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RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, BANGALORE, KARNATAKA. ANNEXURE – II PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION 1. NAME OF THE CANDIDATE AND ADDRESS DR. SURABHI GIGRAS, POST GRADUATE STUDENT, DEPARTMENT OF PERIODONTICS, K.L.E. SOCIETY’S INSTITUTE OF DENTAL SCIENCES, BANGALORE – 560022. 2. NAME OF THE INSTITUTION K.L.E. SOCIETY’S INSTITUTE OF DENTAL SCIENCES, BANGALORE – 560022 3. COURSE OF THE STUDY AND SUBJECT MASTER OF DENTAL SURGERY (MDS) IN PERIODONTICS. 4. DATE OF ADMISSION TO COURSE 29/05/2013 5. TITLE OF THE DISSERTATION GINGIVAL CREVICULAR FLUID LEVELS OF TRIGGERING RECEPTOR EXPRESSED ON MYELOID CELLS, (TREM)-1 IN GINGIVITIS AND CHRONIC PERIODONTITIS BEFORE AND AFTER PHASE –I THERAPY- A CASE-CONTROL STUDY. , BRIEF RESUME OF THE INTENDED WORK: 6.1 NEED FOR THE STUDY: Periodontal diseases are the most common infections in humans caused by complex polymicrobial biofilms attaching on the tooth surface and causing inflammation of the periodontal tissues.1 Gingivitis and chronic periodontitis are the two most common forms of periodontal disease. Gingivitis is a reversible non-destructive periodontal disease that does not involve the loss of periodontal tissues. Chronic periodontitis is characterized by impaired immune responses resulting in connective tissue and alveolar bone destruction. An intermediate mechanism that lies between bacterial stimulation and tissue destruction is the production of pro-inflammatory cytokines which stimulate the inflammatory events.2 Triggering receptor expressed on myeloid cells (TREM)-1 is a cell surface receptor of the immunoglobulin superfamily, which is constitutively expressed on the surface of human polymorphonuclear neutrophils and monocytes.3 This, along with its adaptor signalling molecule DAP12, is activated upon bacterial recognition by host cells via toll like receptors, triggering a number of intracellular signalling events that result in enhanced pro-inflammatory cytokine production.1 Bacterial or fungal infections can cause upregulation of membrane bound TREM-1, as well as release its soluble form, rendering it a useful early inflammatory biomarker for systemic infection.1 Since TREM-1 amplifies the inflammatory response induced by pathogenic bacteria, it may serve as a useful marker for detecting the progression of periodontal lesions.3 Gingival crevicular fluid (GCF) is a transudate originating from the gingival plexus of blood vessels in the gingival connective tissue, close to the epithelial lining of the dento-gingival sulcus.4 It contains an array of measurable inflammatory mediators, which may result from interaction between the host and microbial challenge. Host response mediators in GCF are indicators of the processes occurring in the gingival crevice and adjoining host tissue and may be useful as early risk markers of the disease.5 GCF has been therefore regarded as a promising medium for the detection of periodontal disease activity. The purpose of this study is to estimate GCF levels of TREM-1 in periodontally healthy, gingivitis and chronic periodontitis subjects and evaluate the effect of scaling and root planing (SRP) on the same in periodontal disease. 6.2 REVIEW OF LITERATURE: A study was done to investigate the effect of Porphyromonas gingivalis on the expression of TREM-1/DAP12 pathway, as well as its engagement in pro-inflammatory cytokine production. The results showed that Porphyromonas gingivalis enhanced TREM-1 gene expression by the cells, concomitantly to an increase in soluble TREM-1 (sTREM)-1 secretion. TREM-1 also potentiated pro-inflammatory cytokine responses to Porphyromonas gingivalis as shown by an enhanced IL-1β and IL-6 secretion.6 A Case control study was done to evaluate the presence of TREM-1 in GCF of patients with severe periodontitis. The results showed that sTREM-1 was detected in crevicular fluid and its concentration was higher in pathological sites. They concluded that sTREM-1 could be a marker of periodontal tissue destruction.3 A study was done to investigate the effect of subantimicrobial doses of doxycycline (SDD) on Porphyromonas gingivalis induced TREM-1 expression and secretion by the myelomonocytic cell line MonoMac-6. After 24 