2859.##

In silico Biological Activity of Steroids from the Marine Gastropods Telescopium telescopium Collected from South West Coast of India 2 2 Background: The purpose of this study was to investigate the sterol profiling and predict the pharmacological potential of marine gastropod Telescopium telescopium (T. telescopium), collected from mangrove ecosystem in the South west coast of India. Methods: Sterol fractions were separated from the crude lipids using 15% ethyl acetate. Ethyl acetate fractions were dried under ultrahigh purity N2 and analyzed using GC-MS. The biological activity was predicted using the software CLC-Pred; In silico predictions of cytotoxicity for tumor and non-tumor cell lines and PASS.  Results: This study proved the existence of four sterols, of which cholesterol was abundant. It was found that most of the steroids profiled from T. telescopium displayed activity against reproductive system as well as skin related diseases.  Conclusion: The predicted anti infertility and skin related activity of the steroids identified from the marine gastropod T. telescopium is useful to attract industrial interest towards this species which will be helpful in rising new combinations with added therapeutic and nutritional worth. 158 162 A Ragi Department of Chemical Oceanography, Faculty of Marine Sciences, Cochin University of Science and Technology Department of Chemical Oceanography, Faculty of Marine Sciences, Cochin University of Science and Technology India P Leena Inter University Center for Development of Marine Biotechnology, Cochin University of Science and Technology Inter University Center for Development of Marine Biotechnology, Cochin University of Science and Technology India K Prashob Peter Department of Chemical Oceanography, Faculty of Marine Sciences, Cochin University of Science and Technology Department of Chemical Oceanography, Faculty of Marine Sciences, Cochin University of Science and Technology India S Nair Inter University Center for Development of Marine Biotechnology, Cochin University of Science and Technology Inter University Center for Development of Marine Biotechnology, Cochin University of Science and Technology India CLC-PredPAASTelescopium telescopium 322.pdf Murphy KJ, Mann NJ, Sinclair AJ. Fatty acid and sterol composition of frozen and freeze-dried New Zealand Green Lipped Mussel (Perna canaliculus) from three sites in New Zealand. Asia Pac J Clin Nutr 2003;12(1):50-60.##Zhukova NV. Lipid classes and fatty acid composition of the tropical nudibranch mollusks Chromodoris sp. and Phyllidia coelestis. Lipids 2007;42(12):1169-1175.##Pereira DM, Valentão P, Teixeira N, Andrade PB. Amino acids, fatty acids and sterols profile of some marine organisms from Portuguese waters. Food Chem 2013;141(3):2412-2417.##Ozogul F, Kuley E, Ozogul Y. Sterol content of fish, crustacea and mollusc: effects of cooking methods. Int J Food Prop 2015;18(9):2026-2041.##Kanazawa A. Sterols in marine invertebrates. Fish Sci 2001;67(6):997-1007.##Ozogul Y, Duysak O, Ozogul F, Özkütük AS, Türeli C. Seasonal effects in the nutritional quality of the body structural tissue of cephalopods. Food Chem 2008;108(3):847-852.##Zarai Z, Frikha F, Balti R, Miled N, Gargouri Y, Mejdoub H. Nutrient composition of the marine snail (Hexaplex trunculus) from the Tunisian Mediterranean coasts. J Sci Food Agric 2011;91(7):1265-1270.##Kala KJ, Prashob PKJ, Chandramohanakumar N. Cyto-toxic potential of fucosterol isolated from Turbinaria conoides against Dalton’s Lymphoma Ascites. Int J Pharmacogn Phytochem Res 2015;7:1217-1221.##Thomas A, Prashob PKJ, Chandramohanakumar N. A profiling of anti-tumour potential of sterols in the mangrove fern Acrostichum aureum. Int J Pharmacogn Phytochem Res 2016;8:1828-1832.##Anuradha V, Byju K, Emilda R, Anu G, Nair SM, Chandramohanakumar N, et al. In silico biological activity of steroids from the marine sponge Axinella carteri. Med Chem Res 2013;22(3):1142-1146.##Goad LJ, Akihisa T. Analysis of sterols. 2nd ed. UK: Springer Science & Business Media; 2012. 423 p.##Byju K. Chemistry and bioactivity of natural products from the gorgonian coral Subergorgia reticulate [dissertation]. [India]: Cochin University of Science and Technology. 2015. 289 p.##McCann SE, Freudenheim JL, Marshall JR, Graham S. Risk of human ovarian cancer is related to dietary intake of selected nutrients, phytochemicals and food groups. J Nutr 2003;133(6):1937-1942.##Choi JM, Lee EO, Lee HJ, Kim KH, Ahn KS, Shim BS, et al. Identification of campesterol from Chrysanthemum coronarium L. and its antiangiogenic activities. Phytother Res 2007;21(10):954-959.##Poroikov VV, Filimonov DA, Borodina YV, Lagunin AA, Kos A. Robustness of biological activity spectra predicting by computer program PASS for noncongeneric sets of chemical compounds. J Chem Inf Comput Sci 2000;40(6):1349-1355.##Lagunin A, Filimonov D, Poroikov V. Multi-targeted natural products evaluation based on biological activity prediction with PASS. Curr Pharm Des 2010;16(15):1703-1717.##Stepanchikova AV, Lagunin AA, Filimonov DA, Poroikov VV. 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Arylamine N-acetyltransferase 2 Polymorphisms and the Risk of Endometriosis 2 2 Background: Human arylamine N-acetyltransferase 2 (NAT2) gene has a key role in xenobiotic metabolism through the conjugation of acetyl group to xenobiotic substances. NAT2 has been suggested as a susceptibility factor in endometriosis; however, the results of studies have been controversial. In this study, the association of NAT2 polymorphisms with susceptibility to endometriosis was evaluated in an Iranian population.  Methods: This is an association study and totally 141 women with diagnosis of endometriosis and 158 healthy women as control group were analyzed for NAT2 gene polymorphisms (C481T, A803G, G857A and G590A) by PCR-RFLP methods. Results: The 590 GA genotype was significantly lower (p=0.001; OR=0.42, 95% CI: 0.25-0.71) in the patients (38.3%) than the control group (55.1%). The 590A allele was significantly lower (p=0.033; OR=0.69, 95% CI: 0.49-0.79) in the patients (31.2%) compared with the controls (39.6%). Analysis of haplotypes showed that NAT2 481C, 803A, 590A, 587A combination was significantly different between the case and control women (p= 0.029; OR=3.11, 95% CI: 1.13-8.52). Conclusion: The NAT2 G590A SNP may be associated with susceptibility to endometriosis and the 590A allele may have a protective role in development of endometriosis. The NAT2 481C, 803A, 590A, 587A haplotype was associated with a higher risk of endometriosis in Iranian population. 