RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES,
BANGALORE, KARNATAKA
ANNEXURE –II
PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION
1 / NAME OF THE CANDIDATEAND ADDRESS
(IN BLOCK LETTERS) / DR. IPSITA JAYANTI
POST GRADUATE STUDENT
DEPARTMENT OF PERIODONTICS
RAJARAJESWARI DENTAL COLLEGE AND HOSPITAL, MYSORE ROAD,
BANGALORE – 560060
2 / NAME OF THE INSTITUTION / RAJARAJESWARI DENTAL COLLEGE
AND HOSPITAL, BANGALORE-560060
3 / COURSE OF STUDY AND SUBJECT / MASTER OF DENTAL SURGERY
PERIODONTICS
4 / DATE OF ADMISSION TO THE COLLEGE / 22nd APRIL 2011
5 / TITLE OF THE TOPIC:
EFFECT OF NON-SURGICAL PERIODONTAL THERAPY ON THE LEVELS OF TLR 9 IN CHRONIC AND AGGRESSIVE PERIODONTITIS PATIENTS.
6
/ BRIEF RESUME OF THE INTENDED WORK:
6.1 NEED OF THE STUDY :
Periodontitis, a chronic infection, affects the gingiva and bone supporting the teeth1. The tissue destruction is initiated by the interaction between periodontopathogens and the host cells. A primary challenge to the cells of the innate immune system like monocytes, macrophages and neutrophils is the discrimination of a large number of receptors. This has been met by the evolution of large number of receptors that recognize conserved motifs on pathogens called Toll Like Receptors. Members of TLR family are responsible for the recognition of pathogen associated molecular pattern (PAMP) expressed by a wide spectrum of infectious agents. TLRs activate the signaling pathways that is critical for the induction of the immune response to the given microbial challenge.3
Ten human homologs of the fruit fly drosophila toll–like receptors belong to the interleukin-1 receptor family.4 Neutrophils are the predominant immune system cells in the periodontal tissues which express TLR 1, TLR 2, and TLR 4 to TLR 10. Monocytes or macrophages express toll-like receptor 1,toll-like receptor 2, and toll-like receptor 4 to toll-like receptor 8.5
Various studies have indicated the etiology of periodontitis to be related to
viruses. From certain studies done earlier on viruses and periodontitis it is evident that viruses especially herpes viruses (mainly EBV and HCMV) play a role in the pathogenesis of periodontitis. These studies have demonstrated an association between herpes viruses and periodontal disease by showing their presence in gingival tissue, gingival crevicular fluid and subgingival plaque.6 TLR 9 though not located in the cell membrane, it fulfills its function intracellularly and is expressed in immune cell rich tissues like lymph nodes, bone marrow and peripheral blood leucocytes.7 It recognizes the unmethylated CpG sites on DNA molecule and the signals leads to the activation of the cells initiating pro inflammatory reactions that result in the production of cytokines such as type I interferon and IL -12.11
The present study is aimed at quantitatively analysing the levels of TLR 9 in gingival crevicular fluid of chronic and aggressive periodontitis patients before and after scaling and root planing.
6.2 REVIEW OF LITERATURE:
· In another study on Toll-like receptors and their role in periodontal health and disease it was concluded that periodontal cells actively participate in the innate immune response against dental plaque bacteria. They express different types of Toll-like receptors. Periodontitis, a chronic inflammation of the periodontium, provides a unique opportunity to investigate the host–microbe interactions. Gingival epithelium is continually exposed to at least 500 species of both oral commensal and pathogenic bacteria. The gingival epithelial cells orchestrate their response via Toll-like receptor signaling, and thereby maintain themselves without severe, chronic inflammation is a very interesting issue.1
· In a study done on the association of Toll like receptor 9 haplotypes with chronic periodontitis in Czech population it was concluded that although no significant role of the TLR2 gene in periodontitis was found, but results indicate that TLR9 haplotypes may be associated with chronic periodontitis.5
· In a review study done on the role of Toll-like receptors in immune disorders concluded that TLR agonists may be effective for infectious diseases and vaccination, they can also increase the risk of autoimmune diseases and atherosclerosis. Future studies on TLRs will facilitate new therapeutic approaches for variousdiseases, along with the knowledge of how to modulate this system without adverse side effects.8