hours of challenge, Porphyromonas gingivalis enhanced TREM-1 gene expression by the cells, with a concomitant increase in sTREM-1 release. It was concluded that SDD inhibits bacterially induced TREM-1 and this effect may partly account for its generalized anti-inflammatory properties.7 A cross-sectional study was done to investigate the GCF sTREM-1 levels in chronic periodontitis (CP) or generalized aggressive periodontitis (G-AgP), and their association with the levels of key periodontal pathogens in subgingival plaque. Soluble TREM-1 levels in GCF were found to be higher in CP and G-Agp than healthy sites. Also sTREM-1 levels were positively correlated with the clinical and microbiological parameters.8 An in vitro study was conducted to investigate the effect of Porphyromonas gingivalis on TREM-1 expression and production by primary human Polymorphonuclear neutrophils, and also to evaluate the role of the gingipains in this process. The results showed that after 4 hours of challenge, Porphyromonas gingivalis enhanced TREM-1 expression as identified by quantitative real time polymerase chain reaction (PCR). It was concluded that the differential regulation of TREM-1 by gingipains may present a novel mechanism by which Porphyromonas gingivalis manipulates the host innate immune response helping to establish chronic periodontal inflammation.1 A study was conducted to estimate the sTREM-1 concentration in saliva and serum of patients with chronic or generalized aggressive periodontitis. The results showed that the sTREM-1 concentration, both in saliva and serum was found to be higher in the periodontitis group than the control group. It was concluded that sTREM-1 may serve as a biomarker for periodontitis and may have implications in the association between periodontal infections and systemic inflammatory response.9 NULL HYPOTHESIS: GCF levels of TREM-1 has no value as a biomarker in predicting the progression of periodontal diseases and its response to phase –I therapy. RESEARCH HYPOTHESIS: GCF levels of TREM-1 has a significant value as a biomarker in predicting the progression of periodontal disease and its response to phase –I therapy. 6.3 OBJECTIVES OF THE STUDY: 1. To estimate GCF levels of TREM-1 in periodontal health, Gingivitis and Chronic periodontitis. 2. To compare GCF levels of TREM-1 in different stages of periodontal disease. 3. To compare GCF levels of TREM-1 before and after scaling and root planing (SRP) in Gingivitis and Chronic periodontitis. 4. To correlate GCF levels of TREM-1 with Clinical parameters in periodontal health and disease. 7. MATERIALS AND METHODS: 7.1 Source of data: The study population will consist of 45 subjects diagnosed as periodontally healthy, gingivitis or Chronic periodontitis with 15 subjects in each category, belonging to both genders. All the subjects will be selected from the out-patient division of the department of Periodontics, K.L.E Society’s institute of Dental Sciences, Bangalore, Karnataka, India. A written informed consent (in the local language that can be easily understood by the subject) will be obtained from those who agree to participate voluntarily in the study. 7.2 Method of collection of data: Study design: An in vivo study. Sample size: A total of 45 subjects assigned into 3 groups – One control group and two test groups: Group I (Control group): 15 subjects with clinically healthy periodontium. Group II (Test group 1): 15 subjects with gingivitis. Group III (Test group II): 15 subjects with chronic periodontitis. Inclusion criteria: Systemically healthy subjects, of both the gender in the age group of 20-50 years with at least 20 teeth in the mouth. Subjects who are willing to participate in the study, cooperative and able to attend follow up. Group I(Control group): Absence of clinical signs of gingival inflammation (GI= 0). Probing depth ≤ 3mm. Absence of loss of clinical attachment. Group II(Test group I): Presence of clinical signs of gingival inflammation (GI >1). Probing depth ≤ 3mm. Absence of loss of clinical attachment. Group III(Test group II): Presence of a minimum of three periodontal pockets with probing pocket depth of ≥ 5mm along with clinical signs of gingival inflammation. Clinical attachment loss of ≥ 3mm. Radiographic evidence of alveolar