163 167 Diman Fayez Department of Biology, Faculty of Science, Science and Research Branch, Islamic Azad University Department of Biology, Faculty of Science, Science and Research Branch, Islamic Azad University Iran Kioomars Saliminejad Department of Biology, Faculty of Science, Science and Research Branch, Islamic Azad University Department of Biology, Faculty of Science, Science and Research Branch, Islamic Azad University Iran Shiva Irani Department of Biology, Faculty of Science, Science and Research Branch, Islamic Azad University Department of Biology, Faculty of Science, Science and Research Branch, Islamic Azad University Iran Koorosh Kamali Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Iran Toktam Memariani Central Research Lab, North Khorasan University of Medical Sciences Central Research Lab, North Khorasan University of Medical Sciences Iran Hamid Reza Khorram Khorshid Genetic Research Center, University of Social Welfare and Rehabilitation Sciences Genetic Research Center, University of Social Welfare and Rehabilitation Sciences Iran EndometriosisGenome wide association studyNAT2Polymorphism 10342.pdf Bellelis P, Podgaec S, Abrão MS. 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Curr Opin Obstet Gynecol 2001;13(3):309-314.##Bischoff F, Simpson JL. Genetic basis of endometriosis. Ann N Y Acad Sci 2004;1034:284-299.##Simpson JL, Bischoff FZ, Kamat A, Buster JE, Carson SA. Genetics of endometriosis. Obstet Gynecol Clin North Am 2003;30(1):21-40, vii.##Hickman D, Risch A, Buckle V, Spurr N, Jeremiah S, McCarthy A, et al. Chromosomal localization of human genes for arylamine N-acetyltransferase. Biochem J 1994;297(Pt 3):441-445.##Sim E, Walters K, Boukouvala S. Arylamine N-acetyltransferases: from structure to function. Drug Metab Rev 2008;40(3):479-510.##Hein DW, Boukouvala S, Grant DM, Minchin RF, Sim E. Changes in consensus arylamine N-acetyltransferase gene nomenclature. Pharmacogenet Genomics 2008; 18(4):367-368.##Risch A, Wallace DM, Bathers S, Sim E. Slow N-acetylation genotype is a susceptibility factor in occupational and smoking related bladder cancer. 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Association between endometriosis and N-acetyl transferase 2 polymorphisms in a UK population. Mol Hum Reprod 2001;7(11):1079-1083.##Bischoff FZ, Marquez-Do D, Kosugi Y, Mitchell-Leef D, Malinak LR, Simpson JL, et al. Association of N-acetyltransferase 2 (NAT2) genetic polymorphism resulting in decreased capacity to detoxify aromatic amines in women with endometriosis. J Soc Gynecol Investig 1998;11001(5):111A.##Baranova H, Canis M, Ivaschenko T, Albuisson E, Bothorishvilli R, Baranov V, et al. Possible involvement of arylamine N-acetyltransferase 2, glutathione S-transferases M1 and T1 genes in the development of endometriosis. Hum Reprod 1999;5(7):636-641.##Babu KA, Rao KL, Reddy NG, Kanakavalli MK, Zondervan KT, Deenadayal M, et al. N-acetyl transferase 2 polymorphism and advanced stages of endometriosis in South Indian women. Reprod Biomed Online 2004;9(5):533-540.##Deguchi M, Yoshida S, Kennedy S, Ohara N, Motoyama S, Maruo T. 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Association of WNT3 Variations and Risk of Non-Syndromic Cleft Lip and Palate in a Population of Iranian Infants 2 2 Background: Nonsyndromic cleft lip and/or palate (NSCL/P) is the most common orofacial birth defect, often attributed to ethnic and environmental differences. Up to now, linkage analyses and genome-wide association studies have identified several genomic susceptibility regions for NSCL/P. The WNT genes including WNT3 are strong candidates for NSCL/P, since they are involved in regulating mid-face development and upper lip fusion. This study tested association of the WNT3 polymorphisms, rs3809857 G/T and rs9890413 G/A, with the risk of NSCL/P in a population of Iranian infants. Methods: The allelic and genotypic frequencies for each participant were determined in 113 unrelated Iranian subjects with NSCL/P and 220 control subjects using PCR and restriction fragment length polymorphism (RFLP) methods. A p-value of 0.05 was considered statistically significant.  Results: The WNT3 rs3809857 GT genotype was significantly lower (p=0.039, OR=0.55, 95% CI=0.30-0.97) in the NSCL/P (21.2%) than the control group (30.42%). For the WNT3 rs9890413 G/A polymorphism, neither genotype nor allele frequencies were significantly different between the case and control groups. Conclusion: Our results indicated that the WNT3 rs3809857 GT genotype may have a protective effect against NSCL/P in Iranian population. 168 172 Homa Farrokhi Karibozorg Department of Biochemistry, Islamic Azad University, Damghan Branch Department of Biochemistry, Islamic Azad University, Damghan Branch Iran Nahid Masoudian Department of Biochemistry, Islamic Azad University, Damghan Branch Department of Biochemistry, Islamic Azad University, Damghan Branch Iran Kioomars Saliminejad Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Iran Asghar Ebadifar Dentofacial Deformities Research Center, Research Institute of Dental Sciences, Shahid Beheshti University of Medical Sciences Dentofacial Deformities Research Center, Research Institute of Dental Sciences, Shahid Beheshti University of Medical Sciences Iran Koorosh Kamali Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Iran Hamid Reza Khorram Khorshid Genetic Research Center, University of Social Welfare and Rehabilitation Sciences Genetic Research Center, University of Social Welfare and Rehabilitation Sciences Iran Genome wide association studyCleft lip/palatePolymorphismWNT3 323.pdf Dixon MJ, Marazita ML, Beaty TH, Murray JC. Cleft lip and palate: understanding genetic and environmental influences. Nat Rev Genet 2011;12(3):167-178.##Gorlin RJ, Cohen MM, Hennekam RCM. Syndromes of the head and neck. 4th ed. New York: Oxford University Press; 2001. 1215 p.