Rajiv Gandhi University of Health Sciences s2

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES BANGALORE, KARNATAKA. ANNEXURE II PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION 1. NAME OF THE CANDIDATE AND DR. RAGHAVENDRA N.M. ADDRESS POST GRADUATE STUDENT, DEPARTMENT OF PERIODONTICS, GOVERNMENT DENTAL COLLEGE AND RESEARCH INSTITUTE, BANGALORE.

2. NAME OF THE INSTITUTE GOVERNMENT DENTAL COLLEGE AND RESEARCH INSTITUTE, BANGALORE. 3. COURSE OF STUDY AND SUBJECT MASTER OF DENTAL SURGERY IN PERIODONTICS.

4. DATE OF ADMISSION TO COURSE 02.06.2008

5. TITLE OF THE TOPIC:

“LEVELS OF VISFATIN IN GINGIVAL CREVICULAR FLUID AND SERUM IN PERIODONTAL HEALTH AND DISEASE. ”

6. BRIEF RESUME OF THE INTENDED WORK:

6.1 NEED FOR THE STUDY: -

Periodontitis is an inflammatory disease characterized by destruction of the supporting tissues of the teeth. The imbalance between the plaque biofilm and the host immune system results in the over expression of proinflammatory cytokines and the subsequent destruction of alveolar bone.

Visfatin, which was recently identified as a new adipocytokine, has been recognized as a pre-B-cell colony enhancing factor (PBEF). Visfatin activates human leukocytes and induces cytokine production, such as Tumor necrosis factor –α (TNF-α) 1, Interleukin-1β (IL-1β) and Interleukin-6 (IL-6) which inturn stimulates the production of C-reactive protein (CRP)2 from the liver. Visfatin expression is upregulated during inflammation and it represents a novel clinical biomarker in Rheumatoid arthritis3 and Diabetes Mellitus.4

1 Till date, to our knowledge no study has reported visfatin levels in periodontal health and disease. In this context, the present study is designed to assess the levels of visfatin in gingival crevicular fluid (GCF) and serum in periodontal health and disease.

6.2 REVIEW OF THE LITERATURE: -

Recombinant visfatin was shown to activate human leukocyte and induce cytokine production (IL-1β, TNF-α and IL-6). Circulating IL-6 was found to be induced by visfatin BALB/c mice. Elevated plasma levels of visfatin were detected by using enzyme immunometric assay in patients with inflammatory bowel disease. mRNA expression of visfatin was also found to be significantly increased in the colonic tissue of Crohn’s and Ulcerative colitis patients.1

Serum visfatin levels were assessed by enzyme immuno-assay and found to be positively correlated with the serum levels of IL-6 and slightly related with CRP levels.2 Increased expression of visfatin levels in the serum and arthritic paw was detected in mice with collagen induced arthritis using enzyme linked immuno-assay. Arthritis severity was found to be effectively reduced by a specific competitive inhibitor of visfatin.3

Plasma visfatin levels were found to be elevated in patients with type2 Diabetes Mellitus through ELISA.4 6.3 OBJECTIVES OF THE STUDY: -

1. To estimate the levels of visfatin in GCF and serum in healthy and periodontally affected individuals.

2. To correlate the levels of visfatin in GCF and serum in healthy and periodontally affected individuals.

3. To explore the possibility of visfatin as an “inflammatory biomarker” in periodontal disease progression.

2 7. MATERIALS AND METHODS : -

7.1 SOURCES OF THE DATA :-

The subjects to be studied will be selected from the outpatient section, Department of Periodontics, Government Dental College and Research Institute, Bangalore.

7.2 METHODS OF COLLECTION OF DATA :-

Subjects will be selected randomly and categorized into 3groups based on gingival index (Loe & Silness), probing pocket depth (≥5mm), clinical attachment loss (≥3mm) and bleeding on probing.

80 samples (40 GCF & 40 serum) from 40 subjects will be divided into three groups: Group I (10 subjects with healthy periodontium), Group II (15 subjects with gingivitis), Group III (15 subjects with chronic periodontitis). It will be made clear with the potential subjects that participation will be voluntary and written informed consent will be obtained from those who agree to participate. The grouping is as follows:

Group I : 20 samples (10 GCF and 10 serum) from 10 patients with healthy periodontium. Group II : 30 samples (15 GCF and 15 serum) from 15 patients with gingivitis. Group III : 30 samples (15 GCF and 15 serum) from 15 patients with chronic periodontits (moderate-to-advanced)

INCLUSION CRITERIA: -

1. Age group 23-50 years.

2. Subjects are to have at least 20 natural teeth.

3. Subjects with BMI score ≤ 25kg/m2.

4. Subjects who have not received periodontal therapy, antibiotics or non-steroidal anti-inflammatory drugs within preceding 6 months.

