IFI16 is a Periodontitis Modulating Protein
Phase: NA
Conditions: Severe Periodontitis, Acute Inflammatory Response
Sponsor: University of North Carolina, Chapel Hill

 
 
IFI16 is a Periodontitis Modulating Protein 
 
 
NCT number NCT03513497 
Document Date 03/06/2022 
 

IFI16 is a periodontitis modulating protein 
Version 8.0 
Protocol 
March 6, 2022 
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IFI16 is a Periodontitis Modulating Protein 
NIDCR Protocol Number: 17-068-E  
NIDCR Grant Number: 1K01DE027087-01 
Principal Investigator:  Julie Teresa Marchesan, DDS, PhD 
NIDCR Program Official: Lynn King, PhD 
NIDCR Medical Monitor: Kevin D. McBryde, MD  
 
Version Number:  8.0 
March 6, 2022

IFI16 is a periodontitis modulating protein 
Version 8.0 
Protocol 
March 6, 2022 
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Based on NIDCR Clinical Study (Observational) Protocol Template v4.0 - 20140106 
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STATEMENT OF COMPLIANCE 
The study will be conducted in accordance with the International Conference on 
Harmonisation guidelines for Good Clinical Practice (ICH E6), the Code of Federal 
Regulations on the Protection of Human Subjects (45 CFR Part 46), and the NIDCR 
Clinical Terms of Award.  All personnel involved in the conduct of this study have 
completed human subject’s protection training.  
 The investigator will ensure protection of subject personal data and will not include 
subject names on any forms, reports, publications or in any other disclosures. The 
Investigator will ensure that each study subject, or his/her legally acceptable 
representative, is fully informed about the nature and objectives of the study and 
possible risks associated with participation. The Investigator, or a person designated by 
the Investigator, will obtain written informed consent from each subject, or the subject's 
legally acceptable representative, before any study-specific activity is performed. The 
informed consent form used in this study, and any changes made during the course of 
the study, must be prospectively approved by local Institutional Review Board (IRB) 
before use. The Investigator will retain the original of each subject's signed consent 
form. 
Individuals will be given an Informed Consent document to read and sign. Before 
consent is obtained, the Investigator, or a person designated by the Investigator, will 
provide the subject with ample time and opportunity to inquire about the details of the 
research study, and to decide whether or not to participate. All questions about the 
study should be answered to the satisfaction of the subject. The subject will have all of 
their study related questions answered by the Investigator or designated staff, and if 
they agree, they will sign the Informed Consent document.  
Details on the administrative structure of this study (e.g., principal Investigator, sub- 
Investigator, steering committee, administration, monitoring and evaluation committees, 
institutions, statistician, central laboratory facilities, external service provider (ESP or 
CRO), or clinical study supply management) will be maintained in the Investigator’s 
Master File throughout the study for inclusion in the clinical study report. 

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SIGNATURE PAGE   
The signature below constitutes the approval of this protocol and the attachments, and 
provides the necessary assurances that this study will be conducted according to all 
stipulations of the protocol, including all statements regarding confidentiality, and 
according to local legal and regulatory requirements and applicable US federal 
regulations and ICH guidelines. 
Principal Investigator or Clinical Site Investigator: 
Signed: 
 
Date:  
 
 
Julie Teresa Marchesan, DDS, PhD  
Research Assistant Professor – Dept. of Periodontology 
School of Dentistry – University of North Carolina at Chapel Hill 
115 Brauer Hall – CB# 7450 
Chapel Hill, NC 27599 
Office: (919) 537-3853 
Fax: (919) 843-1798 
Email: julie_marchesan@unc.edu 
 
 
 
 

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TABLE OF CONTENTS 
 
 
CONTENTS 
STATEMENT OF COMPLIANCE .................................................................................................. I 
SIGNATURE PAGE ...................................................................................................................... II 
TABLE OF CONTENTS ............................................................................................................... III 
LIST OF ABBREVIATIONS .......................................................................................................... V 
PROTOCOL SUMMARY ............................................................................................................. VI 
1 
KEY ROLES AND CONTACT INFORMATION .................................................................... 1 
2 
INTRODUCTION:  BACKGROUND INFORMATION AND SCIENTIFIC RATIONALE ........ 3 
2.1 
Background Information ........................................................................................... 3 
2.2 
Rationale .................................................................................................................. 3 
2.3 
Potential Risks and Benefits ..................................................................................... 5 
2.3.1 
Potential Risks .............................................................................................. 6 
2.3.2 
Potential Benefits .......................................................................................... 7 
3 
OBJECTIVES ....................................................................................................................... 8 
3.1 
Study Objectives ...................................................................................................... 8 
3.2 
Study Outcome Measures ........................................................................................ 8 
4 
STUDY DESIGN .................................................................................................................. 9 
5 
STUDY ENROLLMENT AND WITHDRAWAL ................................................................... 10 
5.1 
Subject Inclusion Criteria ........................................................................................ 10 
5.2 
Subject Exclusion Criteria ...................................................................................... 10 
5.3 
Strategies for Recruitment and Retention .............................................................. 11 
5.4 
Subject Withdrawal ................................................................................................. 12 
5.4.1 
Reasons for Withdrawal ............................................................................. 12 
5.4.2 
Handling of Subject Withdrawals ................................................................ 12 
5.5 
Premature Termination or Suspension of Study .................................................... 13 
6 
STUDY SCHEDULE .......................................................................................................... 14 
6.1 
Visit 1: Screening/enrollment .................................................................................. 14 
6.2 
Visit 2: Baseline ...................................................................................................... 15 
6.3 
Visit 3 ...................................................................................................................... 16 
6.4 
Visit 4 ...................................................................................................................... 16 
6.5 
Visit 5 ...................................................................................................................... 17 
6.6 
Visit 6: Final Visit (Post-biopsy follow-up; Prophylaxis; SRP may be completed 
up to visit 8 based on individual needs) ................................................................. 18 
6.7 
Withdrawal Visit ...................................................................................................... 19 
6.8 
Unscheduled Visit ................................................................................................... 19 
7 
STUDY PROCEDURES/EVALUATIONS .......................................................................... 20 
7.1 
Study Procedures/Evaluations ............................................................................... 20 
7.2 
Laboratory Procedures/Evaluations ....................................................................... 22 
7.3 
Study Specific Biospecimens ................................................................................. 22 
7.3.1 
Specimen Collection Procedures ............................................................... 22 