##Rajabian MH, Sherkat M. An epidemiologic study of oral clefts in Iran: analysis of 1669 cases. Cleft Palate Craniofac J 2000;37(2):191-196.##Aldhorae KA, Böhmer AC, Ludwig KU, Esmail AH, Al-Hebshi NN, Lippke B, et al. Nonsyndromic cleft lip with or without cleft palate in arab populations: genetic analysis of 15 risk loci in a novel case-control sample recruited in Yemen. Birth Defects Res A Clin Mol Teratol 2014;100(4):307-313.##Grosen D, Chevrier C, Skytthe A, Bille C, Mølsted K, Sivertsen A, et al. A cohort study of recurrence patterns among more than 54,000 relatives of oral cleft cases in Denmark: support for the multifactorial threshold model of inheritance. J Med Genet 2010;47(3):162-168.##Beaty TH, Taub MA, Scott AF, Murray JC, Marazita ML, Schwender H, et al. Confirming genes influencing risk to cleft lip with/without cleft palate in a case-parent trio study. Hum Genet 2013;132(7):771-781.##Brugmann SA, Goodnough LH, Gregorieff A, Leucht P, ten Berge D, Fuerer C, et al. Wnt signaling mediates regional specification in the vertebrate face. Development 2007;134(18):3283-3295.##Lan Y, Ryan RC, Zhang Z, Bullard SA, Bush JO, Maltby KM, et al. Expression of Wnt9b and activation of canonical Wnt signaling during midfacial morphogenesis in mice. Dev Dyn 2006;235(5):1448-1454.##Mostowska A, Hozyasz KK, Biedziak B, Wojcicki P, Lianeri M, Jagodzinski PP. Genotype and haplotype analysis of WNT genes in non-syndromic cleft lip with or without cleft palate. Eur J Oral Sci 2012;120(1):1-8.##Chiquet BT, Blanton SH, Burt A, Ma D, Stal S, Mulliken JB, et al. Variation in WNT genes is associated with non-syndromic cleft lip with or without cleft palate. Hum Mol Genet 2008;17(1):2212-2218.##Yao T, Yang L, Li PQ, Wu H, Xie HB, Shen X, et al. Association of Wnt3A gene variants with non-syndromic cleft lip with or without cleft palate in Chinese population. Arch Oral Biol 2011;56(1):73-78.## Feng C, Duan W, Zhang D, Zhang E, Xu Z, Lu L. A C392T polymorphism of the Wnt10a gene in nonsyndromic oral cleft in a northeastern Chinese population. Br J Oral Maxillofac Surg 2014;52(8):751-755.##Menezes R, Letra A, Kim AH, Küchler EC, Day A, Tannure PN, et al. Studies with Wnt genes and nonsyndromic cleft lip and palate. Birth Defects Res A Clin Mol Teratol 2010;88(11):995-1000.##Miller SA, Dykes DD, Polesky HF. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 1988;16(3):1215.##Ota M, Fukushima H, Kulski JK, Inoko H. Single nucleotide polymorphism detection by polymerase chain reaction-restriction fragment length polymorphism. Nat Protoc 2007;2(11):2857-2864.##Sivertsen A, Wilcox AJ, Skjaerven R, Vindenes HA, Abyholm F, Harville E, et al. Familial risk of oral clefts by morphological type and severity: population based cohort study of first degree relatives. BMJ 2008;336(7641):432-434.##Mossey PA, Little J, Munger RG, Dixon MJ, Shaw WC. Cleft lip and palate. Lancet 2009;374(9703):1773-1785.##Leslie EJ, Marazita ML. Genetics of cleft lip and cleft palate. Am J Med Genet C Semin Med Genet 2013;163C(4):246-258.##Leslie EJ, Murray JC. Evaluating rare coding variants as contributing causes to non-syndromic cleft lip and palate. Clin Genet 2013;84(5):496-500.##Lu YP, Han WT, Liu Q, Li JX, Li ZJ, Jiang M, et al. Variations in WNT3 gene are associated with incidence of non-syndromic cleft lip with or without cleft palate in a northeast Chinese population. Genet Mol Res 2015;14(4):12646-12653.##Wodarz A, Nusse R. Mechanisms of Wnt signaling in development. 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Development 2009;136(18):3161-3171.##Martinelli M, Girardi A, Cura F, Nouri N, Pinto V, Carinci F, et al. Non-syndromic cleft lip with or without cleft palate in Asian populations: Association analysis on three gene polymorphisms of the folate pathway. Arch Oral Biol 2016;61:79-82.##Shi M, Wehby GL, Murray JC. Review on genetic variants and maternal smoking in the etiology of oral clefts and other birth defects. Birth Defects Res C Embryo Today 2008;84(1):16-29.##Wehby GL, Murray JC. Folic acid and orofacial clefts: a review of the evidence. Oral Dis 2010;16(1):11-19.##

Interleukin-2 and Interferon-Gamma Single Nucleotide Polymorphisms in Iranian Patients with Chronic Heart Failure 2 2 Background: Inflammatory cytokines have been known to be associated with Chronic Heart Failure (CHF). Given the importance of cytokines in the context of the failing heart, the prevalence of Interleukin-2 (IL-2) and Interferon-gamma (IFN-γ) polymorphisms was studied in patients with CHF due to ischemic heart disease in a case-control study. Methods: Fifty-six Iranian patients with CHF were enrolled in this study as the case group and compared with 139 healthy subjects, using polymerase chain reaction with sequence-specific primers method, so as to determine the frequency of alleles, genotypes and haplotypes of IFN-γ (+874 A/T) and IL-2 (-330 G/T, +166 G/T) SNPs.  Results: The GG genotype at IL-2 -330 in patients with CHF was significantly overrepresented in comparison with the control group (p=0.013). Such a positive genotypic association was also observed for IL-2 +166/TT (p=0.022). Meanwhile, the GT genotype frequency at IL-2 -330/GT in the patient group was significantly lower than the one in healthy controls (p=0.049). No significant association was detected between the IFN-γ gene polymorphisms and individuals’ susceptibility to CHF. Conclusion: Certain genotypes in IL-2 gene were overrepresented in patients with CHF, which could render individuals more vulnerable to this disease. 