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RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, BANGALORE, KARNATAKA ANNEXURE II PROFORMA FOR REGISTRATION OF SUBJECT FOR DISSERTATION 1. NAME OF THE MADHUR CANDIDATE AND P.M.N.M. DENTAL COLLEGE AND ADDRESS HOSPITAL BAGALKOT – 587101 KARNATAKA 2. NAME OF THE P.M.N.M. DENTAL COLLEGE AND INSTITUTION HOSPITAL, BAGALKOT – 587101 KARNATAKA 3. 4. COURSE OF THE M.D.S(MASTER OF DENTAL STUDY AND SURGERY) SUBJECT PERIODONTICS DATE OF ADMISSION TO 24-05-2012 THE COURSE 5. TITLE OF THE TOPIC: “EVALUATION OF RORC2 PROTEIN LEVELS IN CHRONIC AND AGGRESSIVE PERIODONTITIS”:AN IMMUNOHISTOCHEMICAL STUDY. 6 BRIEF RESUME OF THE INTENDED WORK 6.1 Need for study Periodontitis is a chronic disease of the tooth supporting tissues which is characterized by gingival inflammation and alveolar bone loss. Although oral bacterial infection is a major factor of periodontitis, its progression and severity depends upon interplay between genetic and environmental factors. Bacterial products such as lipopolysaccharides, leads to the stimulation of host cells resulting in the release of cytokines, which play an important role in mediating and controlling cellular interactions. T cell antigen recognition is a function of the T cell antigen receptor (TCR). CD8 and CD4 are T cell co-receptors, whose recognition of Major Histocompatibility Complex classI & II, respectively on APCs is essential for T cell activation and function. CD4+T lymphocytes represent one of the main components of the adaptive immune response and are the predominant cell type present in periodontitis and gingival tissues. After antigenic stimulation, naive CD4+T cells proliferate and may differentiate into distinct effector subsets, which have been classically divided on the basis of their cytokine production profiles into T helper (Th)1 and (Th)2 cells.6 Th1 cells induce cellular immunity with the production of interleukin IL-2 and interferon γ. In contrast, Th2 cells produce IL-4, IL-5, IL-10 and IL-13 and favor B-cell mediated humoral immunity.1 Recently, two new subsets of CD4+ T lymphocytes have been characterized, the Th17 subset, which follows different polarizing conditions and displays different functional activities than Th1 and Th2 cells, and the regulatory T(Treg) cell subset with suppressor functions. Activated human Th17 cells are phenotypically identified as CCR2+CCR5-, where as human memory CD4+ T cells producing IL-17 and expressing transcription factor related to orphan nuclear receptor C2 (RORC2) mRNA are CCR6+CCR4+.6 RORC2 belongs to the family of RORs that consists of three members: RORA, RORB, and RORC (NF1F3, RORg, RZRg). All ROR genes generate several isoforms, which differ only in their N termini. Proteins of the ROR family display a typical nuclear receptor domain structure consisting of four domains: an N-terminal domain,a highly conserved DNA-binding domain (DBD), a hinge domain, and a C terminal ligand-binding domain.3 RAR-related orphan receptor gamma (RORγ) is a protein that in humans is encoded by the RORC (RAR-related orphan receptor C) gene. Two isoforms are produced from the same RORC gene, probably by selection of alternative promoters- RORγ and RORγt. RORγ (also referred to as RORγ1), which is produced from an mRNA containing exons 1 to 11,7 is a member of the nuclear receptor family of transcription factors which plays an important role in muscle, thymocyte development, heart, liver and maintaining homeostasis. RORγt (also known as RORγ2) is produced from an mRNA identical to that of RORγ , except that the two 5'-most exons are replaced by an alternative exon, located downstream in the gene. This causes a different, shorter N-terminus. This transcription factor is essential for lymphoid organogenesis, in particular lymph nodes and Peyer's patches. But its best understood functionality is in the immune system by inhibiting apoptosis of undifferentiated T cells and promoting their differentiation into Th17 cells, possibly by down regulating the expression of Fas ligand and IL2, respectively.9 RORγt inhibitors are under development for the treatment of autoimmune diseases such as psoriasis and rheumatoid arthritis. Significantly higher levels of RORC2 protein expression was seen in chronic periodontitis patients, which is a master switch that initiates a wide range of phenotypic and functional programming during Th17 cell differentiation.4 It has been established that IL-17, the cytokine of Th17 is present in aggressive periodontitis patients. Till this date, no study has been done revealing the expression of RORC2 protein in aggressive periodontitis patients and comparing RORC2 protein levels in healthy, aggressive and chronic periodontitis patients. This study could add in to the ever challenging growth of research in the field of genetics and periodontal diseases. 