AECS Maaruti College of Dental Sciences and Research Centre

Rajiv Gandhi University of Health Sciences

Bangalore

Master of Dental Surgery (MDS) Periodontology

AECS Maaruti College of Dental Sciences and Research Centre, Bangalore-76. Rajiv Gandhi University of Health Sciences, Karnataka Bangalore

ANNEXURE II

PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION

DR. MUDIGAL VATSALA 1. Name of the candidate and address DEPARTMENT OF PERIODONTOLOGY (in block letters) A.E.C.S. MAARUTI COLLEGE OF DENTAL SCIENCES AND RESEARCH CENTRE, BANGALORE-76.

2. Name of the institution A.E.C.S. MAARUTI COLLEGE OF DENTAL SCIENCES AND RESEARCH CENTRE, BANGALORE-76.

3. Course of study and subject MASTER OF DENTAL SURGERY (MDS) PERIODONTOLOGY.

4. Date of admission to course 10th JUNE 2013

The association of the Receptor for 5. Title of the topic Advanced glycation endproducts (RAGE) with S100 expression in the gingival tissues of type 2 diabetic patients with chronic periodontitis.

BRIEF RESUME OF THE INTENDED WORK: 6.1 Need for the study

The term “diabetes mellitus” is used to identify a group of disorders characterized by elevated levels of glucose in the blood. This elevation is the result of a deficiency in insulin secretion or an increased cellular resistance to the actions of insulin, leading to a variety of metabolic abnormalities involving carbohydrates, fats and proteins. A number of pathological mechanisms related to elevated levels of glucose in the blood have been defined, including the activation of the sorbitol pathway, the formation of Advanced glycation endproducts (AGEs), the damaging effect of oxidative stress and altered lipid metabolism. These mechanisms have been associated with classical clinical complications of diabetes mellitus such as retinopathy, nephropathy, macrovascular disease and poor wound healing.1

The formation of AGEs occurs when excess available glucose is in contact with structural and other proteins. This process is not driven enzymatically and once they are formed, AGEs bind to a specific cellular receptor, known as Receptor for advanced glycation end products (RAGE).1

Periodontal disease is a chronic inflammatory condition characterized by destruction of the periodontal tissues and resulting in loss of connective tissue attachment, loss of alveolar bone and the formation of pathological pockets around the diseased teeth.2

Periodontitis is considered to be the sixth complication of diabetes along with retinopathy, nephropathy, neuropathy, microvascular disease and impaired wound healing. RAGE plays an important role in the pathogenesis of diabetic complications.3

RAGE was first described in 1992 and since then attracted increasing attention due to its involvement in various diseases including diabetic complications, tumor outgrowth, chronic inflammation and neurodegenerative disorders like Alzheimer disease or

Multiple sclerosis.4

RAGE is a member of the immunoglobulin superfamily of cell surface receptors that bind to a broad range of ligands. RAGE is an integral membrane protein composed of three extracellular immunoglobulin-like domains, a single transmembrane - spanning region and a short negatively charged cytosolic tail domain responsible for initiating

RAGE signalling.3

RAGE is found on endothelial cells and monocytes, which is of importance in

1 periodontitis. The binding of AGE to RAGE accelerates vascular disease and contributes to sustained inflammation associated with impaired wound healing. Multiple studies have demonstrated that the AGE–RAGE interaction results in such disorders as diabetes, Rheumatoid arthritis and Sjogren’s syndrome suggesting that the engagement of RAGE by AGE involves inflammatory responses that may trigger chronic diseases.5

In addition to AGEs, other endogenous ligands are also implicated in amplifying

RAGE-dependent pro-inflammatory signalling, prominent among them being, cytokine- like mediators of the S100 family, amphoterin, amyloid β peptide (Aβ), β-sheet fibrils and Mac-1.3

S100 protein family consists of 21 members. S100s are small proteins (9–13 kDa) that bind calcium via EF hand motifs (Calcium-binding motifs composed of two helixes E and F joined by a loop) and are exclusively expressed in vertebrates. The expression of