173 177 Mohammad Jafar Mahmoudi Division of Cardiology, Department of Internal Medicine, Faculty of Medicine, Tehran University of Medical Sciences Division of Cardiology, Department of Internal Medicine, Faculty of Medicine, Tehran University of Medical Sciences Iran Sara Harsini Molecular Immunology Research Center, Tehran University of Medical Sciences Molecular Immunology Research Center, Tehran University of Medical Sciences Iran Elham Farhadi Department of Hematology, Faculty of Allied Medical Science, Iran University of Medical Sciences Department of Hematology, Faculty of Allied Medical Science, Iran University of Medical Sciences Iran Mona Hedayat Division of Immunology, Boston Children's Hospital, Harvard Medical School Division of Immunology, Boston Children's Hospital, Harvard Medical School USA Mohammad Taghvaei Molecular Immunology Research Center, Tehran University of Medical Sciences Molecular Immunology Research Center, Tehran University of Medical Sciences Iran Maryam Mahmoudi Faculty of Nutrition and Dietetics, Tehran University of Medical Sciences Faculty of Nutrition and Dietetics, Tehran University of Medical Sciences Iran Maryam Sadr Molecular Immunology Research Center, Tehran University of Medical Sciences Molecular Immunology Research Center, Tehran University of Medical Sciences Iran Nilufar Esfahanian Molecular Immunology Research Center, Tehran University of Medical Sciences Molecular Immunology Research Center, Tehran University of Medical Sciences Iran Ebrahim Nematipour Tehran Heart Center, Tehran University of Medical Sciences Tehran Heart Center, Tehran University of Medical Sciences Iran Keramat Nourijelyani Department of Epidemiology and Biostatistics, Faculty of Public Health, Tehran University of Medical Sciences Department of Epidemiology and Biostatistics, Faculty of Public Health, Tehran University of Medical Sciences Iran Ali Akbar Amirzargar Molecular Immunology Research Center, Tehran University of Medical SciencesDepartment of Immunology, Faculty of Medicine, Tehran University of Medical Sciences Molecular Immunology Research Center, Tehran University of Medical SciencesDepartment of Immunology, Faculty of Medicine, Tehran University of Medical Sciences IranIran Nima Rezaei Molecular Immunology Research Center, Tehran University of Medical SciencesDepartment of Immunology, Faculty of Medicine, Tehran University of Medical SciencesResearch Center for Immunodeficiencies, Children's Medical Center, Tehran University of Medical SciencesNetwork of Immunity in Infection, Malignancy and Autoimmunity (NIIMA), Universal Scientific Education and Research Network (USERN) Molecular Immunology Research Center, Tehran University of Medical SciencesDepartment of Immunology, Faculty of Medicine, Tehran University of Medical SciencesResearch Center for Immunodeficiencies, Children's Medical Center, Tehran University of Medical SciencesNetwork of Immunity in Infection, Malignancy and Autoimmunity (NIIMA), Universal Scientific Education and Research Network (USERN) IranIranIranIran Heart failureInterferon-gammaInterleukin-2Single-nucleotide polymorphism 10343.pdf Lloyd-Jones DM, Larson MG, Leip EP, Beiser A, D’Agostino RB, Kannel WB, et al. Lifetime risk for developing congestive heart failure the Framingham Heart Study. Circulation 2002;106(24):3068-3072.##Hansson GK, Robertson AK, Söderberg-Nauclér C. Inflammation and atherosclerosis. Annu Rev Pathol 2006;1:297-329.##Kleemann R, Zadelaar S, Kooistra T. Cytokines and atherosclerosis: a comprehensive review of studies in mice. Cardiovasc Res 2008;79(3):360-376.##Lesnik P, Haskell CA, Charo IF. Decreased atherosclerosis in CX3CR1–/–mice reveals a role for fractalkine in atherogenesis. J Clin Invest 2003;111(3):333-340.##Ding R, Gao W, Ostrodci DH, He Z, Song Y, Ma L, et al. Effect of interleukin-2 level and genetic variants on coronary artery disease. Inflammation 2013;36(6):1225-1231.##Fukunaga T, Soejima H, Irie A, Sugamura K, Oe Y, Tanaka T, et al. Expression of interferon-gamma and interleukin-4 production in CD4+ T cells in patients with chronic heart failure. Heart Vessels 2007;22(3):178-183.##Elkind MS, Rundek T, Sciacca RR, Ramas R, Chen HJ, Boden-Albala B, et al. Interleukin-2 levels are associated with carotid artery intima-media thickness. Atherosclerosis 2005;180(1):181-187.##Simon AD, Yazdani S, Wang W, Schwartz A, Rabbani LE. Elevated plasma levels of interleukin‐2 and soluble il‐2 receptor in ischemic heart disease. Clin Cardiol 2001;24(3):253-256.##Frostegård J, Ulfgren AK, Nyberg P, Hedin U, Swedenborg J, Andersson U, et al. Cytokine expression in advanced human atherosclerotic plaques: dominance of pro-inflammatory (Th1) and macrophage-stimulating cytokines. Atherosclerosis 1999;145(1):33-43.##Miyake K, Nakashima H, Akahoshi M, Inoue Y, Nagano S, Tanaka Y, et al. Genetically determined interferon‐γ production influences the histological phenotype of lupus nephritis. Rheumatology (Oxford) 2002;41(5):518-524.##Ohtsuka K, Gray JD, Stimmler MM, Horwitz DA. The relationship between defects in lymphocyte production of transforming growth factor-beta1 in systemic lupus erythematosus and disease activity or severity. Lupus 1999;8(2):90-94.##Amirzargar A, Shahram F, Nikoopour E, Rezaei N, Saeedfar K, Ziaei N, et al. Proinflammatory cytokine gene polymorphisms in Behcet's disease. Eur Cytokine Netw 2010;21(4):292-296.##Mahdaviani SA, Rezaei N, Moradi B, Dorkhosh S, Amirzargar AA, Movahedi M. Proinflammatory cytokine gene polymorphisms among Iranian patients with asthma. J Clin Immunol 2009;29(1):57-62.##Rezaei N, Amirzargar AA, Shakiba Y, Mahmoudi M, Moradi B, Aghamohammadi A. Proinflammatory cytokine gene single nucleotide polymorphisms in common variable immunodeficiency. Clin Exp Immunol 2009;155(1):21-27.##Amirzargar AA, Bagheri M, Ghavamzadeh A, Alimoghadam K, Khosravi F, Rezaei N, et al. Cytokine gene polymorphism in Iranian patients with chronic myelogenous leukaemia. Int J Immunogenet 2005;32(3):167-171.##Amirzargar AA, Rezaei N, Jabbari H, Danesh AA, Khosravi F, Hajabdolbaghi M, et al. Cytokine single nucleotide polymorphisms in Iranian patients with pulmonary tuberculosis. Eur Cytokine Netw 2006;17(2):84-89.##Tahghighi F, Ziaee V, Moradinejad MH, Rezaei A, Harsini S, Soltani S, et al. Tumor necrosis factor-alpha single nucleotide polymorphisms in juvenile systemic lupus erythematosus. Hum Immunol 2015;76(8):533-536.##Mahmoudi M, Tahghighi F, Ziaee V, Harsini S, Rezaei A, Soltani S, et al. Interleukin-4 single nucleotide polymorphisms in juvenile systemic lupus erythematosus. Int J Immunogenet 2014;41(6):512-517.##Rezaei A, Ziaee V, Sharabian FT, Harsini S, Mahmoudi M, Soltani S, et al. Lack of association between interleukin-10, transforming growth factor-beta gene polymorphisms and juvenile-onset systemic lupus erythematosus. Clin Rheumatol 2015;34(6):1059-1064.##Ziaee V, Tahghighi F, Moradinejad MH, Harsini S, Mahmoudi M, Rezaei A, et al. Interleukin-6, interleukin-1 gene cluster and interleukin-1 receptor polymorphisms in Iranian patients with juvenile systemic lupus erythematosus. Eur Cytokine Netw 2014;25(2):35-40.