bone loss. (No delineation will be done within chronic periodontitis based on the extent of alveolar bone loss). Exclusion criteria: Subjects with any systemic diseases like diabetes, hypertension, heart disease and rheumatoid arthritis that can alter the course of periodontal disease. Subjects having taken antibiotic therapy/anti-inflammatory drugs/immunosuppressive drugs in the past 6 months. Subjects who have undergone any form of periodontal therapy in past 6 month Pregnant and lactating mothers. Smokers and subjects using smokeless tobacco. GCF Sampling: The study involves collecting gingival crevicular fluid from all the three groups at baseline, and at 8 weeks following thorough Scaling and Root planing (SRP) in groups II and III to measure TREM-1 levels.10 The GCF samples will be collected using commercially available 1-5µl calibrated micropipettes. A total of 75 GCF samples will be collected during the entire study period. Site Selection:11 Group I: Multiple sites (three to five sites/subject) to ensure collection of an adequate amount of GCF. Group II: The site with the greatest clinical signs of gingival inflammation in the absence of clinical attachment loss. Group III: The site with the greatest clinical signs of gingival inflammation, highest probing depth and clinical attachment loss along with radiographic evidence of alveolar bone loss. Clinical parameters: The following clinical parameters will be recorded at baseline for all the three groups and at follow up visits scheduled at 8 weeks, following SRP in groups II and III:10 Plaque index (Silness and Loe, 1964 ). Gingival index (Loe and Silness, 1963). Probing pocket depth (PD). Clinical attachment level (CAL). Periodontal probing depth (PD) and Clinical attachment level will be recorded using a constant pressure probe (Brock probe TM). Orthopantomogram (OPG) for every patient, showing clinical signs of attachment loss will be taken at baseline. Radiographic bone loss will be recorded dichotomously to differentiate subjects with chronic periodontitis from other groups.11 Study Method: After obtaining informed consent, clinical parameters of each subject will be recorded and OPG of every subject showing clinical signs of attachment loss will be taken. Following this, GCF sampling will be done for all the three groups. After drying the test site gently with air, supragingival plaque will be removed with a sterile curette without touching the marginal gingiva and isolated with cotton rolls to avoid saliva contamination. GCF will be collected by placing a microcapillary pipette at the entrance of the gingival sulcus, gently touching the marginal gingiva .A standard volume of 1µl will be collected with an extracrevicular approach (unstimulated) from each test site. Further, micropipettes that will be suspected of being contaminated with blood or saliva will be discarded. The collected GCF sample will be transferred to airtight plastic vials containing 0.5ml of phosphate buffered saline (PBS) and stored at -700 C until assayed. TREM-1 levels in GCF will be determined by ELISA using specific ELISA kit and reading will be assessed by ELISA microplate reader at 450nm. Subjects with gingivitis and chronic periodontitis will be subjected to thorough scaling and root planing (SRP) and will be recalled after 8 weeks for recording the clinical parameters and collection of GCF samples. Statistical Analysis : Normality assumption of data will be tested using Schapiro-Wilks test. 1. If the data will be normal , Mean comparison between two groups will be done using ANOVA. If ANOVA results will be significant (p< 0.05 values will be considered as significant), further comparison between two groups will be done using Tukey’s test. Intra- group comparison will be done using paired student t test. 2. If the data will not be normal, Mean comparison between two groups will be done using the Kruskal-Wallis test. If Kruskal-Wallis test will be significant , further comparison between the two groups will be done using the Mann-Whitney U test. Intra-group comparison will be made using Wilcoxon Signed Rank test. 7.3 Does the study require any investigation or interventions to be conducted on patients or other humans or animals? If so, please describe briefly? Yes. The study requires collection of gingival crevicular fluid from patients of all the three groups. Scaling and root planing will be done in test groups I and II and during the study, OPG will be taken for every patient showing clinical signs of gingival inflammation. A signed informed consent will be obtained from the patients before the same. 7.4 Has ethical clearance been obtained from your institute in case of 7.3? Yes. A copy of the same has been attached. 