· In a study done on regulation of bone metabolism by innate immune regulators, it was concluded that while the activation of TLRs in committed Osteoclast precursors(OCPs), mature Osteoclasts(OCs) and Osteoblasts(OBs) resulted in increased osteoclastogenesis and is probably the mechanism by which pathogen-induced bone loss occurs. Activation of TLRs in early OCPs exerts an anti-osteoclastogenic effect. This could serve as a mechanism for down-regulating excessive resorption, and as a switch for promoting the differentiation of common precursor cells into inflammatory cells.9
· In another study, the expression and distribution of TLRs (TLR1 – TLR10) were immunohistochemically detected in gingival epithelium and connective tissue. The results showed that the TLR expression was increased towards the basal layer in patients with Periodontitis whereas healthier controls showed more variable cellular TLR expression towards the basal layers of epithelium. In the connective tissue higher TLR expression was found within periodontitis group compared to the healthy group.10
· Expression levels of TLR-5, TLR-7 and TLR-9 mRNAs in gingivitis and periodontitis were investigated and compared in a study. The levels of Interferon-alfa which is important for antiviral response were also assessed. Gene expression was analyzed using quantitative real time polymerase chain reaction and the presence of plasmacytoid dendritic cells was immunohistochemically analyzed. Results showed that the expression levels of TLR-2,TLR-4,TLR-7 and TLR-9 were significantly higher in periodontitis lesions than in gingivitis lesions.11
6.3 AIMS AND OBJECTIVES OF THE STUDY:
1) Quantitative analysis of levels of TLR 9 in GCF of Chronic and Aggressive Periodontitis patients.
2) To compare the levels of TLR 9 in GCF of healthy individuals with that of Chronic and Aggressive Periodontitis patients.
MATERIALS AND METHODS :
7.1 SOURCE OF DATA
7.1.1. Type of study: Randomised Controlled trial
7.1.2. Study population: Patients visiting the Department of Periodontology, Rajarajeswari Dental College and Hospital, Bangalore.
7.2 METHOD OF COLLECTION OF DATA :
The study consists of 45 subjects grouped as follows and a total of 75 GCF samples will be collected from the groups using micropipettes
Group I – 15 GCF samples from healthy controls without periodontitis.
Group II – 15 GCF samples from chronic periodontitis patients.
Group III – 15 GCF samples from aggressive periodontitis patients.
Group IV- 15 GCF samples from Group II 6-8 weeks after treatment.
Group V- 15 GCF samples from Group III 6-8 weeks after treatment.
INCLUSION CRITERIA :
Group 1 : Healthy controls
1. No sites with probing depth ≥4mm or clinical attachment loss ≤1mm.
2. Healthy gingival condition with a gingival index score 1.
Group 2 : Chronic Periodontitis
1. Untreated chronic periodontitis with a probing depth ≥5mm.
2. Clinical attachment loss ≥3mm.
3. Radiographic evidence of alveolar bone loss on atleast two teeth per quadrant
excluding the third molars.
Group 3 : Aggressive Periodontitis
1. Untreated aggressive periodontitis patients with a probing depth ≥5mm.
2. Generalized proximal attachment loss affecting at least 3 teeth other than first molars
and incisors.
3. Amount of microbial deposits inconsistent with disease severity.
EXCLUSION CRITERIA :
1. Patients with history of systemic diseases.
2. Smokers and alcoholics.
3. Patients on any medications taken within a period of 6 months which may alter the clinical parameters of the study.
4. Pregnant women and lactating mothers.
5. Patients who have undergone periodontal treatment within a period of last 1 year.
SCREENING OF PATIENTS :
All the subjects will undergo a full mouth periodontal probing and charting and will be screened for suitability for the study after which an informed consent will be signed. A performa was designed for the present study so as to have a systematic and methodical recording of all the observations and information. The relevant data will be recorded in the proforma. Chronic periodontitis patients were diagnosed based on criteria presented and discussed at the 1999 International Workshop for the Classification of Periodontal Diseases organized by the American Academy of Periodontology.
SCREENING EXAMINATION INCLUDE:
· Gingival Index(GI) (Loe and Silness, 1963)
· Probing Pocket Depth(PPD) measured using a graduated William’s Periodontal probe.
· Clinical Attachment Level(CAL)
SAMPLE COLLECTION :
The patients will receive instructions regarding the study procedure and written informed consent will be obtained. GCF samples of approximately 3 μl will be collected using a graduated micro capillary pipette by an extracrevicular method. The collected GCF will be immediately transferred to plastic vials, wrapped in a tin foil and stored at -70º C until the time of assay. Micro capillary pipette will be procured from Sigma Aldrich, Bangalore, India. The storage of GCF samples was done at Rajarajeswari Dental College and Hospital, Bangalore.