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Specimen Collection Procedures ........................................................................... 22 
7.3.2 
Specimen Preparation, Handling, and Storage .......................................... 23 
7.3.3 
Specimen Shipment ................................................................................... 24 
7.4 
Questionnaire Administration ................................................................................. 24 
8 
ASSESSMENT OF SAFETY .............................................................................................. 25 
8.1 
Specification of Safety Parameters ........................................................................ 25 
8.1.1 
Unanticipated Problems ............................................................................. 25 
8.1.2 
Serious Adverse Events ............................................................................. 25 
8.2 
Reporting Procedures ............................................................................................ 26 
9 
STUDY OVERSIGHT ......................................................................................................... 30 
10 
CLINICAL SITE MONITORING .......................................................................................... 31 
11 
STATISTICAL CONSIDERATIONS ................................................................................... 32 
11.1 
Study Hypotheses .................................................................................................. 32 
11.2 
Sample Size Considerations .................................................................................. 33 
11.3 
Final Analysis Plan ................................................................................................. 33 
12 
SOURCE DOCUMENTS AND ACCESS TO SOURCE DATA/DOCUMENTS .................. 35 
13 
QUALITY CONTROL AND QUALITY ASSURANCE ......................................................... 36 
14 
ETHICS/PROTECTION OF HUMAN SUBJECTS ............................................................. 37 
14.1 
Ethical Standard ..................................................................................................... 37 
14.2 
Institutional Review Board ...................................................................................... 37 
14.3 
Informed Consent Process ..................................................................................... 37 
14.4 
Exclusion of Women, Minorities, and Children (Special Populations) .................... 38 
14.5 
Participant Confidentiality ....................................................................................... 38 
14.6 
Future Use of Stored Specimens and Other Identifiable Data ............................... 38 
15 
DATA HANDLING AND RECORD KEEPING .................................................................... 39 
15.1 
Data Management Responsibilities ........................................................................ 39 
15.2 
Data Capture Methods ........................................................................................... 39 
15.3 
Types of Data ......................................................................................................... 39 
15.4 
Schedule and Content of Reports .......................................................................... 39 
15.5 
Study Records Retention ....................................................................................... 39 
15.6 
Protocol Deviations ................................................................................................ 39 
16 
PUBLICATION/DATA SHARING POLICY ......................................................................... 41 
17 
REFERENCES ................................................................................................................... 42 
SUPPLEMENTAL MATERIALS .................................................................................................. 46 
APPENDIX I:  SCHEDULE OF EVENTS .................................................................................... 47 
APPENDIX II:  GINGIVAL BIOPSY ............................................................................................ 48 
 
 
 

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LIST OF ABBREVIATIONS 
AE 
Adverse Event/Adverse Experience 
BOP 
Bleeding on Probing 
CAL 
Clinical Attachment Level 
CFR 
Code of Federal Regulations 
CRF 
Case Report Form 
FF 
Federal Financial Report 
FWA 
FFR 
Federalwide Assurance 
Federal Financial Report 
FWA 
Federalwide Assurance 
GCP 
Good Clinical Practice 
HIPAA 
Health Insurance Portability and Accountability Act  
ICH 
International Conference on Harmonisation 
IRB 
Institutional Review Board  
N 
Number (typically refers to participants) 
NIDCR 
National Institute of Dental and Craniofacial Research 
NIH 
National Institutes of Health 
OHSP 
Office for Human Research Protections  
PPC 
Periodontal Profile Classes  
PI 
Plaque Index 
PD 
Probing Depth 
SAE 
Serious Adverse Event/Serious Adverse Experience 
SIBO 
Stent-Induced Biofilm Overgrowth 
SOP 
Standard Operating Procedure 
SRP 
Scaling and Root Planing 
US 
United States 
 
 

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PROTOCOL SUMMARY 
Title: 
IFI16 is a periodontitis modulating protein 
Précis: 
A total of 72 subjects 18 years or older will be recruited and 
examined for this 1-8 visit, up to 56 days research study. 
Assuming a 10% dropout in both groups, healthy individuals 
(n=36) and individuals with severe periodontal disease (n=36) 
will be evaluated at 2 main timepoints: baseline and 21-days of 
biofilm overgrowth (visit 5). Visit 1 includes enrollment and 
alginate impressions for a stent fabrication; visit 2 includes 
plaque collection, gingival biopsy and delivery of the stent; visits 
3 and 4 are designed for monitoring disease; visit 5 includes 
collection of gingival biopsies and dental plaque and 
discontinuation of the stent usage. Analysis will include 
characterization of the expression of IFI16 and AIM2 (mRNA 
and protein) in gingival tissues in chronic and acute 
inflammation, and further characterization of the main cells 
expressing these proteins. Clinical outcomes will include 
measurements of periodontal disease, including as CAL, PD, 
BOP, GI, and PI. Plaque samples will be evaluated for the 
presence of periodontal pathogens. Visits 6-8 will be completed 
in 30 days from visit 5 and are for prophylaxis and SRP based 
on individual need. 
Objectives: 
Primary: To characterize the expression of IFI16 and AIM2 in 
gingival tissues in an inflammatory response using the stent-
induced biofilm overgrowth model. 
 
 
Population: 
Adult subjects will be recruited from the patients, students and 
staff at the University of North Carolina, as well as the general 
population in or near Chapel Hill NC. Inclusion criteria for 
subjects includes a diagnosis of a healthy and severe 
periodontal disease according to periodontal and tooth profile 
classification system (1). Individuals will be classified based on 
an algorithm that relies on the periodontal clinical parameters, 
presence of teeth and crowns. Subject exclusion criteria include 
a 
history 
of 
autoimmune 
diseases, 
diabetes, 
immunocompromised subjects, neurologic or psychiatric 
disorders, systemic infections, use of chronic medications 

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known to affect the periodontium, smokers, and untreated dental 
conditions. 
Number of Sites: 
General and Oral Health Center, UNC School of Dentistry 
Study Duration: 
60 Months 
Subject 
Participation 
Duration: 
Up to 56 Days 
Estimated Time to 
Complete 
Enrollment: 
36 Months 
 
 
 

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Figure 1: Schematic of Study Design: 
 
Visit 1 
Screening 
/enrollment 
(If eligible,  
Day -28 to -2) 
 
 
Visit 2  
Baseline 
(Day 0±5) 
 
 
 
 
Visit 3 
(Day 7±5) 
 
 
 
Visit 4 
(Day 14±5) 
 
 
 
 
Visit 5 
(Day 21±5) 
 
 
 
 
Visit 6 
(Day 28±5) 
 
 
 
 
 
 
Initial assessments 
(Comprehensive periodontal examination, collection of plaque samples, gingival 
biopsy. Delivery of acrylic stents for the beginning of the stent-induced biofilm 
overgrowth)  
Follow-up assessments 
(Comprehensive periodontal examination, collection of plaque samples, gingival 
biopsy. End of the stent-induced biofilm overgrowth phase, Scaling and root 
planning initiated in stented quadrant of diseased individuals; treatment must be 
completed within 30 days from visit 5 and take no more than 3 additional visits).  
Screening until 72 adults subjects are enrolled. 
(Obtain informed consent, Inclusion & Exclusion Criteria, Oral exam, 
Comprehensive periodontal examination, Pregnancy Test, Determination of 
Eligibility, Alginate impressions for fabrication of acrylic stents) 
Follow-up assessments 
(Comprehensive periodontal examination; post-biopsy follow up).  
 
Follow-up assessments 
(Comprehensive periodontal examination).  
Post-biopsy follow up  
(Prophylaxis/Scaling and root planning finalized in up to visit 8).  

