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RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES
KARNATAKA, BANGALORE
ANNEXURE-II
PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION
1.
NAME OF THE CANDIDATE
DR.SHRUTHI.S.KRISHNAMURTHY
DEPARTMENT OF ORAL AND
MAXILLOFACIAL PATHOLOGY,
ADDRESS
Dr. SYAMALA REDDY DENTAL
COLLEGE,
HOSPITAL & RESEARCH CENTRE,
MUNNEKOLALA, MARATHALLI
BANGALORE-560037
2.
3.
NAME OF THE INSTITUTION
Dr. SYAMALA REDDY DENTAL
COLLEGE, HOSPITAL & RESEARCH
CENTRE,
BANGALORE-560037
COURSE OF STUDY AND
SUBJECT
MASTER OF DENTAL SURGERY IN
DEPARTMENT OF ORAL AND
MAXILLOFACIAL PATHOLOGY
4.
26-05- 2010
DATE OF ADMISSION TO THE
COURSE
“ ROLE OF TRANSFORMING GROWTH
5.
TITLE OF THE TOPIC
FACTOR BETA IN ORAL SUBMUCOUS
FIBROSIS”
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6. BRIEF RESUME OF THE INTENDED WORK
6.1:NEED FOR THE STUDY
“Oral Submucous Fibrosis (OSF) is an insidious chronic disease affecting
the oral mucosa associated with vesicle formation and juxtraepithelial inflammatory
reaction followed by a fibroelastic change of the lamina propria with epithelial atropy,
leading to stiffness of the oral mucosa and trismus.”1
OSF has also been identified as a pre-cancerous condition.2
The
pathogenesis of the disease is, however, not well established. OSF may be considered a
collagen- metabolic disorder resulting from exposure of mucosa to contents of arecanut. The
important histopathological characteristics of OSF is the deposition of collagen in the oral
sub mucosa. It has been found that alkaloid exposure of buccal mucosal fibroblast results in
accumulation of collagen. Collagen is the major structural component of the connective
tissue and its composition within each tissue needs to be maintained for proper tissue
integrity. The synthesis of collagen is influenced by a variety of mediators, including
growth
factors, hormones, cytokines and lymphokines.
Transforming Growth Factor beta (TGF-β) represents large family of growth and
differentiation factors that mobilize through complex signaling networks to regulate cellular
differentiation, proliferation, motility, adhesion and apoptosis.
A prominent mediator is
TGF-β, it plays a major role in wound repair and fibrosis. It causes deposition of
Extracellular Matrix (EM) by increasing the synthesis of matrix protein like collagen and
decreasing the degradation by stimulating various inhibitor mechanisms. Although TGF-β is
essential for healing, over production leads to scar tissue and fibrosis. Thus TGF- β
signaling pathway might be critical for pathogenesis of OSF. TGF-β stimulates fibroblast
proliferation and EM elaboration suggests the importance of their cytokines in fibroelastic
diseases.
2
Immunohistochemistry or IHC refers to the process of detecting antigens in cells and
antibodies binding specifically to antigen in biological tissues. It takes its name from the
root “immunos” in reference to antibodies used in the procedure and
“ histos” meaning
tissues. . IHC is also widely used in basic research to understand the distribution and
localization of biomarkers and differentially expressed proteins in different parts of a
biological tissue.
In this study it is hypothesized that TGF-β may have a definite role in the
induction and proliferation of fibrosis in OSF. The present study aims to evaluate the
expression of TGF-β immunohistochemically in the paraffin-embedded tissues of OSF and
correlate it to the clinical and histological stages of the disease. It is also planned to
correlate the expression of TGF-β in cases of individuals with habits but no clinical OSF
and cases of normal individuals, compared with cases of fibrosis, scleroderma, kleoid, scar
etc. All estimations are to be done on paraffin embedded H&E stained slides to evaluate
TGF-β for an imunomodulatory role in the fibrosis in Oral Submucous Fibrosis by
immunohistochemistry. It is also envisaged, if feasible, to evaluate genetic alteration
relating to the expression of TGF-β in OSF.