S100 family members is restricted in cell and / or tissue type.4 These proteins are called

S100 because of their solubility in a 100% saturated solution with ammonium sulphate at neutral pH. They were first identified by B. W. Moore in 1965. They are acidic proteins that contain two distinct EF-hands, 4 α-helical segments, a central hinge region of variable length and the N- and C- terminal variable domains. At present at least 25 proteins have been identified as belonging to S100 protein family.6

S100 is a calcium-binding protein specifically expressed by granulocytes that has been shown to exert regulatory effects on inflammatory cells. Extracellular S100 exhibits a wide range of pro-inflammatory functions, including the regulation of cell proliferation and activation, apoptosis and potent chemotactic activity, compared with other chemotactic agents. S100 is involved in Alzheimer’s disease, cardiomyopathy and atherosclerosis. Therefore, it is implicated in the regulation of a variety of cellular activities and plays a pivotal role during inflammation. The binding of S100 to RAGE activates NF- kB, which mediates its pro-inflammatory properties.5 Growing evidence suggests that all secreted S100 proteins act in either an autocrine or paracrine manner through a common receptor, RAGE.4

RAGE is a central cell surface receptor for EN-RAGE (extracellular newly identified

RAGE-binding protein) and related members of the S100/calgranulin superfamily.

Interaction of EN-RAGEs with cellular RAGE on endothelium, mononuclear phagocytes and lymphocytes triggers cellular activation , with generation of key pro- inflammatory mediators. Blockade of EN-RAGE/RAGE quenches delayed-type hypersensitivity and inflammatory colitis in murine models by arresting activation of central signaling pathways and expression of inflammatory gene mediators. These data highlight a novel paradigm in the inflammation and identify roles for EN-RAGE and

RAGE in chronic cellular activation and tissue injury.7

Thus, the RAGE–S100 complex, as well as the RAGE–AGE interaction, participate in important immunological processes and play a role in regulating inflammatory reactions.5 Despite their importance in linking calcium homeostasis and cell function, very little information is available regarding the effects of diabetes on the function of the S100 family of calcium receptor proteins.8

We hypothesize that the severity of periodontitis in diabetic patients may be attributed, at least in part, to the different levels of RAGE and S100 expression in diabetic gingiva.

Hence the objective of this study is to determine the presence and association of RAGE and S100 protein in the gingival tissue samples derived from healthy controls, chronic periodontitis and Type 2 Diabetes mellitus affected subjects. 6.2 Review of literature

This study9 was done to test the hypothesis that activation of RAGE contributes to the pathogenesis of diabetes-associated periodontitis. Diabetes was induced in mice by intraperitoneal injection of Streptozotocin, diluted into sterile citrate buffer. Controls were injected with citrate buffer alone. Diabetic mice were infected with human periodontal pathogen Porphyromonas gingivalis and were treated with soluble RAGE

(sRAGE). sRAGE is the extracellular domain of the receptor, which binds ligand and blocks interaction with and activation of cell-surface RAGE. Blockade of RAGE diminished alveolar bone loss in a dose-dependent manner. Moreover, there was decreased generation of the pro-inflammatory cytokines TNFα and IL-6 in gingival tissue, as well as decreased levels of matrix metalloproteinases. Gingival AGEs were also reduced in mice treated with sRAGE, paralleling the observed suppression in alveolar bone loss. The study concluded that there is link between RAGE and exaggerated inflammatory responses to the pathogenesis of destructive periodontal disease in diabetes.