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1 KEY ROLES AND CONTACT INFORMATION 
 
Principal 
Investigator:   
Julie Marchesan, DDS, PhD  
Research Assistant Professor  
School of Dentistry – University of North Carolina at Chapel Hill 
115 Brauer Hall – CB# 7450 
Chapel Hill, NC 27599 
Office: (919) 537-3853 
Fax: (919) 843-1798 
Email: julie_marchesan@unc.edu 
 
NIDCR Program 
Official:   
Lynn Mertens King, PhD 
Chief, Research Training and Career Development Branch  
NIDCR/NIH 
6701 Democracy Blvd, Room 690 
Bethesda, MD 20892 
Phone: (301) 594-5006 
Fax: (301) 402-7033 
Email: lynn.king@nih.gov  
NIDCR Medical 
Monitor:   
Kevin D. McBryde, MD 
Program Director 
Office of Minority Health Research Coordination (OMHRC) 
National Institute of Diabetes and Digestive and Kidney 
Diseases (NIDDK) 6701 Democracy Blvd, Room 906B 
Bethesda, MD 20892 
Phone: (301) 594-9652 
Fax: (301) 594-9358 
Email: kevin.mcbryde@nih.gov 
 
Institutions: 
 
 
School of Dentistry – University of North Carolina at Chapel Hill 
Manning Dr. & Columbia St., CB# 7450 
Chapel Hill, NC 27599 
(919) 537-3737 
 
 
General and Oral Health Center 
2040 First Dental building  
School of Dentistry, CB# 7455 
Sherrill T. Phillips, RDH-BS 
Dental Research Project Manager 
Phone: (919) 537-3422 
Fax: 919) 537-3469  

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E-mail: Sherrill_phillips@unc.edu 
 
Other Key 
Personnel: 
Carol Culver 
Research Operations Director 
School of Dentistry – University of North Carolina at Chapel Hill 
3502 Koury Bld – CB# 7454 
Chapel Hill, NC 27599 
Office: (919) 537-3176 
Fax: (919) 537-3732 
Email: carol_culver@unc.edu 
 
Kevin Moss 
Application Specialist 
3504 Koury Oral Health Sciences Building 
School of Dentistry, CB 7455 
Chapel Hill, NC  27599 -7454 
(919) 537-3200 
Email: kevin_moss@unc.edu 
 
John Preisser, PhD 
Statistician 
Gillings School of Global Public Health 
University of North Carolina at Chapel Hill 
135 Dauer Dr., CB# 7420 
Chapel Hill, NC 27599 
Office: (919) 966-7265 
Email: john_preisser@unc.edu 
 
 
 
Jennifer Webster-Cyriaque, DDS, PhD 
Research Advisory Committee 
School of Dentistry – University of North Carolina at Chapel Hill 
Koury Building – CB# 7455 
Chapel Hill, NC 27599 
Office: (919) 537-3571 
Email: jwebster@email.unc.edu 
 
Ricardo Padilla, BS, DDS 
Research Advisory Committee 
School of Dentistry – University of North Carolina at Chapel Hill 
Koury Building – CB# 7450 
Chapel Hill, NC 27599 
Office: (919) 966-2751 
Email: ricardo_padilla@unc.edu 

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2 INTRODUCTION:  BACKGROUND INFORMATION AND SCIENTIFIC 
RATIONALE 
2.1 
Background Information 
The host response is recognized as the major contributor to periodontal tissue damage 
by supporting a non-resolving inflammation and sustaining dysbiosis (2). Our group has 
recently shown that interferon, gamma-inducible protein 16 (IFI16) and absent in 
melanoma (AIM2) are potentially involved in the pathogenesis of periodontal disease (3, 
4).  IFI16 and AIM2 are intracellular recognition sensors that modulate inflammatory 
responses against several microorganisms. Our data shows that IFI16 and AIM2 are 
expressed in multiple cells of human gingival tissues (4). In murine experimental 
periodontitis, these proteins significantly increase in comparison to mice with healthy 
gingival tissues. This suggests that pathways of IFI16/AIM2 could be interesting for a 
future therapeutic development to treat periodontal diseases. However, the expression of 
IFI16 and AIM2 has not been fully characterized in human gingival tissues. Thus, we 
propose to characterize the gingival tissue expression of IFI16/AIM2 in distinct periodontal 
conditions represented in healthy and periodontal diseased subjects to elicit chronic and 
acute responses using the stent-induced biofilm overgrowth model. This master protocol 
document describes the procedures for such an experiment that represents the clinical 
portion and the first aim of a larger three aim study of IFI16. 
2.2 
Rationale 
Periodontal disease is a biofilm-induced inflammatory disease that affects the integrity 
of the tissues that surround and support the teeth (5). Severe periodontitis is the 6th 
most prevalent disease worldwide, with an overall prevalence of 11.2% and 734 million 
people affected. In the U.S. Periodontal disease affects 47% of the adult population (6, 
7). Periodontitis is a major cause of tooth loss in adults, having subsequent impact on 
the person’s masticatory dysfunction, quality of life and self-esteem (8). The global cost 
of lost productivity from severe periodontitis alone has been estimated to be 54 billion 
USD/year. Such burden of periodontitis will continue to increase with the growing ageing 
population with increased tooth retention globally (8-10). While treatment of periodontal 
disease is successful in the majority of cases, up to 30% of patients with moderate 
chronic periodontitis respond poorly to treatment (i.e. refractory periodontitis) (11, 12). 
Periodontal disease is a multifactorial disease that has microorganisms and a 
susceptible host as key players. The tooth-associated biofilm is required but not 
sufficient to induce periodontitis because it is the host inflammatory response to this 
microbial challenge that ultimately can cause destruction of the periodontium (13). The 
host response is now recognized as the major contributor to periodontal tissue damage 
by supporting a non-resolving inflammation and sustaining dysbiosis (2). Both innate 
and acquired immune responses are shown to be important for disease initiation and 
progression of periodontal disease (14). A recent consensus by the European 
Workshop on Periodontology recognizes that all current biological approaches to 
improve periodontal treatment have been focusing on direct microbial managements 
(antibiotic treatments) instead of approaches that will assist host response modulation 
(15). Concerns about the ever-increasing development of antimicrobial resistance 

