Evidence Based Medicine and Medical Biotechnology 2 2 Evidence Based Medicine (EBM) can be found as far back as the 1940s. However, it was in 1972 that the concept first came into play, originated by Professor Archie Cochrane, in his book, Effectiveness & Efficiency: Random Reflections on Health Services. This was the foundation for evidence based research, and in 1992 a facility was funded by the UK government, with the aim of performing randomly controlled tests on health services. This is no coincidence since evidence-based medicine suggests a personal responsibility for clinicians to keep abreast of research that would be difficult without the information access that the web provides. Evidence-based medicine is now generally perceived to be the dominant operating system in conventional medicine. The term “evidence-based medicine” first appears in 1991, in a piece by Gordon Guyatt (1). But EBM came to the attention of a wider audience in 1992 with an article by the Evidence-Based Medicine Working Group (2) that boldly proclaimed EBM as a “new paradigm” in medicine. The National Institutes of Health defines “clinical research” as research conducted with human subjects (or on material of human origin such as tissues, specimens and cognitive phenomena) for which an investigator (or colleague) directly interacts with human subjects. Excluded from this definition are in vitro studies that utilize human tissues that cannot be linked to a living individual. Indeed, Clinical trials are important component of evidence based medicine. A clinical trial is a research study that finds new ways to prevent, diagnose or treat disease (3-6). For example, cancer clinical trials test new treatments in people with cancer. These treatments investigate promising new drugs, drug combinations, new approaches to surgery or radiation therapy, and advances in new areas such as gene therapy. Clinical trials are the final step in a long process. There is no doubt that EBM plays an important role in the future of medical biotechnology. 133 133 Shahin Akhondzadeh Psychiatric Research Center, Roozbeh Hospital, South Kargar Street Psychiatric Research Center, Roozbeh Hospital, South Kargar Street Iran Editorial 217.pdf Guyatt, G. Evidence-based Medicine. ACP J Club 1991; 114 (suppl2): A16.##Evidence-Based Medicine Working Group. Evidence-based medicine: a new approach to teaching the practice of medicine. J Am Med Assoc 1992;268(17):2420-2425.##Akhondzadeh S, Kashani L, Fotouhi A, Jarvandi S, Mobaseri M, Moin M, et al. Comparison of Lavandula angustifolia Mill. tincture and imipramine in the treatment of mild to moderate depression: a double-blind, randomized trial. Prog Neuropsychopharmacol Biol Psychiatry 2003;27(1):123-127.##Rezaei V, Mohammadi MR, Ghanizadeh A, Sahraian A, Tabrizi M, Rezazadeh SA, et al. Double-blind, placebo-controlled trial of risperidone plus topiramate in children with autistic disorder. Prog Neuropsychopharmacol Biol Psychiatry 2010;34(7):1269-1272.##Akhondzadeh S, Fallah J, Mohammadi MR, Imani R, Mohammadi M, Salehi B, et al. Double-blind placebo-controlled trial of pentoxifylline added to risperidone: effects on aberrant behavior in children with autism. Prog Neuropsychopharmacol Biol Psychiatry 2010;34(1):32-36.##Akhondzadeh S, Shafiee Sabet M, Harirchian MH, Togha M, Cheraghmakani H, Razeghi S, et al. A 22-week, multicenter, randomized, double blind controlled trial of Crocus sativus in the treatment of mild-to-moderate Alzheimer's disease. Psychopharmacology (Berl) 2010; 207:637-643.##

A Comprehensive Review of Clinical Trials on EGFR Inhibitors Such as Cetuximab and Panitumumab as Monotherapy and in Combination for Treatment of Metastatic Colorectal Cancer 2 2 Metastatic colorectal cancer is the fourth most common cause of death due to cancer after those of lung, stomach, and liver. Anti epidermal growth factor receptor drugs as a targeting therapy seem to be good candidates for curing metastatic colorectal cancer. Two available anti epidermal growth factor receptor monoclonal antibodies are cetuximab and panitumumab which have been approved for metastatic colorectal cancer treatment. Through the available literature on NCBI and clinical trials, 31 clinical trials in which cetuximab or panitumumab as monotherapy or in combination with chemotherapy were used for the treatment of metastatic colorectal cancer patients in different line settings and 12 clinical trials in which bevacizumab was used for being compared with anti epidermal growth factor receptor monoclonal antibodies or chemotherapy were chosen for reviewing and comparing the results of overall survival, progression free survival and adverse effects. Cetuximab and panitumumab are well accepted for the treatment of mCRC patients at all stages in different line settings. Although cetuximab administration in metastatic colorectal cancer patients is mostly associated with better overall survival and panitumumab results in better progression free survival, to confirm the superiority of each of them in the treatment protocol of epidermal growth factor receptor monoclonal antibodies, more clinical trials with larger sample size are needed. Through current available data from clinical studies, it can be concluded that the best treatment outcome is achieved by a combination of anti epidermal growth factor receptor monoclonal antibodies with conventional chemotherapy. 