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6.2 REVIEW OF LITERATURE
1) This study explores the significance of transforming growth factor beta1 (TGF –β1)
in the pathogenesis of oral submucous fibrosis( OSF). TGF-β1 mRNAs in
keratinocytes of the paraffin embedded tissues of 25 OSF cases and 5 normal
patients were
determined by the in situ hybridization technique. The result
showed that there was an expression of TGF-β1 mRNA in keratinocytes of
15OSFs (60%). There was no expression in that of normals. The study suggests
that keratinocytes of OSF tissue may synthesis and release TGF- β1 which may
play an important role in the pathogenesis of OSF and participate as a mediator in
the pathogenesis of OSF.3
2)
In this study, microarray analysis was used to characterize the mRNA changes of
14,500 genes in four OSF and four normal buccal mucosa samples to identify novel
biomarkers of OSF. Five candidate genes with the most differential changes were chosen for
validation. The correlation between clinicopathologic variables of 66 OSF patients and the
expression of each gene was assessed by immunohistochemistry. The microarray analysis
showed that 661 genes were up-regulated and 129 genes were down-regulated in OSF. In
immunohistochemical results, the expression of Loricrin and Excessive Cartilage
Oligomeric Matrix Protein (COMP) showed statistically significant association with
histologic grade of OSF. COMP was found to be overexpressed frequently in patients with
the habit of areca nut. COMP is secreted by fibroblast that subsequently facilitates the
deposition of collagen in OSF and promotes the development of OSF by binding to matrix
components. The increased expression of fibrogenic cytokines, namely TGF-β was found in
OSF tissues. COMP can be induced to overexpress by TGF-β treatment suggesting that
increased TGF-β might also provoke the synthesis of COMP in OSF thus giving indirect
evidence for enhanced TGF-β activity in OSF. 4
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3)
In another study an attempt was made to study the molecular mechanisms and role of
arecoline, an alkaloid in conferring gene expression changes that may lead to the initiation
and progression of oral sub mucous fibrosis. The fibroblasts were treated with arecoline and
a variety of assays including RT-PCR analysis of mRNA of several genes, cell cycle
analysis and other cellular and molecular methods have been employed. The important
inference is the possible paracrine influence of TGF-β isoforms secreted by epithelial cells
on the oral fibroblasts in determining the progression of OSF. 5
4)
Following activation of the TGF-β receptors, intracellular signal transduction is
mediated by a variety of Smad proteins. Smad-3 acts as an activator of signal transduction,
whereas Smad-7 has an inhibitory effect. This study evaluated the biological effect of
neutralizing TGF-β1 antibodies, the relationship between TGF-β1 and Smad-3/7 expression
and changes of collagen synthesis, following inhibition of TGFβ1 activity in irradiated rat
tissue. Following anti-TGF-β1 treatment, an attenuated expression of TGFβ1, a reduction in
EMC synthesis and fibrosis could be observed in irradiated tissue compared to the irradiated
tissue without anti-TGFβ1-treatment. While preoperative RT increases the expression of
Smad-3/7 proteins, an up regulation of Smad-7 from day 3 until day 14 following surgery
and a down regulation of Smad-3 on day 14 after surgery could be observed following antiTGF-β1-treatment. The samples which were irradiated alone displayed a reduced signal for
Smad-7 mRNA and an induction of Smad-3 protein phosphorylation shown by
nucleocytoplasmic shuttling. In contrast, the anti-TGFβ1-treated samples showed an
increase in Smad-7 mRNA and a downregulation of Smad-3 phosphorylation. Prolylhydroxyprolinase-β expression and collagen I synthesis were reduced following antiTGFβ1-treatment. A reduction of Smad-3 proteins in parallel with an increase of Smad-7
may contribute to the inhibitory effect and the reduction of ECM synthesis after blocking of
TGF-β1 activity by treatment with neutralizing antibodies. This may indicate a molecular
mechanism in the therapeutic intervention to reduce fibrosis, hypertrophic scar formation
and chronic wound healing disorders.6
5
5) The objective of this review are to highlight molecular events involved in the
overproduction of insoluble collagen and decrease degradation of collagen occurring via
exposure to betel quid and stimulation of TGF-β pathway and elucidate the cell signaling
that in involved in the etiopathogenesis is of the disease of the process. TGF-β activates the
procollagen , also induces the secretion of Pro-Collagen C- proteinases (PCP) and ProCollagen N –Proteinase (PNP), both of which are required for the conversion of procollagen fibrils. In OSF , there is increased cross- linking of the collagen, resulting in
increased insoluble form. This is facilated by increased activity and production of a key
enzyme – lysyl oxidase (LOX) . PCP / Bone Morphogenetic Protein 1 (BMP1) and
increased copper (Cu)
in Betel Quid (BQ) stimulate LOX activity, a key player in the
pathogenesis of this disease. The flavonoids increase cross-linking in the collagen fibres.
These steps results in increase collagen production. 7
6.3 OBJECTIVES OF THE STUDY:.
1) To identify and correlate the expressions of Transforming Growth Factor-beta ( TGFβ1) in various stages of Oral Submucous Fibrosis in paraffin embedded sections.
2) To identify and correlate the expression of TGF- β1 in the oral mucosa in the individuals
with habits, but without clinical evidence of OSF, in the paraffin sections.
3) To identify and correlate the expression of TGF-β1 in other collagen disorders such as
keloid, scleroderma, scar etc in the paraffin section.
4) To identify and correlate TGF-β1 in paraffin sections of normal oral mucosa.
5) To evaluate if possible the relevant genetic changes associated with TGF-β1 in OSF.