The study10 aimed to test hypothesis that interaction of AGEs with RAGE on endothelial cells enhance vascular activation. AGEs, RAGE, vascular cell adhesion molecule-1, intercellular adhesion molecule-1 and E-selectin are expressed in an overlapping manner in human inflamed rheumatoid synovia, especially within the endothelium. In primary cultures of human saphenous vein endothelial cells, engagement of RAGE by heterogeneous AGEs or N (carboxymethyl) lysine-modified adducts enhanced levels of mRNA and antigen for vascular cell adhesion molecule-1, intercellular adhesion molecule-1 and E-selectin. AGEs increased adhesion of polymorphonuclear leukocytes to stimulated endothelial cells in a manner reduced on blockade of RAGE. The study concluded that AGEs, through RAGE, may prime proinflammatory mechanisms in endothelial cells, thereby amplifying pro-inflammatory mechanisms in atherogenesis and chronic inflammatory disorders.

The study11 aimed to evaluate the presence and distribution of CD1a and S100 protein markers in states of gingival health and chronic periodontitis patients in human subjects.

Gingival tissue samples were derived from from 10 healthy and chronic periodontitis affected human subjects. The presence and distribution of CD1a and S100 protein was assessed using immunohistochemistry and the cell types involved in their expression were assessed. The study concluded that there is translation from CD1a - positive

Langerhans cells to S100 - positive Dendritic cells from epithelium to connective tissue in response to an antigenic challenge. Demonstration of increased number of

S100 - positive DCs in the gingival connective tissue in chronic periodontitis suggests their involvement in bone resorption in addition to their antigen presentation property.

The study was3 was done to investigate a possible link between RAGE expression and the morphological changes seen in the gingiva of diabetic and periodontitis patients.

Gingival biopsies from 15 diabetic patients with periodontitis, 25 non diabetic patients with periodontitis and 10 healthy individuals were collected. Sections were stained with haematoxylin and eosin. Sections were detected for RAGE by immunohistochemistry.

Samples were examined under light microscope and fluorescence microscope. The study concluded that RAGE is strongly expressed in gingiva of diabetic patients with periodontitis and severe periodontitis alone, the later indicating RAGE activation even in absence of hyperglycemia. The aim of this study12 was to investigate the distribution of S100A8 and S100A9 expression in gingival epithelium of mice in the presence and absence of infection. A quantitative analysis of S100A8 and S100A9 mRNA in junctional epithelium (JE) and oral gingival epithelium (OGE) of both germfree mice and conventional mice was performed using laser micro dissection and real-time polymerase chain reaction (PCR).

Confirmation of S100A8 and S100A9 mRNA expression in the JE was conducted by fluorescent immunohistochemistry. The study concluded that S100A8 protein is expressed in JE cells of mice in the presence and in the absence of infection with oral bacteria. S100A9 expression in JE cells in the presence of micro flora is significantly increased compared with the absence of microflora, which suggests that S100A9 expression may be induced by infection of micro flora. The production of calprotectin in gingival epithelial cells may be mediated through S100A9 induction by bacterial infection.

6.3 Objectives of the study

The objective of this study is to determine the presence and association of RAGE and

S100 protein in the gingival tissue samples derived from healthy controls, chronic periodontitis and Type 2 Diabetes mellitus affected subjects. 7. MATERIAL AND METHODS 7.1

Source of data

Patients who have been diagnosed with chronic periodontitis based on the 1999

American Academy of Periodontology Workshop classification13 and Type 2 Diabetes

mellitus based on the criteria of WHO (2011) will be included in the study. 14

Gingival tissue sample collection:

Tissue samples used in this study will be obtained from patients visiting the A.E.C.S

Maaruti College of Dental Sciences and Research Centre, Bangalore during Periodontal

surgery, Surgical extraction or Preprosthetic surgery.

7.2 Method of collection of data:-

Study population:

A total of 80 Indian adults (male) in the age group of 35 to 70 years will be recruited

with written informed consent after being advised about the nature of the study

according to a protocol approved by the Ethics Committee of the A.E.C.S Maaruti

College of Dental Sciences and Research Centre, Bangalore.

The study will be conducted in accordance with the Helsinki Declaration of 1975, as

revised in 2000.