134 142 Mohammad Hossein Yazdi Department of Research and Development, Pasteur Institute of Iran Department of Research and Development, Pasteur Institute of Iran Iran Mohammad Ali Faramarzi Department of Pharmaceutical Biotechnology, Faculty of Pharmacy and Biotechnology Research Center, Tehran University of Medical Sciences Department of Pharmaceutical Biotechnology, Faculty of Pharmacy and Biotechnology Research Center, Tehran University of Medical Sciences Iran Shekoufeh Nikfar Department of Pharmacoeconomics and Pharmaceutical Administration, Faculty of Pharmacy, Tehran University of Medical Sciences Department of Pharmacoeconomics and Pharmaceutical Administration, Faculty of Pharmacy, Tehran University of Medical Sciences Iran Mohammad Abdollahi Anti-EGFR drugsCetuximabMetastatic colorectal cancerPanitumumab 218.pdf Frestad D, Perner A, Pedersen UG. 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Available at: www.Fda.Gov [Last accessed 2007].##Cheng L, Ren W, Xie L, Li M, Liu J, Hu J, et al. Anti-EGFR MoAb treatment in colorectal cancer: limitations, controversies, and contradictories. Cancer Chemother Pharmacol 2014;74(1):1-13.##Karapetis CS, Khambata-Ford S, Jonker DJ, O'Callaghan CJ, Tu D, Tebbutt NC, et al. K-ras mutations and benefit from cetuximab in advanced colorectal cancer. N Engl J Med 2008;359(17):1757-1765.##Amado RG, Wolf M, Peeters M, Van Cutsem E, Siena S, Freeman DJ, et al. Wild-type KRAS is required for panitumumab efficacy in patients with metastatic colorectal cancer. J Clin Oncol 2008;26(10):1626-1634.##Kennecke H, Chen L, Blanke CD, Cheung WY, Schaff K, Speers C. Panitumumab monotherapy compared with cetuximab and irinotecan combination therapy in patients with previously treated KRAS wild-type metastatic colorectal cancer. Curr Oncol 2013;20(6):326-332.##Soeda H, Shimodaira H, Gamoh M, Ando H, Isobe H, Suto T, et al. Phase II trial of cetuximab plus irinotecan for oxaliplatin- and irinotecan-based chemotherapy-refractory patients with advanced and/or metastatic colorectal cancer: evaluation of efficacy and safety based on KRAS mutation status (T-CORE0801). Oncology 2014;87(1):7-20.##Price TJ, Peeters M, Kim TW, Li J, Cascinu S, Ruff P, et al. Panitumumab versus cetuximab in patients with chemotherapy-refractory wild-type KRAS exon 2 metastatic colorectal cancer (ASPECCT): a randomised, multicentre, open-label, non-inferiority phase 3 study. Lancet Oncol 2014;15(6):569-579.##Bokemeyer C, Van Cutsem E, Rougier P, Ciardiello F, Heeger S, Schlichting M, et al. Addition of cetuximab to chemotherapy as first-line treatment for KRAS wild-type metastatic colorectal cancer: pooled analysis of the CRYSTAL and OPUS randomised clinical trials. Eur J Cancer 2012;48(10):1466-1475.##Piessevaux H, Buyse M, Schlichting M, Van Cutsem E, Bokemeyer C, Heeger S, et al. Use of early tumor shrinkage to predict long-term outcome in metastatic colorectal cancer treated with cetuximab. J Clin Oncol 2013;31(30):3764-3775.##Raoul JL, Van Laethem JL, Peeters M, Brezault C, Husseini F, Cals L, et al. Cetuximab in combination with irinotecan/5-fluorouracil/folinic acid (FOLFIRI) in the initial treatment of metastatic colorectal cancer: a multicentre two-part phase I/II study. BMC cancer 2009;9:112.##Ye LC, Liu TS, Ren L, Wei Y, Zhu DX, Zai SY, et al. Randomized controlled trial of cetuximab plus chemotherapy for patients with KRAS wild-type unresectable colorectal liver-limited metastases. J Clin Oncol 2013;31(16):1931-1938.##Primrose J, Falk S, Finch-Jones M, Valle J, Sherlock D, Hornbuckle J, et al. A randomized clinical trial of chemotherapy compared to chemotherapy in combination with cetuximab in k-RAS wild-type patients with operable metastases from colorectal cancer: The new EPOC study. J Clin Oncol 2013;31(15):3504.##Douillard JY, Siena S, Cassidy J, Tabernero J, Burkes R, Barugel M, et al. Randomized, phase III trial of panitumumab with infusional fluorouracil, leucovorin, and oxaliplatin (FOLFOX4) versus FOLFOX4 alone as first-line treatment in patients with previously untreated metastatic colorectal cancer: the PRIME study. J Clin Oncol 2010;28(31):4697-4705.##Pietrantonio F, Perrone F, Biondani P, Maggi C, Lampis A, Bertan C, et al. Single agent panitumumab in KRAS wild-type metastatic colorectal cancer patients following cetuximab-based regimens: Clinical outcome and biomarkers of efficacy. Cancer Biol Ther 2013;14(12):1098-1103.##Garrido-Laguna I, McGregor KA, Wade M, Weis J, Gilcrease W, Burr L, et al. A phase I/II study of decitabine in combination with panitumumab in patients with wild-type (wt) KRAS metastatic colorectal cancer. Invest New Drugs 2013;31(5):1257-1264.##Schwartzberg L, Rivera-Herreros F, M. Karthaus. A randomized phase 2 study of mFOLFOX6 with either panitumumab or bevacizumab as 1st-line treatment in patients with unresectable wild-type KRAS metastatic colorectal cancer (mCRC). J Clin Oncol 2013;30:446.##Price TJ, Peeters M, Kim TW, Li J, Cascinu S, Ruff P, et al. ASPECCT: a randomized, multicenter, open-label, phase 3 study of panitumumab (pmab) vs cetuximab (cmab) for previously treated wild-type (WT) KRAS metastatic colorectal cancer (mCRC). Eur J Cancer 2013;49:18.##Su X, Lacouture ME, Jia Y, Wu S. Risk of high-grade skin rash in cancer patients treated with cetuximab--an antibody against epidermal growth factor receptor: systemic review and meta-analysis. Oncology 2009;77(2):124-133.##Cao Y, Liao C, Tan A, Liu L, Gao F. Meta-analysis of incidence and risk of hypomagnesemia with cetuximab for advanced cancer. Chemotherapy 2010;56(6):459-465.##Cao Y, Liu L, Liao C, Tan A, Gao F. Meta-analysis of incidence and risk of hypokalemia with cetuximab-based therapy for advanced cancer. Cancer Chemother Pharmacol 2010;66:37-42.##Chen P, Wang L, Li H, Liu B, Zou Z. Incidence and risk of hypomagnesemia in advanced cancer patients treated with cetuximab: A meta-analysis. Oncol Lett 2013;5(6):1915-1920.##Seymour MT, Brown SR, Middleton G, Maughan T, Richman S, Gwyther S, et al. Panitumumab and irinotecan versus irinotecan alone for patients with KRAS wild-type, fluorouracil-resistant advanced colorectal cancer (PICCOLO): a prospectively stratified randomised trial. Lancet Oncol 2013;14(8):749-759.