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7. MATERIALS AND METHODS
7.1 SOURCES OF DATA
Paraffin embedded tissues from cases which satisfy the criteria will be included in
the study. Though archival sections would be the target, fresh biopsies would be taken
if the need arises in all the mentioned groups. Routine procedures of consent and
patient information and clearances from the Ethical Research Committee will be
ensured.
7.2 METHOD OF COLLECTION OF DATA
1)
Individual with tobacco and arecanut related habits and clinically proven
cases of OSF in paraffin sections- 50
2) Cases of the individual with tobacco and arecanut related habits but no
clinically proven OSF in paraffin sections – 50
3) Normal individual with no tobacco and arecanut related habits in paraffin
embedded sections- 25
4) Paraffin sections of other collagen disorders such scleroderma etc – Maximum
number attainable
METHODOLOGY:
All the slides will be stained and diagnostically verified by a group of independent
observers.
1.The slides are to be stained with hematoxylin and eosin for routine diagnosi; for
collagen with Van Gieson and/or Masson Tricome.
2. Immunohistochemistry (IHC) kit procured from manufacturer will be utilized for
the expression antigen–antibody reaction. Staining protocol of IHC to be followed as
per the manufacturer’s instruction.
3. All assessment to be done in standard light microscope. Areas of expression of
immunomarker will be assessed visually and by image analysis software
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7.3 Does the study require any investigation or interventions to be conducted on
patients or other human or animals? If so, please describe briefly.
7.4 Has the ethical clearance obtained from your institution in case of 7?
Yes
8. LIST OF REFERENCES
1) .Rajendran. “Oral
Submucous Fibrosis: Etiology, pathogenesis, and future
research”. Bulletin of the World Health Organization, 72 (6); 985-996.
2) World Health Organization- Guide to epidemiology and diagnosis of oral mucosal
disease and conditions. Community dent. Oral Epidemol 1980.8:1-26.
3) Y Gao, T Ling, H Wu. “ Expression of transforming Growth factor beta 1 in
keratinocytes of oral submucous fibrosis tissue” Zhonghua Kou Qiang Yi Xue Zazhi;
1997 July;32(4); 239-41
4) Stephen Schuitz-Mosqau, Marcel A Blaese, Gerhard Grabenbauer, Falk
Wehrhan
,Jurgen Kopp, Kertin Amann, H. Peter Rodemann, Franz Rodel. “ Smad-3 and Smad7 expression following Anti-Transforming Growth Factor beta (TGF β) treatment in
irradiated rat tissue” Radiotherapy & Oncology; March 2004; Vol 70; Issue 3; Page
249-259.
5)
Singh, Thangam Gajan.
“Molecular Action of
Arecoline , An Alkaloid
Implicated In the Manifestation of Oral Submucous Fibrosis” Electronic Theses and
Dissertation of Indian Institute of Science; Issue Date -21-Jun-2010 ; Series/Report
No: G22434
6) Chung- Jung Chiu, Min- Lee, Chun- Pinchiang, Liang-Jiunn, Ling-Ling
Hsieh, and Chein – Jen Chen. “Interaction of collagen related genes and susceptibility
to Betel Quid – Indu oral Submucous Fibrosis” Cancer Epidemology, Biomarkers &
Prevention; voII, July 2002, 646-653.
7) P. Rajlalitha , S. Vali
Molecular Pathogenesis of Oral Submucous fibrosis – a
collagen metabolic disorder. J Oral Pathol Med (2005) 34; 321-8.
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9. SIGNATURE OF THE CANDIDATE
10. REMARKS OF THE GUIDE
11. NAME AND DESIGNATION OF (IN BLOCK LETTERS):
11.1 GUIDE:
Dr. V.V. KAMATH
PROFESSOR AND HEAD,
DEPT. OF ORAL AND
MAXILLOFACIAL PATHOLOGY
Dr SYAMALA REDDY DENTAL
COLLEGE, HOSPITAL & RESEARCH
CENTRE,
BANGALORE-560037
11.2 SIGNATURE:
9
Dr. NAGARAJA . A
11.3 CO-GUIDE, IF ANY:
READER,
DEPT. OF ORAL AND
MAXILLOFACIAL PATHOLOGY
Dr SYAMALA REDDY DENTAL
COLLEGE, HOSPITAL & RESEARCH
CENTRE,
BANGALORE-560037
11.4 SIGNATURE:
Dr. V.V. KAMATH
11.5 HEAD OF THE DEPARTMENT
PROFESSOR AND HEAD,
DEPT. OF ORAL AND
MAXILLOFACIAL PATHOLOGY
Dr SYAMALA REDDY DENTAL
COLLEGE, HOSPITAL & RESEARCH
CENTRE,
BANGALORE-560037
11.6 SIGNATURE
12. REMARKS OF THE CHAIRMAN
AND PRINCIPAL
12.1 SIGNATURE OF THE CHAIRMAN
AND PRINCIPAL
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