Enquiry Questionnaire Medical history will be assessed by interview before the periodontal examination. Each patient will be asked to complete a questionnaire which includes age, gender, oral hygiene habits, diabetes history, smoking history, medications used and family history.

The smoking status of the patient will be assessed and defined as never, former or current smoker.

Assessment of smoking status will be performed according to the criteria established by the Centres for the Disease Control and prevention (CDC).15

 The non smoker’s category will include never smokers and former smokers.

Never smokers are adults who had not smoked 100 cigarettes in their lifetime.

Former smokers are adults who smoked in past, but quit smoking prior to date of interview.

 The smoker’s category will include current smokers.

Current smokers are adults who have smoked at least 100 cigarettes in their lifetime and will be smokers at the time of interview.

The body mass index will be estimated by dividing the body weight (in kilograms) by square of height (in meters). Obesity will be defined as BMI ≥ 30 while being overweight will be defined as BMI 25 – 29.9 and normal weight will be defined as BMI ranging from 20 to 24.9 kg/m2.

Only male patients will be recruited for the study to avoid the possible adverse effects of female ovarian hormones that could increase the gingival inflammation. Patient participation will be limited to data collection and gingival biopsy. Gingival tissue samples (1 per person) will be collected from the following groups.

Group I – 20 healthy individuals without chronic periodontitis or diabetes mellitus/ any systemic disease. (HbA1C levels < 6.5% and FBS levels < 126 mg/dl).

Group II – 20 patients with chronic periodontitis and without diabetes mellitus/ any other systemic disease.

Group III– 20 patients with proven type 2 diabetes mellitus without chronic periodontitis (HbA1C levels ≥6.5% and FBS levels ≥126 mg/dl).

Group IV – 20 patients with chronic periodontitis and proven type 2 diabetes mellitus.

The specimens will be collected under local anesthesia and following strict aseptic procedures from the buccal or lingual or interdental gingival using a surgical blade. All the excised tissue samples will be washed in sterile saline solution to remove blood and will be fixed in buffered formalin solution.

These specimens will be dehydrated in ascending grades of ethyl alcohol, cleared in xylene and embedded in paraffin. Sections of around 4-5µm in thickness will be obtained and collected on positively charged slides. Tissue sections will be deparaffinized and rehydrated before histological staining and immunolabeling.

Inclusion criteria:

Inclusion criteria for the diagnosis of generalized chronic periodontitis will be attachment loss ≥5mm at more than 30% of the sites in patients aged ≥35 years and who will show radiographic evidence of bone loss. Subjects will be included only if they have ≥20 functional teeth.

Periodontally healthy subjects will show no evidence of periodontal disease (probing depth ≤3mm, no attachment loss, without bleeding on probing, with no detectable radiographic alveolar bone loss).

The diagnosis of patients with type 2 diabetes will be based on the criteria of the World

Health Organization as described previously.15 The glycemic status of patients previously diagnosed with T2DM will be confirmed by their glycosylated hemoglobin

(HbA1C) and fasting blood sugar (FBS) levels.

Exclusion criteria

Subjects will be excluded if

1) They suffer from any systemic condition (except diabetes mellitus) that could

affect the progression of periodontal disease (for example immunological

disorders), smokers and former smokers, alcoholics and former alcoholics and

obesity.

2) There is a presence of an active infection other than periodontitis.

3) They are Women, to avoid the possible adverse effects of female ovarian

hormones that could increase the gingival inflammation.

4) There is an intake of antibiotics / corticosteroids and or non- steroidal anti-

inflammatory drugs during the previous 4 weeks.

5) They had received professional periodontal treatment during the 6 month period

prior to the study. 6) Subjects with periapical pathology, orthodontic appliances and multiple systemic

complications of diabetes mellitus will also be excluded from the study.

Clinical Periodontal Examination

A single, calibrated examiner will complete a full mouth assessment of periodontal conditions, except for third molars, using a Williams graduated manual probe (Hu

Friedy, Chicago, IL).