##Alberts SR, Sargent DJ, Nair S, Mahoney MR, Mooney M, Thibodeau SN, et al. Effect of oxaliplatin, fluorouracil, and leucovorin with or without cetuximab on survival among patients with resected stage III colon cancer: a randomized trial. JAMA 2012;307(13):1383-1393.##ClinicalTrials.gov [Internet]. USA: National Institutes of Health; 2004-2013. 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Preliminary Assessment of Various Additives on the Specific Reactivity of Anti- rHBsAg Monoclonal Antibodies 2 2 Background: Antibodies have a wide application in diagnosis and treatment. In order to maintain optimal stability of various functional parts of antibodies such as antigen binding sites, several approaches have been suggested. Using additives such as polysaccharides and polyols is one of the main methods in protecting antibodies against aggregation or degradation in the formulation. The aim of this study was to evaluate the protective effect of various additives on the specific reactivity of monoclonal antibodies (mAbs) against recombinant HBsAg (rHBsAg) epitopes. Methods: To estimate the protective effect of different additives on the stability of antibody against conformational epitopes (S3 antibody) and linear epitopes (S7 and S11 antibodies) of rHBsAg, heat shock at 37°C was performed in liquid and solid phases. Environmental factors were considered to be constant. The specific reactivity of antibodies was evaluated using ELISA method. The data were analyzed using SPSS software by Mann-Whitney nonparametric test with the confidence interval of 95%. Results: Our results showed that 0.25 M sucrose, 0.04 M trehalose and 0.5% BSA had the most protective effect on maintaining the reactivity of mAbs (S3) against conformational epitopes of rHBsAg. Results obtained from S7 and S11 mAbs against linear characteristics showed minor differences. The most efficient protective additives were 0.04 M trehalose and 1 M sucrose. 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The Potential of Brittle Star Extracted Polysaccharide in Promoting Apoptosis via Intrinsic Signaling Pathway 2 2 Background: Anti-cancer potential of marine natural products such as polysaccharides represented therapeutic potential in oncological researches. In this study, total polysaccharide from brittle star [Ophiocoma erinaceus (O. erinaceus)] was extracted and chemopreventive efficacy of Persian Gulf brittle star polysaccharide was investigated in HeLa human cervical cancer cells.   Methods: To extract polysaccharide, dried brittle stars were ground and extracted mechanically. Then, detection of polysaccharide was performed by phenol sulfuric acid, Ultra Violet (UV)-sulfuric acid method and FTIR. The anti proliferative activity of isolated polysaccharide was examined by MTT assay and evaluation of cell death was done through morphological cell changes; Propodium Iodide staining, fluorescence microscopy and caspase-3, -9 enzymatic measurements. To assess its underlying mechanism, expression of Bax, Bcl-2 was evaluated. Results: The polysaccharide detection methods demonstrated isolation of crude polysaccharide from Persian Gulf brittle star. The results revealed that O. erinaceus polysaccharide suppressed the proliferation of HeLa cells in a dose and time dependent manner. Morphological observation of DAPI and Acridine Orange/Propodium Iodide staining was documented by typical characteristics of apoptotic cell death. Flow cytometry analyses exhibited the accumulation of treated cells in sub-G1 region. Additionally, polysaccharide extracted induced intrinsic apoptosis via up-regulation of caspase-3, caspase-9 and Bax along with down-regulation of Bcl-2 in HeLa cells. Conclusion: Taken together, the apoptosis inducing effect of brittle star polysaccharide via intrinsic pathway confirmed the anti tumor potential of marine polysaccharide. Therefore, these findings proposed new insight into anti cancer properties of brittle star polysaccharide as a promising agent in cervical cancer treatment. 151 156 Javad Baharara Elaheh Amini Research Center for Animal Development Applied Biology, Mashhad Branch, Islamic Azad University Research Center for Animal Development Applied Biology, Mashhad Branch, Islamic Azad University Iran ApoptosisBrittle starHeLa cellsPolysaccharides 220.pdf Gandhiappan J, Rengasamy R. Antiproliferative activity of Solanum anguivi against cancer cell lines. Der Pharm Lett 2012;4(3):875-880.##Di Domenico F, Foppoli C, Coccia R, Perluigi M. Antioxidants in cervical cancer: chemopreventive and chemotherapeutic effects of polyphenols. Biochim Biophys Acta 2012;1822(5):737-747.##Sun SY, Hail N Jr, Lotan R. Apoptosis as a novel target for cancer chemoprevention. J Natl Cancer Inst 2004;96 (9):662-672.##Demain AL, Vaishnav P. Natural products for cancer chemotherapy. Microb Biotechnol 2011;4(6):687-699.##Zong A, Cao H, Wang F. Anticancer polysaccharides from natural resources: a review of recent research. Carbohydr Polym 2012;90(4):1395-1410.##Wijesekara I, Pangestuti R, Kim SK. Biological activities and potential health benefits of sulfated polysaccharides derived from marine algae. Carbohydr Polym 2011;84 (1):14-21.##Senni K, Pereira J, Gueniche F, Delbarre-ladrat C, Sinquin C, Ratiskol J, et al. Marine polysaccharides: a source of bioactive molecules for cell therapy and tissue engineering. Mar Drugs 2011;9(9):1664-1681.##Jiménez-Escrig A, Gómez-Ordóñez E, Rupérez P. Seaweed as a source of novel nutraceuticals: sulfated polysaccharides and peptides. Adv Food Nutr Res 2011;64:325-337.##Chen Sh, Xue Ch, Yin L, Tang Q, Yu G, Chai W. Comparison of structures and anticoagulant activities of fucosylated chondroitin sulfates from different sea cucumbers. Carbohydr Polym 2011;83(2):688-696.