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additionally supports methods for modulating the host response instead of drugs 
targeting a broad spectrum of microorganisms.  
The innate immune function depends on recognition of pathogen-associated molecular 
patterns (PAMPS) derived from invading pathogens by germline-encoded pattern-
recognition receptors (PRRs). Activation of PPRs triggers downstream, signaling 
cascades and leads to production of pro-inflammatory cytokines and chemokines (16). 
The balance between pro- and anti-inflammatory mediators determines the severity and 
extent of tissue destruction in the periodontium (17, 18). In order to re-establish the 
balance between mediators, anti-inflammatory pharmacologic agents [non-steroidal 
anti-inflammatory drugs (NSAIDs)] have been used to prevent and slow the progression 
of experimental and human periodontitis (15, 18, 19). While this host-modulation 
approach has shown to be successful, the side effects of NSAIDs have made the usage 
impossible for the routine treatment of periodontitis. Therefore, further understanding of 
the host response is a mean for future therapeutic development. 
Inflammasomes are multimeric protein complexes that assemble in the cytosol after 
sensing PAMPs and danger-associated molecular patterns (DAMPs). Several 
inflammasome sensor molecules can trigger the formation of inflammasomes, including 
the nucleotide-binding domain, leucine-rich repeat containing proteins (NLRs, the 
largest family of inflammasomes), absent in melanoma 2 (AIM2)-like receptors and 
interferon gamma-inducible protein 16 (IFI16) (20). Inflammasome names denote from 
the protein forming the scaffold. While there are differences between inflammasomes 
dependent upon stimuli, classic inflammasomes serve as a scaffold to recruit inactive 
caspase-1. Oligomerization of pro-caspase-1 proteins induces their auto-cleavage into 
active caspase-1, which cleaves precursor cytokines into active IL-1β and IL-18 (21). 
Inflammasomes are shown to play either causative or contributing roles in the initiation 
of 
a 
variety 
of 
auto-inflammatory 
and 
autoimmune 
diseases, 
including 
neurodegenerative diseases (multiple sclerosis, Alzheimer’s disease and Parkinson’s 
disease), and metabolic disorders (atherosclerosis, type 2 diabetes and obesity). 
Expression of several inflammasome components are altered in cells and tissues from 
diseased individuals (22). Recent in vitro and in vivo studies have pointed towards 
promising therapeutics that target inflammasome activity in inflammatory diseases, 
including inhibition of the NLRP3-inflammasome for the reduction of atherosclerosis 
lesions (22, 23).  
AIM2 is the first member of the PYHIN protein family to be linked to the inflammasome 
(24). AIM2 is a cytosolic protein that is a well-characterized sensor of viruses and 
bacteria (25-27). Expression of AIM2 is increased in inflammatory conditions, including 
psoriasis, inflammatory bowel disease, atopic dermatitis and periodontitis, supporting a 
role of this protein in inflammatory diseases (4, 28-32). In vitro, Porphyromonas 
gingivalis (Pg) pro-inflammatory IL-1β response is supported by AIM2-inflammasome 
activation (33). Therefore, AIM2-inflammasome may be important in driving the 
periodontal host response. The role of AIM2-inflammasome in the host response to 
other important periodontal pathogens and in experimental periodontitis has never been 
explored. While AIM2 is shown to be increased in chronic periodontitis (30, 31), the 
effect of this protein in inflammatory bone loss is unknown. 
Recent studies from our group identified IFI16 as a potential protein important for 
periodontal disease pathogenesis (3) and recently published manuscript by the 

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applicant. Our study is the first to demonstrate expression of IFI16 in periodontal tissues 
(4). Expression of IFI16 is observed in multiple cells of the periodontal tissues, including 
epithelial cells, endothelial cells, fibroblasts and leukocytes.  
IFI16 (mouse ortholog ifi204) is a multifunctional protein that has been well characterized 
as a pathogen sensor that leads to inflammasome activation and production of type I 
interferon and a pro-inflammatory response (25, 34, 35). IFI16 is also reported to be 
increased in inflammatory conditions, including psoriatic epidermis, inflammatory bowel 
disease (32, 36). Like other inflammasome proteins, the majority of studies support IFI16 
as a pro-inflammatory molecule. However, IFI16 has also demonstrated an anti-
inflammatory action in vitro by directly binding to AIM2 and suppressing activation of 
caspase-1 and further host response (37, 38) (Figure 2). This suggests that, dependent 
on the type of stimuli and disease, IFI16 can have an anti-inflammatory function by 
dampening the AIM2-mediated inflammatory response. However, these in vitro 
studies did not evaluate IFI16 as a modulator of inflammation upon bacterial infection. In 
addition, currently no in vivo study has evaluated IFI16 as a modulator of inflammation 
via AIM2 inflammasome. We continued our studies by inducing experimental periodontitis 
in Ifi204-/- animals. Analysis of periodontal tissues shows that Ifi204-/- mice have a 
significant increase in the extent of alveolar bone loss in experimental periodontitis 
(p≤0.05). Mean alveolar bone loss is 28.8% for WT and 47.74% for Ifi204ko mice in a 10-
day period of ligature, representing 1.6-fold increase in the amount of bone loss. 
Together, the data suggests that IFI16 acts as an anti-inflammatory protein by altering 
the expression of cytokine/chemokine responses. Better characterization of the cellular 
and molecular biology of inflammasomes will help identify important potential therapeutic 
targets for the treatment and prevention of periodontal diseases (39). The importance of 
IL-1β and IL-18, in periodontal disease pathogenesis is shown in several clinical studies 
(40-45). Currently, therapies targeting IFI16 and AIM2 have not been developed. Studying 
these proteins individually and collectively, along with upstream and downstream events, 
will yield to new approaches to potentially treat periodontal disease severity and 
progression. This project will form the basis of a future project involving inflammasomes 
and alternative therapeutic approaches. 
 
2.3 
Potential Risks and Benefits 
All research involves risks. This may include physical, psychological, social, legal and 
economic risks. There may be uncommon or previously unknown risks that might occur. 
Risks for this study are similar to those associated with other related dental procedures. 
Every participant will be encouraged to report any problems and/or health changes while 
enrolled in this research study. Participants will be given any new information gained 
during the course of the study that might affect their willingness to continue participation.   
In spite of safety measures, a participant might develop a reaction or injury from being in 
this study. If such problems occur, the researchers will help get medical care for the 

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participant, but any costs for the medical care will be billed to the participant. The principal 
investigator or The University of North Carolina at Chapel Hill has not set aside funds to 
pay for any such reactions or injuries, or for the related medical care.  
2.3.1 
Potential Risks 
Subjects may experience short-term discomfort and bleeding of the gums from 
periodontal probing as a result of the periodontal clinical examinations. This exam is not 
different from what would normally be performed in a dental or periodontal office to 
evaluate the condition of the periodontal tissues and extent of disease. No dental 
radiographs will be taken.  
Subjects may experience short-term gingival inflammation, halitosis or bad mouth taste, 
dental plaque accumulation, and bleeding of the gums from refraining from all oral 
hygiene procedures (i.e., tooth brushing, flossing or use of interdental aids and/or 
mouthwashes) in the two selected sextants (up to 6 teeth in each arch) for 21 days. 
Subjects may continue plaque control procedures for the remaining four sextants using 
fluoride dentifrice and toothbrush.  At the completion of the study, adult prophylaxis (for 
orally healthy individuals) or Scaling and Root Planing (SRP) (for individuals with 
periodontal disease) will be completed to restore gingival health eliminating gingival 
bleeding, dental plaque accumulation, and halitosis. Subjects will be monitored for safety 
every week and after the induction of experimental biofilm overgrowth through 21 days. 
Any site undergoing clinical attachment loss of >2mm from the baseline measurement 
will be deemed as “progressing” and participant will exited from the study and given 
scaling and root planing treatment as a rescue therapy at the same day of the visit. 
Subjects may experience mild to moderate discomfort, and bleeding during the scaling 
and planing procedure or adult prophylaxis. Transient bacteremia can occur after dental 
prophylaxis and SRP. Standard of care practices will be utilized for all scaling and root 
planing, adult prophylaxis and dental exams performed. In order to minimize discomfort, 
subjects will receive local anesthesia prior to scaling and root planing procedures. 
Although not required, subjects may receive topical anesthesia prior to and during the 
adult prophylaxis. 
Subjects may experience moderate discomfort following treatment procedures, which will 
be performed under local anesthesia. Topical anesthesia is used initially to minimize the 
discomfort associated with local anesthesia injections. Local anesthesia may cause 
swelling, drooling, dizziness, tachycardia or muscle pain that should be temporary. There 
may be post-operative tenderness, sensitivity to hot or cold, slight bleeding or oozing, or 
development of a transient or permanent temporomandibular joint disorder. Some 
gingival recession or loss of papillae height may occur. Subjects receiving scaling and 
root planing procedures will be encouraged to use over the counter analgesics, according 
to the subject’s medication tolerance, to control any post-operative pain.    
Subjects may experience moderate discomfort following treatment or biopsy 
procedures, which will be performed under local anesthesia. Topical anesthesia is used 
initially to minimize the discomfort associated with local anesthesia injections. Local 