##Amirzargar AA, Naroueynejad M, Khosravi F, Dianat SS, Rezaei N, Mytilineos J, et al. Cytokine single nucleotide polymorphisms in Iranian populations. Eur Cytokine Netw 2008;19(2):104-112.##Miller S, Dykes DD, Polesky HF. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 1988;16(3):1215.##Armstrong PW. Left ventricular dysfunction: causes, natural history, and hopes for reversal. Heart 2000;84 (Suppl 1):i15-17:discussion i50.##Willerson JT, Ridker PM. Inflammation as a cardiovascular risk factor. Circulation 2004;109(21 suppl 1):II2-10.##Mishra A, Srivastava A, Mittal T, Garg N, Mittal B. Role of inflammatory gene polymorphisms in left ventricular dysfunction (LVD) susceptibility in coronary artery disease (CAD) patients. Cytokine 2013;61(3):856-861.##Seaman WE. Natural killer cells and natural killer T cells. 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Association Between rs6759298 and Ankylosing Spondylitis in Iranian Population 2 2 Background: Ankylosing Spondylitis (AS) is a chronic autoinflammatory Spondyloar-thropathy (SpA) which is characterized by sacroiliitis, which progresses to the axial skeleton. It seems that non-Human Leukocyte Antigen (HLA) and also HLA-B27 are associated with the susceptibility and pathogenesis of the disease. The recent Genome-Wide Association Studies (GWASs) have reported intergenic rs6759298 to be associated with AS etiology. The aim of this study was investigation of the rs6759298 polymorphism in Iranian AS patients. In addition, probable correlations with clinical indices and manifestations were considered. Methods: This study included 403 patients with AS. The control group consisted of 506 healthy individuals who were matched for sex, age, and ethnicity with AS group. Genotyping of rs6759298 was determined using the Amplification-Refractory Muta-tion System-Polymerase Chain Reaction (ARMS-PCR). Results: The GG genotype and G allele were found to be significantly more prevalent in the patient group in comparison to the control group [(p=2×10-6 and 7.44×10-9; OR (95% CI) =2.16 (1.56-2.98) and 1.73 (1.43-2.08)], respectively. Conclusion: No associations were found between patients with three genotypes and any disease manifestations or clinical indices. This investigation confirmed a highly significant association of rs6759298 with disease susceptibility, with no effect on disease progress or clinical presentations. Since rs6759298 belongs to the 2p15 gene desert, further studies would elucidate the exact role of this polymorphism in the pathogenesis of AS. 178 182 Mahdi Mahmoudi Rheumatology Research Center, Tehran University of Medical Sciences Rheumatology Research Center, Tehran University of Medical Sciences Iran Masoud Garshasbi Department of Medical Genetics, Faculty of Medical Sciences, Tarbiat Modares University Department of Medical Genetics, Faculty of Medical Sciences, Tarbiat Modares University Iran Amir Ashraf-Ganjouei Rheumatology Research Center, Tehran University of Medical SciencesStudents’ Scientific Research Center, Tehran University of Medical Sciences Rheumatology Research Center, Tehran University of Medical SciencesStudents’ Scientific Research Center, Tehran University of Medical Sciences IranIran Ali Javinani Rheumatology Research Center, Tehran University of Medical SciencesStudents’ Scientific Research Center, Tehran University of Medical Sciences Rheumatology Research Center, Tehran University of Medical SciencesStudents’ Scientific Research Center, Tehran University of Medical Sciences IranIran Mahdi Vojdanian Rheumatology Research Center, Tehran University of Medical Sciences Rheumatology Research Center, Tehran University of Medical Sciences Iran Masoumeh Saafi Department of Genetics, Islamic Azad University, Tabriz Branch Department of Genetics, Islamic Azad University, Tabriz Branch Iran Nooshin Ahmadzadeh Rheumatology Research Center, Tehran University of Medical Sciences Rheumatology Research Center, Tehran University of Medical Sciences Iran Ahmadreza Jamshidi Rheumatology Research Center, Tehran University of Medical Sciences Rheumatology Research Center, Tehran University of Medical Sciences Iran Ankylosing spondylitisHLA-B27Iran 324.pdf Akgul O, Ozgocmen S. Classification criteria for spondyloarthropathies. World J Orthop 2011;2(12):107-115.##Shahlaee A, Mahmoudi M, Nicknam MH, Farhadi E, Fallahi S, Jamshidi AR. Gender differences in Iranian patients with ankylosing spondylitis. Clin Rheumatol 2015;34(2):285-293.##Nicknam MH, Mahmoudi M, Amirzargar AA, Ganjalikhani Hakemi M, Khosravi F, Jamshidi AR, et al. Determination of HLA-B27 subtypes in Iranian patients with ankylosing spondylitis. Iran J Allergy Asthma Immunol 2008;7(1):19-24.##Nicknam MH, Mahmoudi M, Amirzargar AA, Jamshidi AR, Rezaei N, Nikbin B. HLA-B27 subtypes and tumor necrosis factor alpha promoter region polymorphism in Iranian patients with ankylosing spondylitis. Eur Cytokine Netw 2009;20(1):17-20.##Brewerton DA, Hart FD, Nicholls A, Caffrey M, James DC, Sturrock RD. Ankylosing spondylitis and HL-A 27. Lancet 1973;1(7809):904-907.##Jamshidi AR, Shahlaee A, Farhadi E, Fallahi S, Nicknam MH, Bidad K, et al. Clinical characteristics and medical management of Iranian patients with ankylosing spondylitis. Mod Rheumatol 2014;24(3):499-504.##Soleimanifar N, Amirzargar AA, Mahmoudi M, Pourfathollah AA, Azizi E, Jamshidi AR, et al. Study of programmed cell death 1 (PDCD1) gene polymorphims in Iranian patients with ankylosing spondylitis. Inflammation 2011;34(6):707-712.##Mahmoudi M, Amirzargar AA, Jamshidi AR, Farhadi E, Noori S, Avraee M, et al. Association of IL1R polymorphism with HLA-B27 positive in Iranian patients with ankylosing spondylitis. Eur Cytokine Netw 2011;22(4):175-180.##Mahmoudi M, Jamshidi AR, Amirzargar AA, Farhadi E, Nourijelyani K, Fallahi S, et al. Association between endoplasmic reticulum aminopeptidase-1 (ERAP-1) and susceptibility to ankylosing spondylitis in Iran. Iran J Allergy Asthma Immunol 2012;11(4):294-300.##Hacquard-Bouder C, Chimenti MS, Giquel B, Donnadieu E, Fert I, Schmitt A, et al. Alteration of antigen-independent immunologic synapse formation between dendritic cells from HLA-B27-transgenic rats and CD4+ T cells: selective impairment of costimulatory molecule engagement by mature HLA-B27. Arthritis Rheum 2007;56(5):1478-1489.