6.2 Review of literature A study was undertaken in the human system Manel et al which showed that similar to mouse naive T cells, forced over-expression of RORC in human naive T cells induced a Th17-like phenotype, by inducing IL-17A, IL-17F, IL-26 and CCR6 expression and down-regulating IFN-γ secretion.8 A study was carried out which showed the coordinated temporal downregulation of Gata3 with the increased expression of RORC2 done under Th17 skewing conditions, indicating that RORC2 can provide direct regulation of Th2 responses.The differentiation of T cell subsets is dependent upon the expression of specific transcription factors. The expression of RORC2 that demarcates Th17 cells was significantly upregulated in the presence of Dll4. The simultaneous decrease in Gata3 expression, the signature regulatory transcription factor for Th2 cytokines, displayed no additional alteration in the presence of Dll4 under Th17 skewing conditions. It demonstrated that Dll4 down-regulates Gata3 under Th2 skewing conditions.5 One more study demonstrated repression of FOXP3 by the Th17 cellpromoting transcription factor RORC2 and thus identified a novel role for RORC2 in T cell polarization. ELISA-based system was used to detect the binding of RORC2 to RORE 2942 and to a lesser extent to RORE+115. ROREs consist of the DNA sequence GGTCA as a core motive and are often preceded by an A/T-rich sequence. Using a ChIP assay, they demonstrated that interaction of RORC2 and the promoter region of FOXP3 occurs in vivo. Experiments revealed that repression of FOXP3 by RORC2 involved physical binding of RORC2 to two ROREs on its promoter, a mechanism commonly observed for ROR family members. Overexpression of RORC2 decreased the levels of FOXP3 on both the mRNA and protein levels. This relationship was confirmed by siRNA-mediated knockdown of RORC2, which increased FOXP3 expression, indicating an inverse correlation of the two factors.3 A study was underlined to know the elevated levels of IL-17 in the systemic circulation of patients with periodontitis. IL-17 was present in sera from a significant proportion of systemically healthy patients with aggressive periodontitis compared with healthy controls. Secretion of Th 17 cytokines such as IL-17 and IL-22 subsequently induces production of inflammatory cytokines, chemokines and matrix metalloproteinases that contribute to pathology typical of affected tissues. So, it is reasonable to speculate that Th17 cells and IL-17 may play a role in the pathology of aggressive periodontitis patients. 2 Recently Adibrad et al. (2012) demonstrated a significant increase in the number of some specific markers of Th17 cells in patients suffering from periodontal disease in comparison with normal control subjects. Study was done to evaluate protein levels of IL-17A and RORC2 by immunohistochemistry which showed a positive correlation between the SID score of IL-17A and RORC2 in periodontal lesion sites. It also showed a positive correlation between gene expression of IL-17 and RANKL with RORC2 in periodontal lesions.1 6.3 Aims & Objectives 1. To evaluate the protein levels of RORC2 in gingival tissues of subjects with healthy periodontium, with moderate to severe chronic periodontitis and with aggressive periodontitis subjects. 2. To compare the protein levels of RORC2 among periodontally healthy, moderate to severe chronic and aggressive periodontitis patients. 7 MATERIALS AND METHODS 7.1 Source of data Samples will be randomly selected from the out patient section of Department of Periodontics, PMNM Dental College and Hospital, Bagalkot 7.2 Methods of collection of data This case control study will be carried out on randomly selected 30 subjects with healthy periodontium, 30 patients having chronic periodontitis and 30 patients having aggressive periodontitis. Gingival tissue samples from the selected subjects will be obtained for the analysis of RORC2 protein levels. Samples will be grouped as follows: Group I: 30 gingival tissue samples from subjects with healthy periodontium. Group II :30 gingival tissue samples from patients with chronic periodontitis subjects. Group III:30 gingival tissue samples from patients with aggressive periodontitis subjects. CLINICAL EXAMINATION TO ASSESS THE PERIODONTAL CONDITION: 1) Gingival index (Loe & Silness,1963) 2) Clinical attachment loss (CAL) 3) Probing Pocket Depth (PPD) Clinical assessments using the above mentioned parameters will be performed. Samples will be collected on the subsequent day. Gingival tissue specimens for group I will be obtained during extraction of tooth due to orthodontic treatment reasons and during third molar extraction. For groups II and III, specimens will be obtained from periodontally compromised teeth indicated for extraction. Specimens obtained will be placed in buffer solution and will be sent to laboratory for immunohistochemical evaluation of RORC2 protein levels. INCLUSION CRITERIA Group 1: Healthy controls 1. Good oral hygiene. 