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anesthesia may cause swelling, drooling or muscle pain that should be temporary There 
may be post-operative tenderness, sensitivity to hot or cold, slight bleeding or oozing, or 
development of a transient or permanent temporomandibular joint (TMJ) disorder. Some 
gingival recession or loss of papillae height may occur. Subjects receiving biopsy and/or 
scaling and root planing procedures will be encouraged to use over the counter 
analgesics, according to the subject’s medication tolerance, to control any post-
operative pain.    
In this study, the following events will not be considered reportable AEs: 
• Short-term discomfort, bleeding or oozing of the gums, tooth sensitivity, cheek 
biting, gingival soreness,mild gingival inflammation, mild gingival bleeding, mild 
tooth mobility, mild gingival abrasion or irritation,as a result of the periodontal 
assessments, local anesthesia or periodontal treatment. 
• Gingival recession or loss of gingival papillae 
• Short term jaw soreness resulting from periodontal assessments or periodontal 
treatment  
2.3.2 
Potential Benefits 
This study will provide mechanistic insight into the underlying causes and molecular level 
pathogenesis of periodontal diseases. We will identify key mechanisms that potentially 
confer risk and protection. Ultimately this may lead to new and improved diagnostics and 
therapeutics. Since IFI16 and AIM2 are reported to be involved in other diseases, 
clarifying the role of these proteins in periodontal disease will also provide insights into 
the role of these proteins in other inflammatory conditions. 
Orally healthy individuals will receive adult prophylaxis that is recommended for healthy 
individuals every 6 months. Subjects with periodontal conditions will have therapeutic 
benefit from the treatment. Diseased subjects will be offered referral to the UNC School 
of Dentistry graduate clinics for additional care at reduced fees, based upon the UNC 
School of Dentistry graduate student fee schedule, to treat their periodontal condition 
beyond initial periodontal therapy.  

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3 OBJECTIVES 
3.1 
Study Objectives 
We will establish 2 new cohorts of individuals: healthy with acute inflammation (SIBO) 
and chronic periodontitis with acute inflammation (SIBO) to study the role of IFI16 and 
AIM2 in the pathogenesis of periodontitis. Studying these proteins individually and 
collectively, along with upstream and downstream events, will yield to new approaches to 
potentially treat periodontal disease severity and progression. This project will form the 
basis of a future project involving inflammasomes and alternative therapeutic approaches. 
The study objectives are to: 
1) (Primary) compare the expression of AIM2 and IFI16 between healthy (PPC-A, PPC-
B) and severe periodontitis (PPC-E, PPC-G) subjects during an acute inflammatory 
response using the stent-induced biofilm overgrowth model (SIBO). 
2) (Exploratory) correlate the expression of these proteins with periodontal pathogens 
3.2 
Study Outcome Measures 
Primary study outcomes measured for individuals in each group (healthy and severe 
periodontitis):  
• Expression of AIM2 and IFI16 levels will be quantified by IHC and qRT-PCR 
• Periodontal clinical measurements  
• Composition of oral microbiota by 16S rDNA (Illumina) 
 
 
 

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4 STUDY DESIGN 
A total of 72 subjects will be enrolled. Specifically, a sufficient number of adults 18 years 
and older will be screened until 36 healthy (PPC-A, PPC-B) subjects and 36 subjects with 
severe periodontal disease (PPC-E, PPC-G) will be enrolled based on the algorithm 
previously defined on Morelli et al. 2017. Eligibility for study participation will be 
determined during the screening session. Barring dropout, subject participation will 
include 1 to 8 visits lasting over a maximum period of 56 days. The last 3 visits (visit 5-8) 
will depend on the individual need of the subject for providing SRP. Clinical data and 
medical history data will be collected at the screening visit to ascertain eligibility. All 
subjects will have dental plaque and a gingival biopsy collected at baseline. Enrolled 
subjects will be included in an experimental gingivitis model (SIBO) for 21 days. 
Individuals will return for safety checks every week during the 21-day period. At 21-days, 
plaque samples and a gingival biopsy will be collected. For individuals with severe 
periodontal disease, SRP will initiate during the 21-day visit at the SIBO quadrant. Visit 6 
(28 days) will include the post-biopsy follow up, prophylaxis (for healthy individuals) and 
SRP (diseased individuals). Subjects receiving SRP may have 2 additional visits (visit 7 
at day 35 and visit 8 at day 42) for completing the SRP of all quadrants and will be 
dependent on individual need. Medical histories, demographics, height and weight, 
clinical and biological data described above will be recorded and stored on a secure 
server located at the University of North Carolina.  Each participant enrolled into the study 
will have a unique identification number that has been stripped of any information that 
could be used by non-study members to identify the subject.  
 
 
 

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5 STUDY ENROLLMENT AND WITHDRAWAL 
All subjects will be seen in the General and Oral Health Center, which is a dental research 
clinic at the University of North Carolina at Chapel Hill School of Dentistry. The General 
and Oral Health Center is affiliated with the UNC CTSA. There are no collaborating sites 
where human subject’s research will be performed.  
5.1 
Subject Inclusion Criteria 
 
In order to be eligible to participate in this study, an individual must meet all of the 
following criteria: 
• Subjects must have read, understood and signed an informed consent form.  
• Subjects must be able and willing to follow study procedures and instructions.  
• Subjects must be adult males or females with a minimum of 18 years (inclusive). 
• Subjects must present with at least 20 teeth in the functional dentition, excluding 
third molars.  
• Subjects must have at least 2 natural adjacent teeth in two posterior sextants that 
will be selected for baseline gingival biopsy and SIBO gingival biopsy. 
• Subjects must be in good general health, as evidenced by medical hx (exclusion 
conditions defined under subject exclusion criteria below). 
• Females of child-bearing capacity must be willing to have pregnancy test to 
confirm they are not pregnant. 
• Subjects must be in the healthy (PPC-A, PPC-B) or severe periodontitis (PPC-E, 
PPC-G) categories according to the PPC (1).  
5.2 
Subject Exclusion Criteria 
An individual who meets any of the following criteria will be excluded from participation in 
this study: 
• If the sextants identified for the analysis has implants 
• 
All individuals who meet criteria for anti-infective prophylaxis prior to dental procedures 
should be excluded from this study. 
• Chronic disease with oral manifestations including diabetes mellitus. 
• Current smoker or one that has stopped smoking less than 2 years prior to 
enrollment (self-report). 
• Gross oral pathology other than the periodontal disease.  