The following parameters will be assessed at six sites per tooth (mesio-buccal, mid- buccal, disto-buccal, mesio-lingual, mid-lingual, disto-lingual).

1) Gingival index16(LOE AND SILNESS,1963)

2) Probing pocket depth (PD) in mm from gingival margin to the base of the

gingival sulcus.

3) Clinical attachment level (CAL) in mm from CEJ to the base of the gingival

sulcus.

Fasting Plasma Glucose (FPG) and HbA1c levels

Venous blood samples will be collected from all the patients prior to obtaining the biopsy to confirm their diabetic or non-diabetic status.

10ml of venous blood sample will be collected from 8 a.m. to 10 a.m. and will be analyzed by a single laboratory technician.

Glucose levels will be measured by an automated enzymatic method and will be expressed in milligrams per deciliter (mg/dl). The HbA1c values will be measured by high performance liquid chromatography and will be expressed as a percentage.

Histological examination:

A Set of sections from all the four groups will be stained with Heamotoxylin and Eosin solutions ( Sigma, St. Louis, MO, USA), dehydrated, mounted and examined in a light microscope ( BX41 Olympus, Tokyo, Japan) equipped with digital camera and image analysis software for histologic evaluation of morphologic changes.

Sectioned areas of embedded samples will be randomly selected, at least 3 different places in one sample block will be examined by two independent observers.

Immunohistochemical staining :

Another set of deparaffinized sections will be labelled for localization of RAGE

(monoclonal Anti - RAGE antibody, Chemicon International, Inc, Tamecula, CA,

USA) and S100 ( Anti human S100 protein, polyclonal, AR058; Biogenix Co

Duiven, The Netherlands) in the gingival tissue using an indirect immunoperoxidase technique according to the manufacturer’s instructions. Normal mouse IgG and normal goat IgG antibodies will be co-incubated for a negative control in place of the primary antibodies.

Histomorphometric analysis:

Image analysis will be performed using microscope equipped with digital camera

ProgRes C3 and software. The image analyzer will be calibrated automatically to convert the measurement units (pixels) produced by the image analysis program into actual micrometer units.

In the hematoxylin and eosin stained sections, the image analysis system will be used to count the number of inflammatory cells using 40x objective lens to assess the degree of inflammation observed during microscopic examination.

In the immunohistochemically labelled sections, the image analysis will be used to assess the optical density of the S100 and RAGE immunoreactivity in the epithelial and inflammatory cells using 40x objective lens.

The specimens will be scored by counting the number of immunoreactive cells over 5 different fields of vision in one section. All immunolabelled sections will be examined in a masked fashion by two independent observers (oral pathologists) and scored. The scores given by the observers for each section will be averaged and used in data analysis. The examination will include the presence or absence of immunoreactiviy, the intensity of immunoreactiviy and the homogenous or heterogenous characterization of staining.

Statistical analysis:

Data obtained from histomorphometric analysis will be statistically described in terms of mean ± SD. Statistical analysis will be performed using SPSS (Statistical pakage for social science ; SPSS Inc, Chicago, IL, USA) version 15 for Microsoft windows.

Differences among RAGE and S100 expressing cells will be analysed using ANOVA followed by Bonferroni test. Differences will be considered singinficant at P ≤0.05.

The percentage of immunoreactive cells will be calculated as number of both RAGE and S100 expressing cells divided by the total number of cells. Differences amongst

RAGE / S100 expressing cells will be analysed using the student T test.

7.3 Does the study require any investigation or intervention to be conducted on patients or other humans or animals (If so please describe briefly):

Gingival biopsies will be obtained for estimation of RAGE and S100.

Venous blood samples will be obtained.

Clinical parameters will be assessed. 7.4 Has ethical clearance been obtained from your institution in case of 7.3

7.5 Any trial test has been carried out?

8. LIST OF REFERENCES

1) Lamster I B, Lalla E, Borgnakke W S, Taylor G W. The relationship

between oral health and diabetes mellitus. JADA 2008;139:19S-24S.