##Mear JP, Schreiber KL, Munz C, Zhu X, Stevanovic S, Rammensee HG, et al. Misfolding of HLA-B27 as a result of its B pocket suggests a novel mechanism for its role in susceptibility to spondyloarthropathies. J Immunol 1999;163(12):6665-6670.##Fussell H, Nesbeth D, Lenart I, Campbell EC, Lynch S, Santos S, et al. Novel detection of in vivo HLA-B27 conformations correlates with ankylosing spondylitis association. Arthritis Rheum 2008;58(11):3419-3424.##Reeves E, Elliott T, James E, Edwards CJ. ERAP1 in the pathogenesis of ankylosing spondylitis. Immunol Res 2014;60(2-3):257-269.##Australo-Anglo-American Spondyloarthritis Consortium (TASC),Reveille JD, Sims AM, Danoy P, Evans DM, Leo P, et al. Genome-wide association study of ankylosing spondylitis identifies non-MHC susceptibility loci. Nat Genet 2010;42(2):123-127.##Robinson PC, Claushuis TA, Cortes A, Martin TM, Evans DM, Leo P, et al. Genetic dissection of acute anterior uveitis reveals similarities and differences in associations observed with ankylosing spondylitis. Arthritis Rheumatol 2015;67(1):140-151.##International Genetics of Ankylosing Spondylitis Consortium (IGAS), Cortes A, Hadler J, Pointon JP, Robinson PC, Karaderi T, et al. Identification of multiple risk variants for ankylosing spondylitis through high-density genotyping of immune-related loci. Nat Genet 2013;45(7):730-738.##Deodhar A. Axial spondyloarthritis criteria and modified NY criteria: issues and controversies. Clin Rheumatol 2014;33(6):741-747.##Abtahi S, Farazmand A, Mahmoudi M, Ashraf-Ganjouei A, Javinani A, Nazari B, et al. IL-1A rs1800587, IL-1B rs1143634 and IL-1R1 rs2234650 polymorphisms in Iranian patients with systemic sclerosis. 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Menstrual Blood-derived Stromal Stem Cells Augment CD4+ T Cells Proliferation 2 2 Background: It is more than sixty years that the concept of the fetal allograft and immunological paradox of pregnancy was proposed and in this context, several regulatory networks and mechanisms have been introduced so far. It is now generally recognized that mesenchymal stem cells exert potent immunoregulatory activity. In this study, for the first time, the potential impact of Menstrual blood Stem Cells (MenSCs), as surrogate for endometrial stem cells, on proliferative capacity of CD4+ T cells was tested. Methods: MenSCs and Bone marrow Mesenchymal Stem Cells (BMSCs) were isolated and assessed for their immunophenotypic features and multi-lineage differentiation capability. BMSCs and MenSCs with or without IFNγ pre-stimulation were co-cultured with purified anti-CD3/CD28-activated CD4+ T cells and the extent of T cell proliferation at different MenSCs: T cell ratios were investigated by CSFE flow cytometry. IDO activity of both cell types was measured after stimulation with IFNγ by a colorimetric assay. Results: MenSCs exhibited dual mesenchymal and embryonic markers and multi-lineage differentiation capacity. MenSCs significantly increased proliferation of CD4+ cells at ratios 1:2, 1:4 and 1:8. IFNγ pre-treated BMSCs but not MenSCs significantly suppressed CD4+ T cells proliferation. Such proliferation promoting capacity of MenSCs was not correlated with IDO activity as these cells showed the high IDO activity following IFNγ treatment. Conclusion: Although augmentation of T cell proliferation by MenSCs can be a basis for maintenance of endometrial homeostasis to cope with ascending infections, this may not fulfill the requirement for immunological tolerance to a semi-allogeneic fetus. However, more investigation is needed to examine whether or not the immunomodulatory properties of these cells are affected by endometrial microenvironment during pregnancy. 183 191 Mehdi Aleahmad Department of Immunology, Faculty of Public Health, Tehran University of Medical Sciences Department of Immunology, Faculty of Public Health, Tehran University of Medical Sciences Iran Alireza Ghanavatinejad Department of Immunology, Faculty of Public Health, Tehran University of Medical Sciences Department of Immunology, Faculty of Public Health, Tehran University of Medical Sciences Iran Mahmood Bozorgmehr Reproductive Immunology Research Center, Avicenna Research Institute, ACECROncopathology Research Center, Iran University of Medical Sciences Reproductive Immunology Research Center, Avicenna Research Institute, ACECROncopathology Research Center, Iran University of Medical Sciences IranIran Mohammad-Reza Shokri Department of Immunology, Faculty of Medicine, Iran University of Medical Sciences Department of Immunology, Faculty of Medicine, Iran University of Medical Sciences Iran Shohreh Nikoo Immunology Research Center (IRC), Iran University of Medical Sciences Immunology Research Center (IRC), Iran University of Medical Sciences Iran Maryam Tavakoli Reproductive Immunology Research Center, Avicenna Research Institute, ACECR Reproductive Immunology Research Center, Avicenna Research Institute, ACECR Iran Somaieh Kazemnejad Reproductive Biotechnology Research Centre, Avicenna Research Institute, ACECR Reproductive Biotechnology Research Centre, Avicenna Research Institute, ACECR Iran Fazel Shokri Department of Immunology, Faculty of Public Health, Tehran University of Medical SciencesDepartment of Hybridoma, Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR Department of Immunology, Faculty of Public Health, Tehran University of Medical SciencesDepartment of Hybridoma, Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR IranIran Amir-Hassan Zarnani Department of Immunology, Faculty of Public Health, Tehran University of Medical SciencesReproductive Immunology Research Center, Avicenna Research Institute, ACECR Department of Immunology, Faculty of Public Health, Tehran University of Medical SciencesReproductive Immunology Research Center, Avicenna Research Institute, ACECR IranIran EndometriumImmunological toleranceMenstrual blood stem cellsPregnancyProliferationT lymphocytes 10363.pdf Verma S, Hiby SE, Loke YW, King A. 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Human bone marrow stromal cells suppress T-lymphocyte proliferation induced by cellular or nonspecific mitogenic stimuli. Blood 2002;99(10):3838-3843.