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• Treatment with antibiotics for any medical or dental condition within 1 month 
prior to the screening examination. 
• Chronic treatment (i.e., two weeks or more) with any medication known to affect 
periodontal status (e.g., phenytoin, calcium antagonists, cyclosporin, 
coumadin, non-steroidal anti-inflammatory drugs, aspirin) within one month of 
the screening examination. 
• Ongoing medications initiated less than three months prior to enrollment (i.e., 
medications for chronic medical conditions must be initiated at least three 
months prior to enrollment). 
• Significant organ disease including impaired renal function, heart murmur, 
history of rheumatic fever or valvular disease, or any bleeding disorder. 
• 
Individuals with prosthetic material used for intra-cardiac repair (e.g. for congenital 
heart disease), or intra-cardiac devices, cardiac transplant, infective endocarditis and 
individuals who have had previous infectious complications of prosthetic joint 
infections 
• Infectious diseases such as hepatitis, HIV or tuberculosis. 
• Anemia or other blood dyscrasias. 
• Anticoagulant therapy or drugs, such as heparin or warfarin. 
• Severe unrestored caries, or any condition that is likely to require antibiotic 
treatment over the trial. 
• Pregnant, or expect to become pregnant within the next several months. 
• Females of child-bearing capacity not using any form of contraceptive methods 
• Anything that would place the individual at increased risk or preclude the 
individual’s full compliance with or completion of the study. 
5.3 
Strategies for Recruitment and Retention 
Before initiation of the study, the investigator will obtain written approval of the research 
protocol and informed consent form from the UNC IRB complying with the provisions 
specified in 21 CFR Part 56 and applicable pertinent governmental regulations. 
All recruitment plans including advertising, email postings, and flyers will be submitted to 
the IRB and must be approved before study initiation. Our plan is to use campus-wide list 
server postings, website postings, our IRB approved patient registry, newspaper ads and 
postings within UNC including the dental school and the local community. Our dental 
school has a lottery-based patient acceptance program and many individuals are on 
waiting lists to become patients within the dental school clinics. While waiting to be 
accepted at the dental school, subjects become patients of the General and Oral Health 

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Center and are able to receive dental care per study protocol as well as referrals to other 
dental clinics within the school as appropriate. 
Monetary compensation of $200 after completion of 2 biopsy procedures will be provided 
at the end of visit 5. Free parking vouchers will be given to each subject for every 
completed visit. Periodontal treatment that includes scaling and root planning may be 
offered to each study participant according to his/her level of need. Study participants will 
be called by phone and/or emailed, depending upon their indicated preferred contact 
method, to be reminded of planned study visits or to reschedule missed study visits.    
5.4 
Subject Withdrawal 
Subjects may withdraw from the study at any time at their own request, or they may be 
withdrawn at any time at the discretion of the Investigator for safety, behavioral or 
administrative reasons (i.e. protocol compliance). If a participant withdraws consent, no 
further evaluations will be performed on that participant and no attempts will be made to 
collect additional data regarding that participant. If a participant fails to present for a 
scheduled study visit, study staff will attempt three times to reschedule the participant 
utilizing the means of contact provided by the participant. 
5.4.1 
Reasons for Withdrawal 
The Investigator may discontinue a subject if, in the opinion of the Investigator, the subject 
is no longer a suitable candidate for the study.  
Possible reasons for the discontinuation of a subject are including but not limited to:  
 Adverse events (i.e. delayed healing) 
 Protocol deviations (i.e. subject compliance with protocol) 
 Failure to meet or maintain eligibility 
5.4.2 
Handling of Subject Withdrawals 
All participants that prematurely discontinue from the study, regardless of cause will be 
given the opportunity to be seen for a final evaluation and complete a dental prophylaxis. 
Withdrawn subjects with unresolved oral disease will be offered referral for dental care 
within the UNC School of Dentistry. Subjects will only be compensated after two biopsy 
procedures are completed.   
Any participant with an AE that is ongoing at the time of discontinuation will be followed 
until the event returns to baseline, resolves, or stabilizes. If the AE does not meet these 
outcomes within 30 days after discontinuation, the participant will be offered a referral to 
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5.5 
Premature Termination or Suspension of Study 
 
This study may be suspended or prematurely terminated if there is sufficient reasonable 
cause.  Written notification, documenting the reason for study suspension or termination, 
will be provided by the suspending or terminating party.  If the study is prematurely 
terminated or suspended, the principal investigator will promptly inform the IRB and will 
provide the reason(s) for suspension or termination. No interim analyses will be done to 
determine futility. It is expected that valuable results will be obtained from the 200 
participants in the five-week SIBO experiment even if there is an unanticipated finding 
that no biomarkers vary across the four groups under strong error rate control.  Therefore, 
futility analyses for potential early stopping of the trial are not applicable.   
 
Circumstances that may warrant termination include, but are not limited to: 
 Determination of unexpected, significant, or unacceptable risk to subjects. 
 Insufficient adherence to protocol requirements. 
 
 

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6 STUDY SCHEDULE 
 
6.1 
Visit 1: Screening/enrollment  
(Day -28 +/- 2 days) 
• Informed Consent: Before recruitment and enrollment, each prospective candidate 
will be given a full explanation of the study, and allowed to read the informed 
consent form that has been approved by the Institutional Review Board. Qualified 
study personnel will directly ask the subject if he/she understands the implications 
of participating in the study. Subject will have the opportunity to ask questions and 
have answers related to the study participation. No protocol procedures will be 
performed prior to confirmation that the subject understands the procedures and 
risks. Once the subject confirms this understanding verbally, the subject will be 
asked to give consent to participate in the study by signing the informed consent 
form. The study personnel will provide copies of the signed informed consent forms 
to the subject. 
• Study personnel will obtain medical history and demographics related to the 
participant, height and weight 
• Study personnel will record the subject’s vital signs (blood pressure and pulse). 
• Female participants capable of child bearing will be given a urine-based pregnancy 
test. 
• A dental examiner (calibrated dentist or dental hygienist) will perform an intraoral 
examination to verify no presence of oral pathology.  
• Dental examiner will perform a comprehensive periodontal screening examination 
that includes Plaque Index (PI), Gingival Index (GI), Pocket Depth (PD), Bleeding 
on Probing (BOP) and Clinical Attachment Levels (CAL). 
• Subjects in the categories of healthy (PPC-A, PPC-B) or severe disease (PPC-E, 
PPC-G) will be considered eligible for the study.  
• If the participant meets eligibility criteria during the clinic visit, the participant will 
be invited to participate in the research study at that time. If determined non-
eligible, the individual will be informed at that visit, or by telephone, to avoid an 
unnecessary visit. All subjects that complete visit 1 will receive a free parking 
voucher. 
• If eligible, an alginate impression will be taken for stent fabrication  