##Wang W, Hong JK, Lee CO, Shin S, Jung JH, Cho HY. Bioactive metabolite from the brittle star Ophioplocus japonicus. Nat Prod Sci 2004;10(6):253-261.##Czarkwiani A, Dylus DV, Oliveri P. Expression of skeletogenic genes during arm regeneration in the brittle star Amphiura filiformis. Gene Expr Patterns 2013;13(8): 464-472.##Utkina NK, Denisenko VA. Ophiuroidine, the first indolo [2,1-b] quinazoline alkaloid from the Caribbean brittle star Ophiocoma riisei. Tetrahedron Lett 2007;48 (25):4445-4447.##Kuppusamy P, Yusoff MM, Maniam GP, Govindan N. A case study- Regulation and functional mechanisms of cancer cells and control its activity using plants and their derivatives. J Pharm Res 2013;6(8):884-892.##Nam KS, Shon YH. Chemopreventive effects of polysaccharides extract from Asterina pectinifera on HT-29 human colon adenocarcinoma cells. BMB Rep 2009;31;42(5):277-280.##Albalasmeh AA, Berhe AA, Ghezzehei TA. A new method for rapid determination of carbohydrate and total carbon concentrations using UV spectrophotometry. Carbohydr Polym 2013;97(2):253-261.##Ratna D, Simanjuntak P, Abdillah S, Heffen WL. Apoptosis of human breast cancer cells induced by ethylacetate extracts of propolis. Am J Biotechs 2010;6(2):84-88.##Sarfaraj H, Sheeba F, Ansari S, Sajid Khan M. Marine natural products: A lead for anticancer. Indian J Mar Sci 2012;41(1):27-39.##Mousavi SH, Tayrani Najaran Z, Hersey P. Apoptosis: from signalling pathways to therapeutic tools. Iran J Basic Med Sci 2008;11(3):121-142.##Portt L, Norman G, Clapp C, Greenwood M, Greenwood MT. Anti-apoptosis and cell survival: a review. Biochim Biophys Acta 2011;1813(1):238-259.##Cao W, Li XQ, Wang X, Fan HT, Zhang XN, Hou Y, et al. A novel polysaccharide, isolated from Angelica sinensis (Oliv.) Diels induces the apoptosis of cervical cancer HeLa cells through an intrinsic apoptotic pathway. Phytomedicine 2010;17(8-9):598-605.##Lavi I, Friesem D, Geresh S, Hadar Y, Schwartz B. An aqueous polysaccharide extract from the edible mushroom Pleurotus ostreatus induces anti-proliferative and pro-apoptotic effects on HT-29 colon cancer cells. Cancer Lett 2006;244(1):61-70. ##Chen G, Zhang P, Huang T, Yu W, Lin J, Li P, et al. Polysaccharides from Rhizopus nigricans mycelia induced apoptosis and G2/M arrest in BGC-823 cells. Carbohydr Polym 2013;97(2):800-808.##Wu J, Zhou J, Lang Y, Yao L, Xu H, Shi H, et al. A Polysaccharide from Armillaria mellea exhibits strong in vitro anticancer activity via apoptosis-involved mechanisms. Int J Biol Macromol 2012;51(4):663-667.##Chen J, Yao D, Yuan H, Zhang S, Tian J, Guo W, et al. Dipsacus asperoides polysaccharide induces apoptosis in osteosarcoma cells by modulating the PI3K/Akt pathway. Carbohydr Polym 2013;95(2):780-784.##Gamal-Eldeen AM, Ahmed EF, Abo-zeid MA. In vitro cancer chemopreventive properties of polysaccharide extract from the brown alga, Sargassum latifolium. Food Chem Toxicol 2009;47(6):1378-1384.##Lee K, Shin J, Nam K. Cancer chemopreventive effects of starfish polysaccharide in human breast cancer cells. Biotech Bioprocess Eng 2011;16:987-991.##Wang Z, Lu C, Wu C, Xu M, Kou X, Kong D, et al. Polysaccharide of Boschniakia rossica induces apoptosis on laryngeal carcinoma Hep2 cells. Gene 2014;536 (1):203-206.##Lu X, Liu W, Wu J, Li M, Wang J, Wu J, et al. A poly-saccharide fraction of adlay seed (Coix lachryma jobi L.) induces apoptosis in human non-small cell lung cancer A549 cells. Biochem Biophys Res Commun 2013;430 (2):846-851.##Thangam R, Sathuvan M, Poongodi A, Suresh V, Pazhanichamy K, Sivasubramanian S, et al. Activation of intrinsic apoptotic signaling pathway in cancer cells by Cymbopogon citratus polysaccharide fractions. Carbohydr Polym 2014;107:138-150.##

Interferences in the Optimization of the MTT Assay for Viability Estimation of Proteus mirabilis 2 2 Background: The chromogenic assay based on MTT bioreduction was adapted to Proteus mirabilis viability estimations. We primarily intended to use the assay for the evaluation of novel antimicrobial compounds, including structures with possible permeabilizing activity. Therefore, the influence of basic permeabilizing agents like Triton X-100 and EDTA upon the MTT assay was studied. Methods: 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) was used as a substrate for the whole-cell dehydrogenase activity estimations. The amount of formazan product was evaluated in the end-point reactions terminated with acidic isopropanol or in the continuous reactions run in the presence of low detergent concentrations. Results: The generally established procedure of the end product dissolution with acidic isopropanol caused absorbance instability which strongly affected the results accuracy. The disadvantage was especially pronounced when the assay was conducted in Mueller-Hinton Broth. PBS with 0.01% Triton X-100 used as the reaction medium allowed to omit the formazan dissolution step and follow the microbial MTT reduction in a continuous mode. It was observed that in Proteus mirabilis with a compromised outer membrane the assay score was artificially increased above the untreated control. Conclusion: The dependence of the assay results on the cell integrity might be a major drawback of the MTT assay application for the evaluation of novel antimicrobials against Gram-negative microorganisms. On the other hand, the MTT reduction could be conveniently used to assay the permeabilization degree in biotechnological protocols. 