2) Loe H. Periodontal disease : The sixth complication of diabetes mellitus.

Diabetes care 1993;16:329-334.

3) Abbassa M M, Koranya N S, Salama A H , Dmytryk J J, Ahmed H S,

Mroczka B S. The relationship between receptor for Advanced Glycation end

products expression and the severity of periodontal disease in the gingiva of

diabetic and non-diabetic periodontitis patients. Arch Oral Biol 2012;57:1342- 1354.

4) Leclerc E, Fritz G, Stefan W V, Heizmann C W. Binding of S100 proteins to

RAGE : An update. Biochim Biopys Acta 2009;1793:993-1007.

5) Takeichi O, Hatori K, Kamimoto A, Oka S, Ogiso B, Saito I. Receptors for

advanced glycation end products (RAGE) expressing Endothelial cells co-

express AGE and s100 in human periapical granulomas. J Dent 2011;39: 679-

6) Sedgath F, Notpoulos A. S100 protein family and its applications in clinical

practice. Hippokratia 2008;12:198-204.

7) Marion A, Dury S, Caifeng F et al. RAGE mediates a Novel proinflammatory

axis, A central cell surface receptor for s100/calgranulin polypeptides. Cell

1999; 97:889-901.

8) Danna B, Zimmer , chessher J, Glenn L, Wilson and Zimmer W E. S100A1

and S100B Expression and Target Proteins in Type I Diabetes S100A1 and

S100B Expression and Target Proteins in Type I Diabetes. Endocrinology

1997;138:5176-5183.

9) Lalla E, Lamster I B, Feit M et al. Blockade of RAGE suppresses

periodontitis-associated bone loss in diabetic mice. J Clin Invest 2000;105:1117-

10) Basta G, Lazzererin G, Massaro M et al. Advanced Glycation End Products

Activate Endothelium Through Signal-Transduction Receptor RAGE A Mechanism for Amplification of Inflammatory Responses. Circulation 2002;

105:816-822.

11) R Anjana, L D Joseph , R Suresh. Imunohistochemical localization of CD1a

and S100 in gingival tissues of healthy and chronic periodontitis. Oral Dis

2012;18:778- 785.

12) Nishii K, Yamamoto G, Yajima et al. The distribution and expression of

S100A8 and S100A9 in gingival epithelium of mice. J Periodontal Res 2013;

48:235–242.

13) Flemmig T F. Periodontitis. Ann Periodontol 1999;4:32-38.

14) World Health Organization. Use of Glycated Haemoglobin (HbA1c) in the

Abbreviated report of a WHO consultation. WHO/NMH/CHP/CPM/11.1.

Geneva, World Health Organization, 2011.

15) Schoenborn CA, Vickerie JL, Barnes PM. Cigarette smoking behavior of

adults: United States, 1997-98. Centers for disease control (CDC)2003; 331:1-

16) Loe, H. & Silness, J. Periodontal disease in pregnancy I. Prevalence and

severity. Acta Odontol Scand 1963;21:533–551. 9 Signature of candidate

10. Remarks of guide This is a genuine study which will be carried out by the post graduate student under my supervision and guidance.

11. Name and designation (in block letters)

11.1 Guide DR. ASHISH S.NICHANI PROFESSOR DEPARTMENT OF PERIODONTOLOGY A.E.C.S. MAARUTI COLLEGE OF DENTAL SCIENCES AND RESEARCH CENTRE, BANGALORE-76. 11.2 Signature

11.3 Co – Guide (if any) DR. RAJESH NAIK PROFESSOR DEPARTMENT OF PERIODONTOLOGY A.E.C.S. MAARUTI COLLEGE OF DENTAL SCIENCES AND RESEARCH CENTRE, BANGALORE-76. 11.4 Signature

11.5Head of department DR. V. RANGANATH

11.6 Signature

12. 12.1Remarks of chairman principal

12.2 Signature