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• The interproximal region of a tooth will be selected by a dental examiner for the 
first gingival biopsy (occurs at visit 2).  
• The subject will receive a parking voucher for the Dogwood deck and be scheduled 
for visit 2 
6.2 
Visit 2: Baseline 
(Day 0 +/- 5 days) 
• Study personnel will update medical history, including concomitant medication, 
enquire about potential adverse events related to the dental exam performed 
during screening and ask continuance criteria questions. 
• Study personnel will record the subject’s vital signs (blood pressure and pulse). 
• A dental examiner will verify the previously performed intraoral exam and assess 
for potential adverse events. 
• Dental examiner will perform a comprehensive periodontal screening examination 
that includes Plaque Index (PI), Gingival Index (GI), Pocket Depth (PD), Bleeding 
on Probing (BOP) and Clinical Attachment Levels (CAL). 
• The dental examiner will collect two separate subgingival plaque samples from the 
interdental sulcus of the teeth that are selected for a gingival biopsy (see appendix 
II). Dental implant sites will not be considered for subgingival plaque samples. 
• 
A small gingival tissue biopsy sample will be removed from underneath the 
papillae, buccally and lingually, to include the col area of depth of the osseous 
crest. Biopsies will be collected by a study dentist and performed under local 
anesthesia. Absorbable sutures will be used to close the interproximal papillae.  
• The dental examiner will deliver 1 customized acrylic stent that will be used in a 
different sextant from the one that the biopsy was performed. The sextant selected 
will be the right posterior sextant (maxillary or mandibular), except when fewer than 
two teeth are present and adjacent without significant diastema.  
• The participant will be instructed to defer oral hygiene measures in the 
experimental sextant, to wear the customized stents during brushing and to 
discontinue flossing throughout his/her mouth. The participant will continue to use 
his/her standard toothpaste and toothbrush. Participants will be informed to avoid 
crunchy or sticky food (e.g. apples and carrots), and use of mouthwashes during 
baseline through visit 5 (Day 21). 
• Subjects will be monitored for safety every week during the induction of 
experimental biofilm overgrowth through 21 days. Any site undergoing CAL 

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increase of >2 mm from the baseline measurement will be deemed as 
“progressing.” That site will not be included in data analysis, and will receive 
scaling and root planing treatment as a rescue therapy.  
• Subjects will be given parking vouchers for the UNC Dogwood parking deck and 
scheduled to return for visit 3. 
6.3 
Visit 3 
(Day 7 +/- 5 days) 
Visit 3 may occur within 7 days of visit 2 (+/-5 days). The following procedures will be 
performed: 
• Study personnel will update medical history, including concomitant medication, 
enquire about potential adverse events related to the dental procedures and ask 
continuance criteria questions. 
• Study personnel will record the subject’s vital signs (blood pressure and pulse). 
• A dental examiner will verify the previously performed intraoral exam and assess 
for potential adverse events. 
• Dental examiner will perform a comprehensive periodontal screening examination 
that includes Plaque Index (PI), Gingival Index (GI), Pocket Depth (PD), Bleeding 
on Probing (BOP) and Clinical Attachment Levels (CAL). 
• A follow-up for verifying the healing of the gingival biopsy will be completed. 
• The participant will be re-instructed to defer oral hygiene measures in the selected 
sextant and to continue wearing the customized stents during brushing of the non-
selected sextants. 
• Adverse events will be monitored and recorded as necessary.  
• The subject will be given a parking voucher for the Dogwood deck, and Visit 4 will 
be scheduled.  
6.4 
Visit 4 
(Day 14 +/- 5 days) 
Visit 4 may occur within 7 days +/-5 days of day 7. The following procedures will be 
performed: 
• Study personnel will update medical history, including concomitant medication, 
enquire about potential adverse events related to the dental procedures and ask 
continuance criteria questions. 
• Study personnel will record the subject’s vital signs (blood pressure and pulse). 

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• A dental examiner will verify the previously performed intraoral exam and assess 
for potential adverse events. 
• Dental examiner will perform a comprehensive periodontal screening examination 
that includes Plaque Index (PI), Gingival Index (GI), Pocket Depth (PD), Bleeding 
on Probing (BOP) and Clinical Attachment Levels (CAL). 
• The participant will be re-instructed to defer oral hygiene measures in the selected 
sextant and to continue wearing the customized stents during brushing of the non-
selected sextants. 
• Adverse events will be monitored and recorded as necessary. 
• The subject will receive a parking voucher to the Dogwood parking deck, and Visit 
5 will be scheduled.  
6.5 
Visit 5 
(Day 21 +/- 5 days) 
Visit 5 may occur within 7 days +/-5 days of day 14. The following procedures will be 
performed: 
• Study personnel will update medical history, including concomitant medication, 
enquire about potential adverse events related to the dental procedures and ask 
continuance criteria questions. 
• Study personnel will record the subject’s vital signs (blood pressure and pulse). 
• A dental examiner will verify the previously performed intraoral exam and assess 
for potential adverse events. 
• Dental examiner will perform a comprehensive periodontal screening examination 
that includes Plaque Index (PI), Gingival Index (GI), Pocket Depth (PD), Bleeding 
on Probing (BOP) and Clinical Attachment Levels (CAL). 
• The dental examiner will collect two separate subgingival plaque samples from the 
interdental sulcus of the teeth that are selected for a gingival biopsy (see appendix 
II). Dental implant sites will not be considered for subgingival plaque samples. 
• A small tissue biopsy sample will be removed from underneath the papillae, 
buccally and lingually, to include the col area of depth of the osseous crest of a 
tooth included in the SIBO model. The site for biopsy will be selected based on the 
tooth (included in the SIBO model) with the highest GI score. The biopsy will be 
collected by a study dentist and performed under local anesthesia. Absorbable 
sutures will be used to close the interproximal papillae.  

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• SRP may be initiated in individuals with severe periodontitis and will include the 
quadrant that received the gingival biopsy to allow optimal healing 
• Adverse events will be monitored and recorded as necessary 
• The subject will receive compensation for study participation, a parking voucher to 
the Dogwood deck. and visit 6 will be scheduled.  
6.6 
Visit 6: Final Visit (Post-biopsy follow-up; Prophylaxis; SRP may be 
completed up to visit 8 based on individual needs) 
(Day 28 +/- 5 days) 
Participants will be seen for a final visit at day 28 (+/-5 days). The following procedures 
will be performed at this visit: 
• Study personnel will update medical history, including concomitant medication, 
enquire about potential adverse events related to the dental procedures and ask 
continuance criteria questions. 
• Study personnel will record the subject’s vital signs (blood pressure and pulse). 
• A dental examiner will verify the previously performed intraoral exam and assess 
for potential adverse events. 
• Dental examiner will perform a comprehensive periodontal screening examination 
that includes Plaque Index (PI), Gingival Index (GI), Pocket Depth (PD), Bleeding 
on Probing (BOP) and Clinical Attachment Levels (CAL). 
• A follow-up for verifying the healing of the gingival biopsy will be done 
• Prophylaxis will be performed on healthy individuals and prophylaxis/SRP will be 
performed on individuals with severe periodontal disease where the need is 
clinically indicated. Participants will be compensated for their participation in the 
study. 
• Adverse events will be monitored and recorded as necessary. 
 