159 165 Ewa Grela Department of Bioorganic Chemistry, Faculty of Chemistry, Wrocław University of Technology Department of Bioorganic Chemistry, Faculty of Chemistry, Wrocław University of Technology Poland Adam Ząbek Department of Bioorganic Chemistry, Faculty of Chemistry, Wrocław University of Technology Department of Bioorganic Chemistry, Faculty of Chemistry, Wrocław University of Technology Poland Agnieszka Grabowiecka Department of Bioorganic Chemistry, Faculty of Chemistry, Wrocław University of Technology Department of Bioorganic Chemistry, Faculty of Chemistry, Wrocław University of Technology Poland MTT assayProteus mirabilisViability 221.pdf Wang S, Yu H, Wickliffe JK. Limitations of the MTT and XTT assays for measuring cell viability due to superoxide formation induced by nano-scale TiO2. Toxicol in Vitro 2011;25(8):2147-2151.##Tsukatani T, Higuchi T, Suenaga H, Akao T, Ishiyama M, Ezoe T, et al. Colorimetric microbial viability assay based on reduction of water-soluble tetrazolium salts for antimicrobial susceptibility testing and screening of antimicrobial substances. Anal Biochem 2009;393(1):117-125.##Tachon S, Michelon D, Chambellon E, Cantonnet M, Mezange C, Henno L, et al. Experimental condition affect the site of tetrazolium violet reduction in the electron transport chain in Lactococcus lactis. Microbiology 2009;155(Pt 9):2941-2948.##Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immun Methods 1983;65(1-2):55-63.##Twentyman PR, Luscombe M. A study of some variables in a tetrazolium dye (MTT) based assay for cell growth and chemosensitivity. Br J Cancer 1987;56(3):279-285.##Kim E, Jeon IS, Kim JW, Kim J, Jung HS, Lee SJ. An MTT-based method for quantification of periodontal ligament cell viability. Oral Dis 2007;13(5):495-499.##Trafny EA, Lewandowski R, Zawistowska-Marciniak I, Stępińska M. Use of MTT assay for determination of the biofilm formation capacity of microorganisms in metalworking fluids. World J Microbiol Biotechnol 2013;29(9):1635-1643.##Carmichael J, DeGraff WG, Gazdar AF, Minna JD, Mitchell JB. Evaluation of a tetrazolium-based semiautomated colorimetric assay: assessment of chemosensitivity testing. Cancer Res 1987;47(4):936-942.##Denizot F, Lang R. Rapid colorimetric assay for cell growth and survival. Modifications to the tetrazolium dye procedure giving improved sensitivity and reliability. J Immunol Methods 1986;89(2):271-277.##Ciapetti G, Cenni E, Pratelli L, Pizzoferrato A. In vitro evaluation of cell biomaterial interaction by MTT assay. Biomaterials 1993;14(5):359-364.##Young FM, Phungtamdet W, Sanderson BJ. Modification of MTT assay conditions to examine the cytotoxic effects of amitraz on the human lymphoblastoid cell line, WIL2NS. Toxicol in Vitro 2005;19(8):1051-1059.##Wang H, Cheng H, Wang F, Wei D, Wang X. An improved 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction assay for evaluating the viability of Escherichia coli cells. J Microbiol Methods 2010;82(3):330-333.##Wu T, Hu W, Guo L, Finnegan M, Bradshaw DJ, Webster P, et al. Development of a new model system to study microbial colonization of dentures. J Prosthodont 2013;22(5):344-350.##Plumb JA. Cell sensitivity assays: the MTT assay. Methods Mol Med 2004;88:165-169.##Niles AL, Moravec RA, Riss LT. Update on in vitro cytotoxicity assays for drug development. Expert Opin Drug Discov 2008;3(6):655-669.##Tominaga H, Ishiyama M, Ohseto F, Sasamoto K, Hamamoto T, Suzuki K, et al. A water soluble tetrazolium salt useful for colorimetric cell viability assay. 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Association of Transforming Growth Factor Alpha Polymorphisms with Nonsyndromic Cleft Lip and Palate in Iranian Population 2 2 Background: Cleft lip with or without cleft palate (CL/P) is one of the most common congenital anomalies and the etiology of orofacial clefts is multifactorial. Transforming growth factor alpha (TGFA) is expressed at the medial edge epithelium of fusing palatal shelves during craniofacial development. In this study, the association of two important TGFA gene polymorphisms, BamHI (rs11466297) and RsaI (rs3732248), with CL/P was evaluated in an Iranian population. Methods: The frequencies of BamHI and RsaI variations were determined in 105 unrelated Iranian subjects with nonsyndromic CL/P and 218 control subjects using PCR and RFLP methods, and the results were compared with healthy controls. A p-value of <0.05 was considered statistically significant. Results: The BamHI AC genotype was significantly higher (p=0.016) in the patients (12.4%) than the control group (5.0%). The BamHI C allele was significantly higher (p=0.001; OR=3.4, 95% CI: 1.6-7.4) in the cases (8.0%) compared with the control group (2.5%). Conclusion: Our study showed that there was an association between the TGFA BamHI variation and nonsyndromic CL/P in Iranian population. 168 172 Asghar Ebadifar Dentofacial Deformities Research Center, Research Institute of Dental Sciences, Department of Orthodontic, Faculty of Dentistry, Shahid Behehsti University of Medical Sciences Dentofacial Deformities Research Center, Research Institute of Dental Sciences, Department of Orthodontic, Faculty of Dentistry, Shahid Behehsti University of Medical Sciences Iran Roya Hamedi Hamid Reza Khorram Khorshid Genetic Research Centre, University of Social Welfare and Rehabilitation Sciences Genetic Research Centre, University of Social Welfare and Rehabilitation Sciences Iran Kioomars Saliminejad Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Iran Koorosh Kamali Department of Public Health, Faculty of Public Health, Zanjan University of Medical Sciences Department of Public Health, Faculty of Public Health, Zanjan University of Medical Sciences Iran Fatemeh Aghakhani Moghadam Medical Laboratory Sciences, University of Social Welfare and Rehabilitation Sciences Medical Laboratory Sciences, University of Social Welfare and Rehabilitation Sciences Iran Nazanin Esmaeili Anvar Genetic Research Centre, University of Social Welfare and Rehabilitation Sciences Genetic Research Centre, University of Social Welfare and Rehabilitation Sciences Iran Nazila Ameli Department of Orthodontic, Faculty of Dentistry, Semnan University of Medical Sciences Department of Orthodontic, Faculty of Dentistry, Semnan University of Medical Sciences Iran Association StudyCleft lip/palatePolymorphismTransforming Growth Factor Alpha 222.pdf Mijiti A, Ling W, Guli, Moming A. Association of single-nucleotide polymorphisms in the IRF6 gene with non-syndromic cleft lip with or without cleft palate in the Xinjiang Uyghur population. Br J Oral Maxillofac Surg 2015;53(3):268-274.