##Le Blanc K, Tammik L, Sundberg B, Haynesworth SE, Ringdén O. Mesenchymal stem cells inhibit and stimulate mixed lymphocyte cultures and mitogenic responses independently of the major histocompatibility complex. Scand J Immunol 2003;57(1):11-20.##Tse WT, Pendleton JD, Beyer WM, Egalka MC, Guinan EC. Suppression of allogeneic T-cell proliferation by human marrow stromal cells: implications in transplantation. Transplantation 2003;75(3):389-397.##Yañez R, Lamana ML, García-Castro J, Colmenero I, Ramírez M, Bueren JA. Adipose tissue‐derived mesenchymal stem cells have in vivo immunosuppressive properties applicable for the control of the graft‐versus‐host disease. Stem Cells 2006;24(11):2582-2591.##Lee JM, Jung J, Lee HJ, Jeong SJ, Cho KJ, Hwang SG, et al. Comparison of immunomodulatory effects of placenta mesenchymal stem cells with bone marrow and adipose mesenchymal stem cells. Int Immunopharmacol 2012;13(2):219-224.##Chan RW, Schwab KE, Gargett CE. Clonogenicity of human endometrial epithelial and stromal cells. Biol Reprod 2004;70(6):1738-1750.##Schwab KE, Chan RW, Gargett CE. Putative stem cell activity of human endometrial epithelial and stromal cells during the menstrual cycle. Fertil Steril 2005;84 Suppl 2:1124-1130.##Schwab KE, Hutchinson P, Gargett CE. Identification of surface markers for prospective isolation of human endometrial stromal colony-forming cells. Hum Reprod 2008;23(4):934-943.##Gargett CE, Schwab KE, Zillwood RM, Nguyen HP, Wu D. Isolation and culture of epithelial progenitors and mesenchymal stem cells from human endometrium. Biol Reprod 2009;80(6):1136-1145.##Patel AN, Park E, Kuzman M, Benetti F, Silva FJ, Allickson JG. Multipotent menstrual blood stromal stem cells: isolation, characterization, and differentiation. Cell transplant 2008;17(3):303-311.##Cho NH, Park YK, Kim YT, Yang H, Kim SK. Lifetime expression of stem cell markers in the uterine endometrium. Fertil Steril 2004;81(2):403-407.##Matthai C, Horvat R, Noe M, Nagele F, Radjabi A, van Trotsenburg M, et al. Oct-4 expression in human endometrium. Mol Hum Reprod 2006;12(1):7-10.##Nikoo S, Ebtekar M, Jeddi-Tehrani M, Shervin A, Bozorgmehr M, Kazemnejad S, et al. Effect of menstrual blood‐derived stromal stem cells on proliferative capacity of peripheral blood mononuclear cells in allogeneic mixed lymphocyte reaction. J Obstet Gynaecol Res 2012;38(5):804-809.##Bozorgmehr M, Moazzeni SM, Salehnia M, Sheikhian A, Nikoo S, Zarnani AH. Menstrual blood-derived stromal stem cells inhibit optimal generation and maturation of human monocyte-derived dendritic cells. Immunol lett 2014;162(2):239-246.##Kazemnejad S, Zarnani AH, Khanmohammadi M, Mobini S. Chondrogenic differentiation of menstrual blood-derived stem cells on nanofibrous scaffolds. Methods Mol Biol 2013;1058:149-169.##Darzi S, Zarnani AH, Jeddi-Tehrani M, Entezami K, Mirzadegan E, Akhondi MM, et al. Osteogenic differentiation of stem cells derived from menstrual blood versus bone marrow in the presence of human platelet releasate. Tissue Eng Part A 2012;18(15-16):1720-1728.##Djouad F, Bony C, Häupl T, Uzé G, Lahlou N, Louis-Plence P, et al. Transcriptional profiles discriminate bone marrow-derived and synovium-derived mesenchymal stem cells. Arthritis Res & Ther 2005;7(6):R1304-1315.##Rahimi M1, Zarnani AH, Mohseni-Kouchesfehani H, Soltanghoraei H, Akhondi MM, Kazemnejad S. Comparative evaluation of cardiac markers in differentiated cells from menstrual blood and bone marrow-derived stem cells in vitro. Mol Biotechnol 2014;56(12):1151-1162.##Bishara A, Malka R, Brautbar C, Barak V, Cohen I, Kedar E. Cytokine production in human mixed leukocyte reactions performed in serum-free media. J Immunol Methods 1998;215(1):187-190.##Krampera M. Mesenchymal stromal cell ‘licensing’: a multistep process. Leukemia 2011;25(9):1408-1414.##Groh ME, Maitra B, Szekely E, Koç ON. Human mesenchymal stem cells require monocyte-mediated activation to suppress alloreactive T cells. Exp Hematol 2005;33(8):928-934.##Rozenberg A, Rezk A, Boivin MN, Darlington PJ, Nyirenda M, Li R, et al., Human mesenchymal stem cells impact Th17 and Th1 responses through a prostaglandin E2 and myeloid‐dependent mechanism. Stem Cells Transl Med 2016;5(11):1506-1514.##Cutler AJ, Limbani V, Girdlestone J, Navarrete CV. Umbilical cord-derived mesenchymal stromal cells modulate monocyte function to suppress T cell proliferation. J Immunol 2010;185(11):6617-6623.##Wira CR, Rodriguez-Garcia M, Patel MV. The role of sex hormones in immune protection of the female reproductive tract. 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Endometrial stromal cells undergoing decidualization down-regulate their properties to produce proinflammatory cytokines in response to interleukin-1β via reduced p38 mitogen-activated protein kinase phosphorylation. J Clin Endocrinol Metabol 2003;88(5):2236-2241.##Polchert D, Sobinsky J, Douglas G, Kidd M, Moadsiri A, Reina E, et al. IFN‐γ activation of mesenchymal stem cells for treatment and prevention of graft versus host disease. Eur J Immunol 2008;38(6):1745-1755.##Sheng H, Wang Y, Jin Y, Zhang Q, Zhang Y, Wang L, et al. A critical role of IFNγ in priming MSC-mediated suppression of T cell proliferation through up-regulation of B7-H1. Cell Res 2008;18(8):846-857.##Chinnadurai R, Copland IB, Patel SR, Galipeau J. IDO-independent suppression of T cell effector function by IFNγ-licensed human mesenchymal stromal cells. J Immunol 2014;192(4):1491-1501.##Krampera M, Glennie S, Dyson J, Scott D, Laylor R, Simpson E, et al. 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TNFRSF13B/TACI Alterations in Turkish Patients with Common Variable Immunodeficiency and IgA Deficiency 2 2 Background: The Transmembrane Activator and Calcium modulator ligand Interactor (TACI), encoded by TNFRSF13B/TACI gene, is mutated in some patients with Common Variable Immunodeficiency (CVID) and IgA Deficiency (IgAD). The purpose of the study was to investigate for the first time in Turkish patients the prevalence of TNFRSF13B alterations in CVID, selective and partial IgAD patients.  Methods: Forty two CVID, 36 selective IgAD, 34 partial IgAD and 25 healthy controls were included. All patients were examined for TNFRSF13B gene mutations by PCR.  