8. LIST OF REFERENCES: 1. Bostanci N, Thurnheer T, Aduse-Opoku J, Curtis MA, Zinkernagels AS, Belibasakis GN. Porphyromonas gingivalis regulates TREM-1 in human polymorphonuclear neutrophils via its gingipains. PLoS ONE 2013;8:1-8. 2. Graves D. Cytokines that promote periodontal tissue destruction. J Periodontol 2008;79:1585-1591. 3. Bisson C, Massin F, Lefevre PA, Thilly N, Miller N, Gibot S. Increased gingival crevicular fluid levels of soluble triggering receptor expressed on myeloid cells (sTREM ) -1 in severe periodontitis. J Clin Periodontol 2012;39:1141-1148. 4. Buduneli N, Kinane DF. Host – derived diagnostic markers related to soft tissue destruction and bone degradation in periodontitis. J Clin Periodontol 2011;3:85105. 5. Lamster IB. Evaluation of components of gingival crevicular fluid as diagnostic tests.Ann Periodontol 1997;2:123-137. 6. Bostanci N, Thurnheer T, Belibasakis GN. Involvement of the TREM-1/DAP-12 Pathway in the innate immune responses to porphyromonas gingivalis. Mol Immunol 2011;49:387-394 7. Bostanci N, Belibasakis GN. Doxycycline inhibits TREM-1 induction by Porphyromonas gingivalis. FEMS Immunol Med Microbiol 2012;66:37-44. 8. Belibasakis GN, Ozturk VO, Emingil G, Bostanci N. Soluble triggering receptor expressed on myeloid cells (sTREM ) -1 in gingival crevicular fluid: Association with clinical and microbiological parameters. J periodontol 2013;84:1-11. 9. Bostanci N, Ozturk VO, Emingil G, Belibasakis GN. Elevated oral and systemic levels of soluble triggering receptor expressed on myeloid cells -1 in periodontitis. J Dent Res 2013;92:161-165. 10. Segelnick SL, Weinberg MA. Reevaluation of initial therapy:when is the appropriate time? J Periodontol 2006;77:1598-1601. 11. Pradeep AR, Happy D, Parag H, Garima G, Manojkumar T. Correlation of gingival crevicular fluid interleukin-18 and monocyte chemoattractant protein-1 levels in periodontal health and disease. J periodontol 2009;80:1454-1461. 9. SIGNATURE OF THE CANDIDATE 12. REMARKS OF THE GUIDE 13. NAME & DESIGNATION OF (IN BLOCK LETTERS) 11.1 GUIDE Dr. SUDHIR R PATIL, PROFESSOR AND HOD, DEPARTMENT OF PERIODONTICS, K.L.E. SOCIETY’S INSTITUTE OF DENTAL SCIENCES, BANGALORE-22. 11.2 SIGNATURE 11.3 CO-GUIDE (IF ANY ) Dr. VEENA HR, READER, DEPARTMENT OF PERIODONTICS, K.L.E. SOCIETY’S INSTITUTE OF DENTAL SCIENCES, BANGALORE-22. 11.4 SIGNATURE 11.5 HEAD OF THE DEPARTMENT 1.6 SIGNATURE DR.SUDHIR R PATIL, PROFESSOR & HOD, DEPARTMENT OF PERIODONTICS, K.L.E. SOCIETY’S INSTITUTE OF DENTAL SCIENCES, BANGALORE-560022. KARNATAKA. 12 12.1 REMARKS OF THE CHAIRMAN & PRINCIPAL 12.2 SIGNATURE K.L.E. SOCIETY’S INSTITUTE OF DENTAL SCIENCES, YESHWANTHPUR SUBURB 2AND STAGE,BANGALORE- 560022 DEPARTMENT OF PERIODONTICS STUDY TITLE GINGIVAL EXPRESSED CREVICULAR ON FLUID MYELOID LEVELS CELLS -1 OF IN TRIGGERRING GINGIVITIS AND RECEPTOR CHRONIC PERIODONTITIS BEFORE AND AFTER PHASE –I THERAPY- A CASE CONTROL STUDY. INFORMED CONSENT PATIENT’S NAME- DATE- AGE/SEX- OP NO.- ADDRESS- CONTACT NO.- I ...........................................................................,hereby, authorize Dr.SURABHI GIGRAS at the department of Periodontics, K.L.E Society’s Institute Of Dental Sciences, Bangalore, to perform examination/investigation/treatment or any other necessary procedure required for the research purpose titled as above. I have been explained the risk and benefits of the treatment procedure and neither the dentist nor the assistants will be held responsible, if any untoward incident happens. I have no objection to the data collected being used for research purpose entitiled above. I also agree to cooperate for the further appointments needed for the scheduled follow up of the procedure being conducted on me. PATIENT’S SIGNATURE DATEPLACE- TREATING DOCTOR’S SIGNATURE- DATEPLACE- NAME OF THE GUIDE AND SIGNATURE- DATEPLACE-