##Niranjane PP, Kamble RH, Diagavane SP, Shrivastav SS, Batra P, Vasudevan SD, et al. Current status of presurgical infant orthopaedic treatment for cleft lip and palate patients: A critical review. Indian J Plast Surg 2014;47(3):293-302.##Ranganathan K, Vercler CJ, Warschausky SA, MacEachern MP, Buchman SR, Waljee JF. Comparative effectiveness studies examining patient-reported outcomes among children with cleft lip and/or palate: a systematic review. Plast Reconstr Surg 2015;135(1):198-211.##Crockett DJ, Goudy SL. Cleft lip and palate. Facial Plast Surg Clin North Am 2014;22(4):573-586.##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.##Rajabian MH, Sherkat M. An epidemiologic study of oral clefts in Iran: analysis of 1,669 cases. Cleft Palate Craniofac J 2000;37(2):191-196.##Kalaskar R, Kalaskar A, Naqvi FS, Tawani GS, Walke DR. Prevalence and evaluation of environmental risk factors associated with cleft lip and palate in a central Indian population. Pediatr Dent 2013;35(3):279-283.##Ibarra-Lopez JJ, Duarte P, Antonio-Vejar V, Calderon-Aranda ES, Huerta-Beristain G, Flores-Alfaro E, et al. Maternal C677T MTHFR polymorphism and environmental factors are associated with cleft lip and palate in a Mexican population. J Investig Med 2013;61(6):1030-1035.##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.##Brissenden JE, Derynck R, Francke U. Mapping of transforming growth factor alpha gene on human chromosome 2 close to the breakpoint of the Burkitt's lymphoma t(2;8) variant translocation. Cancer Res 1985;45(11 Pt 2):5593-5597.##Tricoli JV, Nakai H, Byers MG, Rall LB, Bell GI, Shows TB. The gene for human transforming growth factor alpha is on the short arm of chromosome 2. Cytogenet Cell Genet 1986;42(1-2):94-98.##Nemo R, Murcia N, Dell KM. Transforming growth factor alpha (TGF-alpha) and other targets of tumor necrosis factor-alpha converting enzyme (TACE) in murine polycystic kidney disease. Pediatr Res 2005;57(5 Pt 1):732-737.##Mydlo JH, Michaeli J, Cordon-Cardo C, Goldenberg AS, Heston WD, Fair WR. Expression of transforming growth factor alpha and epidermal growth factor receptor messenger RNA in neoplastic and nonneoplastic human kidney tissue. Cancer Res 1989;49(12):3407-3411.##Beaty TH, Hetmanski JB, Zeiger JS, Fan YT, Liang KY, VanderKolk CA, et al. Testing candidate genes for non-syndromic oral clefts using a case-parent trio design. Genet Epidemiol 2002;22(1):1-11.##Dixon MJ, Ferguson MW. The effects of epidermal growth factor, transforming growth factors alpha and beta and platelet-derived growth factor on murine palatal shelves in organ culture. Arch Oral Biol 1992;37(5):395-410.##Mossey PA, Little J, Munger RG, Dixon MJ, Shaw WC. Cleft lip and palate. Lancet 2009;374(9703):1773-1785.##Mitchell LE. Transforming growth factor alpha locus and nonsyndromic cleft lip with or without cleft palate: a reappraisal. Genet Epidemiol 1997;14(3):231-240.##Vieira AR, Orioli IM. Candidate genes for nonsyndromic cleft lip and palate. ASDC J Dent Child 2001;68(4):272-279.##Shiang R, Lidral AC, Ardinger HH, Buetow KH, Romitti PA, Munger RG, et al. Association of transforming growth-factor alpha gene polymorphisms with nonsyndromic cleft palate only (CPO). Am J Hum Genet 1993;53(4):836-843.##Machida J, Yoshiura Ki, Funkhauser CD, Natsume N, Kawai T, Murray JC. Transforming growth factor-alpha (TGFA): genomic structure, boundary sequences, and mutation analysis in nonsyndromic cleft lip/palate and cleft palate only. Genomics 1999;61(3):237-242.##Qian JF, Feingold J, Stoll C, May E. Transforming growth factor-alpha: characterization of the BamHI, RsaI, and TaqI polymorphic regions. Am J Hum Genet 1993;53(1):168-175.##Vanderas AP. Incidence of cleft lip, cleft palate, and cleft lip and palate among races: a review. Cleft Palate J 1987;24(3):216-225.##Holder SE, Vintiner GM, Farren B, Malcolm S, Winter RM. Confirmation of an association between RFLPs at the transforming growth factor-alpha locus and non-syndromic cleft lip and palate. J Med Genet 1992;29 (6):390-392.##Stoll C, Qian JF, Feingold J, Sauvage P, May E. Genetic variation in transforming growth factor alpha: possible association of BamHI polymorphism with bilateral sporadic cleft lip and palate. Hum Genet 1993;92(1):81-82.##Jugessur A, Lie RT, Wilcox AJ, Murray JC, Taylor JA, Saugstad OD, et al. Cleft palate, transforming growth factor alpha gene variants, and maternal exposures: assessing gene-environment interactions in case-parent triads. Genet Epidemiol 2003;25(4):367-374.##Basart AM, Qian JF, May E, Murray JC. A PCR method for detecting polymorphism in the TGFA gene. Hum Mol Genet 1994;3(4):678.##Jara L, Blanco R, Chiffelle I, Palomino H, Carreño H. Evidence for an association between RFLPs at the transforming growth factor alpha (locus) and nonsyndromic cleft lip/palate in a South American population. Am J Hum Genet 1995;56(1):339-341.##Jara L, Blanco R, Chiffelle I, Palomino H, Curtis D. [Cleft lip and palate in the Chilean population: association with BamH1 polymorphism of the transforming growth factor alpha (TGFA) gene]. Rev Med Chil 1993;121(4):390-395. Spanish.##Tanabe A, Taketani S, Endo-Ichikawa Y, Tokunaga R, Ogawa Y, Hiramoto M. Analysis of the candidate genes responsible for non-syndromic cleft lip and palate in Japanese people. Clin Sci (Lond) 2000;99(2):105-111.