LABORATORY ANALYSIS :
Quantitative analysis of expression of TLR-9 in the GCF samples will be assessed using an Enzyme Linked Immunosorbent Assay kit before and 6-8 weeks after treatment.
STATISTICAL ANALYSIS:
· Descriptive statistics will be used to analyse demographic data.
· Student’s Paired-t test will be used to compare the level of TLR 9, before and after scaling and root planning.
· Analysis of Variant (ANOVA) test will be used to compare TLR 9 level in GCF in healthy, Chronic, Aggressive Periodontitis.
· Pearson correlation test was used to correlate GCF levels of TLR 9 with clinical parameters.
Duration of the study: 2 years.
7.3 DOES THE STUDY REQUIRE ANY INVESTIGATION OR INTERVENTION TO BE CONDUCTED ON PATIENTS OR OTHER HUMANS, ANIMALS? IF SO PLEASE DESCRIBE BRIEFLY?
Yes.
Patients will be informed and written consent will be obtained.
7.4 HAS ETHICAL CLEARANCE BEEN OBTAINED FROM YOUR INSTITUTION IN CASE OF 7.3?
Yes.
Ethical clearance letter has been attached.
LIST OF REFERENCES:
1. Rangsini Mahanonda and Sathit Pichyankul. Toll-like receptors and their role in periodontal health and disease. Periodontol 2000. 2007; 43: 41-45.
2. Hayashi F, Means TK, Luster AD. Toll-like receptors stimulate human neutrophil function. Blood 2003; 102: 2660-69.
3. O’Neil LA. Signal transduction pathways activated by the IL-1 Receptor/Toll-like
receptor superfamily. Current Top Microbiol Immunol 2002; 270: 47-61.
4. Chuang TH, Ulevitch RT. Identifiction of Htlr 10: A novel human Toll-like receptor preferentially expressed in immune cells. Biochim Biophys Acta 2001; 1518: 157-61.
5. Iwasaki A, Matzhitov R. Toll-like rceptor control of the adaptive immune response. Nat Immunol 2004; 3: 987-95.
6. Shivaprasad B, Sachin Mangalekar, Dilip Sharma, Ashok Prabhakar, Sridhar Reddy, Nagaraj Kalburgi, et al. Herpes viruses in Chronic and Aggressive Periodontitis patients in Indian population. Journal of Oral Science 2009; 51: 79-86.
7. Holla LI, Vokurka J, Hrdlickova B, Augustin P, Fassmann A. Association of Toll like receptor 9 haplotypes with chronic periodontitis in Czech population. J Clin Periodontol 2010; 37: 152-59.
8. Satoshi Uematsu, Shizuo Akira. The role of Toll-like receptors in immune disorders. Expert Opin. Biol. Ther. 2006; 6: 203-14
9. Zvi bar-shavit. Taking a Toll on the bones: Regulation of bone metabolism by innate immune regulators. Autoimmunity, May 2008; 41: 195-203.
10. A Beklen, M. Hukkanen, R. Richardson, Y. T. Konttinen. Immunohistochemical localization of Toll-like receptors 1-10 in periodontitis. Oral Microbiol Immunol; 23; 5: 425-31.
11. Kajita K, Honda T, Amanuma R. Quantitative messenger RNA expression of Toll-like receptors and Interferon-α 1 in gingivitis and periodontitis. Oral Microbiol Immunol 2007; 2: 398-402.
9 / SIGNATURE OF THE CANDIDATE
10 /
REMARKS OF THE GUIDE
11 / 11.1 NAME & DESIGNATION OFGUIDE
(in block letters) /
DR. SAVITA S. MDS
PROFESSOR AND HEADDEPARTMENT OF PERIODONTICS
RAJARAJESWARI COLLEGE OF DENTAL SCIENCES AND HOSPITAL, BANGALORE
11.2 SIGNATURE OF GUIDE
11.3 CO-GUIDE (if any)
11.4 SIGNATURE
11.5 HEAD OF THE DEPARTMENT /
DR. SAVITA S. MDS
PROFESSOR AND HEADDEPARTMENT OF PERIODONTICS
RAJARAJESWARI COLLEGE OF DENTAL SCIENCES AND HOSPITAL, BANGALORE
11.6 SIGNATURE
12 / 12.1 REMARKS OF THE
CHAIRMAN & PRINCIPAL
12.2 SIGNATURE