2. Probing depth (PD) ≤ 3mm. 3. Clinical attachment loss (CAL) ≤ 1mm. 4. Healthy gingival condition with a gingival index (GI) score <1. Group 2: Chronic Periodontitis 1. Presence of at least 20 natural teeth. 2. Minimum of six teeth with periodontal pockets of PD >5mm. 2. Minimum of six teeth with CAL ≥3mm. 3. Radiographic evidence of alveolar bone loss on at least 14 teeth excluding the third molars. Group3: Aggressive periodontitis 1. Absence of large accumulations of plaque and calculus. 2. Rapid rate of disease progression in an otherwise healthy individual. 3. Generalized interproximal attachment loss of > 5mm affecting at least 3 permanent teeth other than first molars and incisors, bone loss of > 50% of the root length as assessed by radiographs. EXCLUSION CRITERIA Smoker Systemic diseases like diabetes mellitus, hepatitis, and HIV infection etc known to influence the periodontal disease. Diseases of oral hard and soft tissue except caries and periodontitis. Use of antibiotics and analgesics within three months prior to study. Pregnant and lactating female. Periodonal therapy 6 months prior to study. All potential participants will be explained about the need and design of the study. Only those subjects who will give written consent for the study will be included. DURATION OF STUDY: The study will be carried for 1 ½ years. LABORATORY INVESTIGATIONS Gingival tissue specimens will be sent to the laboratory for detecting protein levels of RORC2 through immunehistochemical technique. STATISTICAL ANALYSIS The data so gathered from clinical examination and lab investigation will be subjected to statistical analysis. The results will be statistically analyzed using the following methods: One way ANOVA- It is used to compare values among three groups. t-test or Mann-Whitney test- It is used to compare values among two groups. Chi-square test (if required) Other statistical methods may be used accordingly, if required. 7.3 DOES THE STUDY REQUIRE ANY INVESTIGATIONS OR INTERVENTIONS TO BE CONDUCTED IN THE PATIENTS? Yes, gingival tissue samples have to be obtained from the patients. 7.4 HAS ETHICAL CLEARANCE BEEN OBTAINED FROM THE INSTITUTION? Yes, the copy of ethical clearance certificate is attached. 8. REFERENCES 1) Adibrad M, Deyhimi P, Ganjalikhani HM, Behfarnia P, Shahabuei M, Rafiee L. Signs of the presence of Th17 cells in chronic periodontal disease. J Periodontal Res 2012;47:525–531. 2) Schenkein HA, Koertge TE, Brooks CN, Sabatini R, Purkall DE, Tew JG. IL-17 in Sera from Patients with Aggressive Periodontitis. J Dent Res 2010;89(9):943-947. 3) Burgler S, Mantel P, Bassin C, Ouaked N, Akdis CA, Schmidt Weber CB.RORC2 Is Involved in T Cell Polarization through Interaction with the FOXP3 Promoter.J Immunol 2010;184:61616169. 4) Unutmaz D. RORC2: The master of human Th17 cell programming. Eur J Immunol 2009;39:1452–1455. 5) Mukherjee S, Schaller MA, Neupane R , Kunkel SL, Lukacs NW. Regulation of T Cell Activation by Notch Ligand, DLL4 promotes IL-17 Production and Rorc Activation. J Immunol 2009;182:73817388. 6) Hernandez M, Vernal R, Sorsa T, Tervahartiala T, Mantyla P, Gamonal J. Pathogenesis and treatment of Periodontitis. Chile,Finland:Intech:2012.p.33-54. 7) Medvedev A, Chistokhina A, Hirose T, Jetten AM. Genomic Structure and Chromosomal Mapping of the Nuclear Orphan Receptor RORg (RORC) Gene. Genomics 1997 Nov;46(1):93–102. 8) Manel N, Unutmaz D and Littman DR. The differentiation of humanT(H)-17 cells requires transforming growth factor-beta and induction of the nuclear receptor RORγt. Nat. Immunol 2008;9:641– 649. 9) He YW, Deftos ML, Ojala EW, Beva MJ. RORγt, a Novel Isoform of an Orphan Receptor Negatively Regulates Fas Ligand Expression and IL-2 Production in T Cells. Immunity 1998 Dec;9(6):797–806. 9. SIGNATURE OF THE CANDIDATE: 10. REMARKS OF THE GUIDE: 10.1 NAME & DESIGNATION OF GUIDE: Dr. SHIVARAJ WARAD PROFESSOR AND HOD, DEPT. OF PERIODONTICS, P.M.N.M. DENTAL COLLEGE AND HOSPITAL, BAGALKOT, KARNATAKA 11.1 SIGNATURE OF GUIDE : 11.2 NAME & DESIGNATION OF HEAD: Dr. SHIVARAJ WARAD PROFESSOR AND HEAD, DEPT. OF PERIODONTICS, P.M.N.M. DENTAL COLLEGE AND HOSPITAL, BAGALKOT, KARNATAKA 11.3 SIGNATURE OF HEAD: 12. REMARKS OF THE PRINCIPAL: 12.1 SIGNATURE OF PRINCIPAL: Dr. SHREENIVAS S. VANAKI PRINCIPAL, P.M.N.M.DENTAL COLLEGE & HOSPITAL BAGALKOT, KARNATAKA