##Vieira AR. Association between the transforming growth factor alpha gene and nonsyndromic oral clefts: a HuGE review. Am J Epidemiol 2006;163(9):790-810.##Jawdat NG, Adnan FN, Akeel HA. Simple salting – out method for genomic DNA extraction from whole blood. Tikrit J Pure Sci 2011;16(2):9-11.##Ardinger HH, Buetow KH, Bell GI, Bardach J, VanDemark DR, Murray JC. Association of genetic variation of the transforming growth factor-alpha gene with cleft lip and palate. Am J Hum Genet 1989;45(3):348-353.##Chenevix-Trench G, Jones K, Green AC, Duffy DL, Martin NG. Cleft lip with or without cleft palate: associations with transforming growth factor alpha and retinoic acid receptor loci. Am J Hum Genet 1992;51(6):1377-1385.##Lidral AC, Murray JC, Buetow KH, Basart AM, Schearer H, Shiang R, et al. Studies of the candidate genes TGFB2, MSX1, TGFA, and TGFB3 in the etiology of cleft lip and palate in the Philippines. Cleft Palate Craniofac J 1997;34(1):1-6.##

Association between Serum Paraoxonase 1 Activities (PONase/AREase) and L55M Polymorphism in Risk of Female Infertility 2 2 Background: The risk of developing female infertility has been associated with gene polymorphisms that decrease the activity of enzymes involved in systemic Oxidative Stress (OS). In this study, PON1 L55M polymorphism for association with susceptibility to infertility was investigated among Iranian female population. Methods: Samples from 120 Iranian females [20 endometriosis; 30 Polycystic Ovary Syndrome (PCO); 70 controls] were analyzed and PCR-RFLP assay was used to determine the PON1 rs854560 (L55M) frequencies. The paraoxonase (PONase) and arilesterase (AREase) activities of PON1 enzyme were also assessed in order to investigate the association between serum PON1 activities, female infertility, and PON1 L55M polymorphism. Results: The women with a MM genotype (p=0.021; OR=2.55) showed more possibilities of experiencing infertility than those with a LM genotype (p=0.039; OR=1.91). According to LSD test, endometriosis subjects had significantly lower paraoxonase enzyme activity compared to control group (p=0.0024; CI=95%). No significant difference was found in women with PCOS for both PONase and AREase activity in comparison with control group (p=0.469; CI=95%). Furthermore, PON1 activities were the highest in LL genotype followed by LM and then MM genotype (MM<LM<LL) in both patients and controls. Conclusion: PON1 L55M polymorphism may be associated with serum PON1 activity and the risk of developing female infertility. 173 178 Majid Motovali-Bashi Saeid Sedaghat Genetics Division, Department of Biology, Faculty of Sciences, University of Isfahan Genetics Division, Department of Biology, Faculty of Sciences, University of Isfahan Iran Fariba Dehghanian Genetics Division, Department of Biology, Faculty of Sciences, University of Isfahan Genetics Division, Department of Biology, Faculty of Sciences, University of Isfahan Iran EndometriosisInfertilityPolycystic ovary syndrome 223.pdf Gnoth C, Godehardt E, Frank-Herrmann P, Friol K, Tigges Jr, Freundl G. Definition and prevalence of subfertility and infertility. Hum Reprod 2005;20(5): 1144-1147.##Deligeoroglou E, Kouskouti C, Christopoulos P. The role of genes in the polycystic ovary syndrome: predisposition and mechanisms. Gynecol Endocrinol 2009;25(9):603-609.##Richter RJ, Jarvik GP, Furlong CE. Paraoxonase 1 status as a risk factor for disease or exposure. Adv Med Biol 2010;660:29-35.##Bragatto FB, Barbosa CP, Christofolini DM, Peluso C, dos Santos AA, Mafra FA, et al. There is no relationship between Paraoxonase serum level activity in women with endometriosis and the stage of the disease: an observational study. Reprod Health 2013;10:32.##Augoulea A, Alexandrou A, Creatsa M, Vrachnis N, Lambrinoudaki I. Pathogenesis of endometriosis: the role of genetics, inflammation and oxidative stress. Arch Gynecol Obstet 2012;286(1):99-103.##Ji G, Gu A, Wang Y, Huang C, Hu F, Zhou Y, et al. Genetic variants in antioxidant genes are associated with sperm DNA damage and risk of male infertility in a Chinese population. Free Rad Biol Med 2012;52(4):775-780.##Yeon Lee J, Baw C-K, Gupta S, Aziz N, Agarwal A. Role of oxidative stress in polycystic ovary syndrome. Curr Womens Health Rev 2010;6(2):96-107.##H Sekhon L, Gupta S, Kim Y, Agarwal A. Female infertility and antioxidants. Curr Womens Health Rev 2010;6(2):84-95.##Goswami B, Tayal D, Gupta N, Mallika V. Paraoxonase: a multifaceted biomolecule. Clin Chim Acta 2009;410 (1):1-12.##bin Ali A, Zhang Q, Lim YK, Fang D, Retnam L, Lim SK. Expression of major HDL-associated antioxidant PON-1 is gender dependent and regulated during inflammation. Free Radic Biol Med 2003;34(7):824-829.##Camps J, Marsillach J, Joven J. The paraoxonases: role in human diseases and methodological difficulties in measurement. Crit Rev Lab Sci 2009;46(2):83-106.##Précourt LP, Amre D, Denis MC, Lavoie JC, Delvin E, Seidman E, et al. The three-gene paraoxonase family: physiologic roles, actions and regulation. Atherosclerosis 2011;214(1):20-36.##Kokouva M, Koureas M, Dardiotis E, Almpanidou P, Kalogeraki A, Kyriakou D, et al. Relationship between the paraoxonase 1 (PON1) M55L and Q192R polymorphisms and lymphohaematopoietic cancers in a Greek agricultural population. Toxicology 2013;307:12-16.##Ferk P, Gersak K. Association of -108 C>T PON1 polymorphism with polycystic ovary syndrome. Biomed Rep 2014;2(2):255-259.##Abu Farha M, Fawzy M, Abdel Wahab A, Rashed L, Farouq A. Relation between paraoxonase 1 (PON1) gene polymorphism insulin resistance in polycystic ovary syndrome (PCOS). Kasr Al-Aini J Obstet & Gynecol 2011;1(2):75-82.