Download RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES,
BANGALORE, KARNATAKA
ANNEXURE II
PROFORMA FOR REGISTRATION OF SUBJECT FOR
DISSERTATION
1.
NAME OF THE CANDIDATE
MADHUR
AND ADDRESS
P.M.N.M. DENTAL COLLEGE AND
HOSPITAL
BAGALKOT – 587101
KARNATAKA
2.
NAME OF THE INSTITUTION
P.M.N.M. DENTAL COLLEGE AND
HOSPITAL, BAGALKOT – 587101
KARNATAKA
3.
4.
COURSE OF THE STUDY
M.D.S(MASTER OF DENTAL
AND
SURGERY)
SUBJECT
DATE OF ADMISSION TO
PERIODONTICS
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.
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 class-I & 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. 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.1 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.2
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+.1
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
RORC gene produces two isoforms - RORγ and RORγt. RORγ (also
referred to as RORγ1), which is produced from an mRNA containing exons
1 to 11,4 is a member of the nuclear receptor family of transcription factors
and is encoded in humans by the RORC (RAR-related orphan receptor C)
gene. 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.5
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.6 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.7
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.8
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. 9
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.2
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 subjects, 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 immunohistochemical technique. The method can
be changed depending upon the availability of antibody.
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) Hernandez M, Vernal R, Sorsa T, Tervahartiala T, Mantyla P,
Gamonal J. Pathogenesis and treatment of Periodontitis.
Chile,Finland:Intech:2012.p.33-54.
2) 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.
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) 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.
5) 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.
6) Unutmaz D. RORC2: The master of human Th17 cell programming.
Eur J Immunol 2009;39:1452–1455.
7) Manel N, Unutmaz D and Littman DR. The differentiation of
human T(H)-17 cells requires transforming growth factor-beta
and induction of the nuclear receptor RORγt. Nat Immunol
2008;9:641–649.
8) 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;182:7381- 7388.
9) 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.
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