3 EXCLUSION CRITERIA: -

1. Use of tobacco in any form 2. Diabetes Mellitus 3. Rheumatoid arthritis 4. Pulmonary diseases 5. Multiple sclerosis 6. Bacterial, viral and fungal systemic infections 7. Aggressive periodontitis 8. Inflammatory bowel diseases

The Gingival index, probing pocket depth, bleeding on probing and clinical attachment loss will be measured after GCF collection to avoid contamination of the sample with blood. The radiographs will be done to confirm site assessment. The clinical measurements will be carried out by the same examiner, using William's graduated periodontal probe. The site showing the most severe inflammatory status (in case of gingivitis) and a probing pocket depth of ≥5mm with clinical attachment loss of ≥3mm, (in case of periodontitis) will be selected for GCF sample collection.

GCF collection will be done using micro capillary pipettes at the initial visit in Group I, Group II and Group III and samples will be stored at −70 oC till the assay procedure.

Blood collection: 2 ml of blood will be collected from the antecubital fossa by venipuncture using 20-guage needle with 2 ml syringes and immediately transferred to the laboratory. The Serum will be extracted from blood and stored at −70 oC till the assay procedure.

Estimation of “VISFATIN” levels will be done by using ‘Human VISFATIN ELISA KIT’ obtained from ‘BIOVISION (Axxora)’ USA.

Duration of the study will be approximately three months from the sample collection till the assay procedure.

The following statistical tests will be carried out:-

1. To test the hypothesis of equality of means for all the groups Kruskal- Wallis rank non parametric test is done.

2. Mann-Whitney U test to find out which pair or pairs will differ Significantly.

4 3. Spearman’s rank correlation will be done to find the correlation between GCF and serum visfatin concentrations.

4. Confidence interval with probability of 0.95 in GCF and serum to explore visfatin as an inflammatory biomarker.

7.3 Does the study require any investigations or interventions to be conducted on patients or other humans or animals?

a) GCF collection will be done by using micro capillary pipettes.

b) 2 ml of blood sample will be drawn from each patient by venipuncture at the antecubital fossa.

7.4 Has ethical clearance been obtained from your institution in case of 7.3?

LIST OF REFERENCES:-

1. Moschen A R, Kaser A, Enrich B, Mosheimer B, Theurl M, Niederegger H, Tilg H. Visfatin, an Adipocytokine with Proinflammatory and Immunomodulating Properties. J Immunol 2008; 178:1748-1758.

2. Oki K, Yamane K, Nojima H and Kohono N. Circulating visfatin level is correlated with inflammation, but not with insulin resistance. Clin Endocrinol 2007; 67:796-800.

3. Busso N, Karababa M, Nobile M, Rolaz A, Gool F V, Galli M, Leo O, So A, Smedt D. Pharmacological Inhibition of Nicotinamide Phosphoribosyltransferase / Visfatin Enzymatic Activity Identifies a New Inflammatory Pathway Linked to NAD. Plosone 2008;3:e2267

4. Chen M, Chung F-M, Chang D, C.-R.Tsai Jack, Huang, Shin S-J and Lee Y-J. Elevated Plasma Level of Visfatin/Pre-B cell Colony Enhancing Factor in Patients with type 2 Diabetes Mellitus. J Clin Endocrinol metab 2006; 91:295-299

5 6 9. SIGNATURE OF THE CANDIDATE

10. REMARKS OF THE GUIDE

11 NAME AND DESIGNATION OF DR. M.V.RAMACHANDRA PRASAD 11.1 GUIDE ASSISTANT PROFESSOR, DEPARTMENT OF PERIODONTICS, GOVERNMENT DENTAL COLLEGE AND RESEARCH INSTITUTE, BANGALORE.

11.2 SIGNATURE

11.3 CO-GUIDE

11.4 SIGNATURE

11.5 HEAD OF THE DEPARTMENT DR. A.R. PRADEEP PROFESSOR, HEAD AND Ph.D GUIDE DEPARTMENT OF PERIODONTICS, GOVERNMENT DENTAL COLLEGE AND RESEARCH INSTITUTE, BANGALORE. 11.6 SIGNATURE

12. 12.1 REMARK OF THE CHAIRMAN AND PRINCIPAL

12.2 SIGNATURE