##Homburg R. The management of infertility associated with polycystic ovary syndrome. Reprod Biol Endocrinol 2003;1:109.##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.##Sumegova K, Nagyova Z, Waczulikova I, Zitnanova I, Durackova Z. Activity of paraoxonase 1 and lipid profile in healthy children. Physiol Res 2007;56(3):351-357.##Augoulea A, Mastorakos G, Lambrinoudaki I, Christodoulakos G, Creatsas G. The role of the oxidative-stress in the endometriosis-related infertility. Gynecol Endocrinol 2009;25(2):75-81.##Gur M, Aslan M, Yildiz A, Demirbag R, Yilmaz R, Selek S, et al. Paraoxonase and arylesterase activities in coronary artery disease. Eur J Clin Invest 2006;36(11): 779-787.##Marsillach J, Camps J, Beltran-Debon R, Rull A, Aragones G, MaestreMartnez C, et al. Immunohistochemical analysis of paraoxonases-1 and 3 in human atheromatous plaques. Eur J Clin Invest 2011;41(3):308-314.##Ozenoglu A, Balci H, Ugurlu S, Caglar E, Uzun H, Sarkis C, et al. The relationships of leptin, adiponectin levels and paraoxonase activity with metabolic and cardiovascular risk factors in females treated with psychiatric drugs. Clinics (Sao Paulo) 2008;63(5):651-660.##Ikeda Y, Suehiro T, Inoue M, Nakauchi Y, Morita T, Arii K, et al. Serum paraoxonase activity and its relationship to diabetic complications in patients with non-insulin-dependent diabetes mellitus. Metabolism 1998;47(5): 598-602.##Elkiran ET, Mar N, Aygen B, Gursu F, Karaoglu A, Koca S. Serum paraoxonase and arylesterase activities in patients with lung cancer in a Turkish population. BMC cancer 2007;7(1):48.##Ginsberg G, Neafsey P, Hattis D, Guyton KZ, Johns DO, Sonawane B. Genetic polymorphism in paraoxonase 1 (PON1): Population distribution of PON1 activity. J Toxicol Environ Health B Crit Rev 2009;12(5-6):473-507.##Agarwal A, Gupta S, Sharma R. Oxidative stress and its implications in female infertility-a clinician's perspective. Reprod Biomed Online 2005;11(5):641-650.##Jackson LW, Schisterman EF, Dey-Rao R, Browne R, Armstrong D. Oxidative stress and endometriosis. Hum Reprod 2005;20(7):2014-2020.##Agarwal A, Gupta S, Sharma RK. Role of oxidative stress in female reproduction. Reprod Biol Endocrinol 2005;3:28.##Verit FF, Erel O, Celik N. Serum paraoxonase-1 activity in women with endometriosis and its relationship with the stage of the disease. Hum Reprod 2008;23(1): 100-104.##Dursun P, Demirtaş E, Bayrak A, Yarali H. 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Construction of CTLA-4-Ig Fusion Gene in pBudCE4.1 Expression Vector 2 2 Background: CTLA-4 inhibitory signals prevent cell cycle progression and IL-2 production, leading to a halt on an ongoing immune response. CTLA4-Ig fusion proteins contain the extracellular domain of CTLA-4 and Fc fragment of human IgG antibody. In this study we aimed to fuse the ctla-4 gene encoding the extracellular domain of CTLA-4 molecule with igg1 gene encoding Fc region of human IgG. Methods: After primer design, PCR reaction was performed using pfu polymerase enzyme and specific primers. PCR amplified fragment was ligated into the vector containing the human igg1 gene. The resulting fusion fragment of ctla-4 and human igg1 genes was ligated to pBudCE4.1 expression vector. Results: Extracellular domain of ctla-4 gene was ligated to igg1 gene and then ctla4-ig fragment was cloned into pBudCE4.1 vector. Construction of the expression vector was confirmed by restriction pattern analysis and sequencing. Conclusion: By confirming the construct, in the next step, the recombinant DNA will be used to produce CTLA4-Ig recombinant protein for the clinical uses. 179 181 Mahsa Yazdanpanah-Samani Elham Mahmoudi Maymand Tayebeh Jahangeerfam Abbas Ghaderi AbataceptCTLA-4 antigenCTLA4-IgRecombinant DNA 224.pdf Zhu S, Liu S, Wan L, Yang G, Yang H, Cheng J, et al. Molecular cloning, expression and characterization of the functional domain of CTLA4 from the rhesus monkey, Macaca mulatta. Dev Comp Immunol 2011;35(7):736-744.##Fife BT, Bluestone JA. Control of peripheral T-cell tolerance and autoimmunity via the CTLA-4 and PD-1 pathways. Immunol Rev 2008;224:166-182.##Grosso JF, Jure-Kunkel MN. CTLA-4 blockade in tumor models: an overview of preclinical and translational research. Cancer Immun 2013;13:5.##Chambers CA, Kuhns MS, Egen JG, Allison JP. CTLA-4-mediated inhibition in regulation of T cell responses: mechanisms and manipulation in tumor immunotherapy. Annu Rev Immunol 2001;19:565-594.##Chikuma S, Bluestone JA. CTLA-4 and tolerance: the biochemical point of view. Immunol Res 2003;28 (3):241-253.##Egen JG, Kuhns MS, Allison JP. CTLA-4: new insights into its biological function and use in tumor immunotherapy. Nat immunol 2002;3(7):611-618.##Bluestone JA, St Clair EW, Turka LA. CTLA4Ig: bridging the basic immunology with clinical application. Immunity 2006;24(3):233-238.##Riella LV, Liu T, Yang J, Chock S, Shimizu T, Mfarrej B, et al. Deleterious effect of CTLA4-Ig on a Treg-dependent transplant model. Am J Transplant 2012;12 (4):846-855.##Sambrook J, Russell DW. Molecular Cloning: A Laboratory Manual. 3rd ed. New York:Cold Spring Harbor Laboratory Press; 2001. P. 2100.##Silva MV, Machado JR, Rocha LP, Castellano LR, Reis MA, Corrêa RR. CD28 family and chronic rejection: "to belatacept...And beyond!". J Transplant 2012;2012: 203780.##Thompson RH, Allison JP, Kwon ED. Anti-cytotoxic T lymphocyte antigen-4 (CTLA-4) immunotherapy for the treatment of prostate cancer. Urol Oncol 2006;24(5):442-447.##Ippoliti G, D'Armini AM, Lucioni M, Marjieh M, Viganò M. Introduction to the use of belatacept: a fusion protein for the prevention of posttransplant kidney rejection. Biologics 2012;6:355-362.##Chistiakov DA, Turakulov RI. CTLA-4 and its role in autoimmune thyroid disease. J Mol Endocrinol 2003;31 (1):21-36.##Janeway Ch. Immunobiology Five: Garland Pub.; 2001.##