Results: The percentages of TNFRSF13B mutations in CVID, selective and partial IgAD patients were 7.1, 2.7 and 2.9%, respectively. No disease causing TNFRSF13B mutation in healthy controls was found. Patients with TACI mutations had recurrent respiratory tract infections. None of them experienced autoimmunity, bronchiectasis or granulomatous disease. In conclusion, TNFRSF13B mutations were present not only in CVID patients, but also in IgAD cases. Conclusion: Modifier genes as well as their combination with other genetic or environmental factors may play an important role in the development of the immunodeficiency phenotype. 192 195 Neslihan Karaca Department of Pediatric Immunology, Faculty of Medicine, Ege University Department of Pediatric Immunology, Faculty of Medicine, Ege University Turkey Ezgi Severcan Department of Pediatric Immunology, Faculty of Medicine, Ege University Department of Pediatric Immunology, Faculty of Medicine, Ege University Turkey Burcu Guven Department of Pediatric Immunology, Faculty of Medicine, Ege University Department of Pediatric Immunology, Faculty of Medicine, Ege University Turkey Elif Azarsiz Department of Pediatric Immunology, Faculty of Medicine, Ege University Department of Pediatric Immunology, Faculty of Medicine, Ege University Turkey Guzide Aksu Department of Pediatric Immunology, Faculty of Medicine, Ege University Department of Pediatric Immunology, Faculty of Medicine, Ege University Turkey Necil Kutukculer Department of Pediatric Immunology, Faculty of Medicine, Ege University Department of Pediatric Immunology, Faculty of Medicine, Ege University Turkey Common variable immunodeficiencyIgA deficiencyTACIMutationRespiratory tract infection 10346.pdf Schäffer AA, Pfannstiel J, Webster AD, Plebani A, Hammarström L, Grimbacher B. Analysis of families with common variable immunodeficiency (CVID) and IgA deficiency suggests linkage of CVID to chromosome 16q. Hum Genet 2006;118(6):725-729.##Pan-Hammarstrom Q, Salzer U, Du L, Björkander J, Cunningham-Rundles C, Nelson DL, et al. Reexamining the role of TACI coding variants in common variable immunodeficiency and selective IgA deficiency. Nat Genet 2007;39(4):429-430.##Speletas M, Mamara A, Papadopoulou-Alataki E, Iordanakis G, Liadaki K, Bardaka F, et al. TNFRSF13B/TACI alterations in Greek patients with antibody deficiencies. J Clin Immunol 2011;31(4):550-559.##Pulvirenti F, Zuntini R, Milito C, Specchia F, Spadaro G, Danieli MG, et al. Clinical associations of biallelic and monoallelic TNFRSF13B vriants in Italian primary antibody deficiency syndromes. J Immunol Res 2016;2016:8390356.##Conley ME, Notarangelo LD, Etzioni A. Diagnostic criteria for primary immunodeficiencies. Representing PAGID (pan-American group for immunodeficiency) and ESID (European society for Immunodeficiencies). Clin Immunol 1999;93(3):190-197.##Martinez-Gallo M, Radigan L, Almejún MB, Martínez-Pomar N, Matamoros N, Cunningham-Rundles C. TACI mutations and impaired B-cell function in subjects with CVID and healthy heterozygotes. J Allergy Clin Immunol 2013;131(2):468-476.##

Polymorphisms in the Cholinergic Receptors Muscarinic (CHRM2 and CHRM3) Genes and Alzheimer’s Disease 2 2 Background: Disruption of the cholinergic neurotransmitter pathway which is important for cognition, memory and learning abilities has been reported in Alzheimer’s Disease (AD) patients. The receptors involved include the Cholinergic Receptors Muscarinic (CHRM). CHRM2 gene has been associated with intelligence, personality traits, substance dependence and depression. CHRM3 has been found to heterodimerize with CHRM2. Methods: DNA samples from 240 AD patients with SNPs rs6962027 of CHRM2 gene and rs7511970 of CHRM3 gene were amplified using PCR and genotyped using Restriction Fragment Length Polymorphism (RFLP). Chi-squared test was done to check if the genes are in Hardy-Weinberg equilibrium.  Results and Conclusion: Although the results did not show significant associations, these data denote plausible interaction between TT in SNP rs6962027 in CHRM2 gene and TT in SNP rs7511970 in CHRM3 gene affecting AD risk. SNP rs7511970 of CHRM3 gene may also exert an influence on late-onset AD. 196 199 Lim Ya Chee Pengiran Anak Puteri Rashidah Sa'adatul Bolkiah Institute of Health Sciences, Universiti Brunei Darussalam, Jalan Tungku Link, BE 1410 Pengiran Anak Puteri Rashidah Sa'adatul Bolkiah Institute of Health Sciences, Universiti Brunei Darussalam, Jalan Tungku Link, BE 1410 Brunei Darussalam Alaistair Cumming Pengiran Anak Puteri Rashidah Sa'adatul Bolkiah Institute of Health Sciences, Universiti Brunei Darussalam, Jalan Tungku Link, BE 1410 Pengiran Anak Puteri Rashidah Sa'adatul Bolkiah Institute of Health Sciences, Universiti Brunei Darussalam, Jalan Tungku Link, BE 1410 Brunei Darussalam Alzheimer diseaseGenesGenetic polymorphism 10347.pdf Zou YM, Lu D, Li-ping Liu, Hui-hong Zhang, Yu-ying Zhou. Olfactory dysfunction in Alzheimer ’ s disease. Neuropsychiatr Dis Treat 2016;12:869-875.##Luo X, Kranzler HR, Zuo L, Wang S, Blumberg HP, Gelernter J. CHRM2 gene predisposes to alcohol dependence, drug dependence and affective disorders: results from an extended case-control structured association study. Hum Mol Genet 2005;14(16):2421-2434.##Bock A, Schrage R, Mohr K. Allosteric modulators targeting CNS muscarinic receptors. Neuropharmacology 2017. pii: S0028-3908(17)30441-0.##Gosso MF, de Geus EJ, Polderman TJ, Boomsma DI, Posthuma D, Heutink P. Exploring the functional role of the CHRM2 gene in human cognition: results from a dense genotyping and brain expression study. BMC Med Genet 2007;8:66.##Volpicelli LA, Levey AI. Muscarinic acetylcholine receptor subtypes in cerebral cortex and hippocampus. Prog Brain Res 2004;145:59-66.##Goin JC, Nathanson NM. Quantitative analysis of muscarinic acetylcholine receptor homo- and heterodimerization in live cells: regulation of receptor down-regulation by heterodimerization. J Biol Chem 2006;281(9):5416-5425.##Gustincich S, Manfioletti G, Del Sal G, Schneider C, Carninci P. A fast method for high-quality genomic DNA extraction from whole human blood. Biotechniques 1991;11(3):298-300.##Radu BM, Osculati AMM, Suku E, Banciu A, Tsenov G, Merigo F, et al. All muscarinic acetylcholine receptors (M1-M5) are expressed in murine brain microvascular endothelium. Sci Rep 2017;7(1):5083.##