Scaling and root planing may involve up to 2 additional visits (visit 7 and 8) 
that must be completed within 4 weeks of visit 6 (+/-5 days) 
 
 
 
 
 

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6.7 
Withdrawal Visit 
Subjects will be exited from the experimental component of the research protocol 
following the first visit or following visit 6. All subjects will be exited from the study and 
assessed by the General and Oral Health Center dental personnel for additional 
periodontal care within the General and Oral Health Center or for referral to another dental 
clinic as appropriate. 
6.8 
Unscheduled Visit 
Unscheduled study visits will be documented in the electronic data capturing tool (CDART 
2.0 which is the clinical research data management system designed and developed by 
UNC's Collaborative Studies Coordinating Center (CSCC) and NC TraCS), utilized for 
this research study. This documentation will be recorded in the clinical notes section of 
the participant and will include date of visit, reason for visit, procedures performed during 
the visit, if a follow up visit will be required and the initials of the study staff recording the 
clinical note.   
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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7 STUDY PROCEDURES/EVALUATIONS 
 
7.1 
Study Procedures/Evaluations 
Prior to the first subject study visit, study examiner(s) will be trained and calibrated for 
accuracy and repeatability in using the UNC Modified Gingival Index (GI); Loe and 
Sillness, UNC Modified Plaque Index (Green & Vermillion), periodontal pocket depths, 
and clinical attachment levels using a periodontal probe. All examiners have a kappa > 
0.90 for all calibration sessions. 
Calibration training will be conducted on an annual basis according to the guidelines set 
forth in this document including:  Periodontal determination of plaque (PI), gingival 
inflammation (GI), gingival bleeding (BOP), pocket probing depths (PD) and clinical 
attachment levels (CAL). These parameters are customarily used in clinical dental studies 
to measure the disease status of gingivitis and periodontitis. Probe penetration and depth 
may vary with the degree of inflammation, probing force, angulation, position and 
instrument tip diameter.  
Other confounding factors include patient discomfort, accuracy of probe markings, 
anatomical differences in tooth crown and roots, and technique variability within and 
between examiners. Thus, all of these measurements are important to consider in 
periodontal clinical studies and this training session is designed to minimize the inter-
examiner variance.  
Stent-Induced Biofilm Overgrowth (SIBO) Model  
A customized stent will be fabricated for each subject. The stent will be fabricated to 
resemble an acrylic mouth-guard but extended to cover approximately 2mm over gingival 
margins. The stent will form a seal and rest on the gingiva, but will be relieved on the 
tooth and tissue side except for the occlusal surfaces to avoid disturbing plaque or gingival 
tissues. The acrylic stent will cover the area in one sextant  where no brushing and 
flossing teeth is to occur during 21 days. The acrylic stent will be worn only during tooth 
brushing. Subjects will be monitored for safety every week and after the induction of 
experimental biofilm overgrowth through 21 days. 
 
The SIBO model only induces a transient gingival inflammation without any addition of 
further periodontal attachment loss. The model was previously used and results support 
the safety of the model with no further loss of periodontal attachment (47-49). If test sites 
demonstrate a clinical attachment loss of more than 2 mm during the study, subjects will 
be removed from the study and receive rescue therapy (Scaling and Root Planing) at the 
same day of the visit. Clinical attachment loss will be automatically calculated by the entry 
software used in the study (Dental ToolKit) and compared longitudinally to the baseline 
clinical attachment level measurement. Previous publications from our group 

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demonstrated that the SIBO model is safe by having no subjects required to withdrawn 
previous SIBO studies due to clinical attachment loss (47-49).  
Clinical Assessments and Data Collection 
UNC Modified Gingval Index (GI), Loe and Silness 
Full mouth gingival scores shall be visually assessed by segmenting marginal and 
papillary units, 6 sites per tooth: distobuccal, buccal, mesiobuccal and distolingual, 
lingual, mesiolingual surfaces.  
 
0 = Normal gingiva 
1 = Mild inflammation; slight change in color, slight edema. No bleeding on probing. 
2 = Moderate inflammation:redness, edema, and glazing. Bleeding on probing 
3= Severe inflammation; marked redness and edema. Ulceration. Tendency for 
spontaneous bleeding.  
 
UNC Modified Plaque Index (Green & Vermillion) 
Full mouth plaque assessment shall be assessed using the UNC Modified Plaque Index 
(Greene and Vermillion).  Plaque scores shall be visually assessed at 6 sites per tooth 
(distobuccal, buccal, mesiobuccal and distolingual, lingual, mesiolingual surfaces) on a 
scale of 0-3.   
 
0 =No debris or stain present on the clinical crown. 
1 =Soft debris covering not more than 1/3 of the clinical crown (cervical 3rd), or presence 
of extrinsic stains without other debris regardless of surface area covered. 
2 =Soft debris covering more than 1/3, but not more than 2/3 (middle 3rd) of the clinical 
crown. 
3 =Soft debris covering more than 2/3 of the clinical crown 
 
Pocket depth (PD): Linear distance from the gingival margin (GM) to base of the pocket. 
If a PD reading falls between two millimeter readings, the rule shall be to round down and 
the lower of the two readings will be recorded.  
 
Bleeding on Probing (BOP): Presence or absence of bleeding to manual probing 
recorded as a dichotomous variable. 
0- No bleeding within 10 seconds after probing. 
1- Bleeding within 10 seconds after probing. 
 

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Clinical Attachment Level (CAL): Linear distance from the cemento-enamel junction 
(CEJ) to base of the pocket. If a CAL reading falls between two millimeter readings, the 
rule shall be to round down and the lower of the two readings will be recorded.  
 
 
7.2 
Laboratory Procedures/Evaluations 
Dental plaque samples and gingival tissue samples will be collected at the dental 
research clinic of the UNC School of Dentistry (General and Oral Health Center). All bar 
coded de-identified biological samples will be transported to the laboratory of the principal 
investigator for processing with a chain of custody recording for each sample.  All 
information collected for this study will only be used for the study, and not as part of 
another study. Additional grant request may be submitted in the future to complete 
additional analyses on stored biological specimens.  
All measurements will be conducted using standard techniques that will be carried out in 
the investigator’s laboratory and in collaborations previously established by the 
investigator (Histology Research Core Facility, Microbiome Core Facility at UNC). 
7.3 
Study Specific Biospecimens 
7.3.1 
Specimen Collection Procedures 
Specimen Collection Procedures 
Plaque: The dental examiner will collect two separate, facial, sublingual plaque samples, 
from the interdental sulculs of the teeth that are selected for a gingival biopsy .Dental 
implants will not be considered for plaque samples. Subgingival plaque samples will be 
obtained with a sterile Gracey dental curette. The curette tip shall be inserted into to the 
depth of the sulcus and plaque shall be collected in a single coronal stroke movement. 
After plaque collection, the curette is then placed in an Eppendorf tube containing 500 ul 
of TE buffer (10mM Tris-HCl, 0.1 mM EDTA, pH 7.6) and shaken for a few seconds to 
release the bacterial cells from the curette into the solution. The plaque sample can then 
remain at the clinic until the end of the day, when all samples are taken to the laboratory, 
where they are stored at -80 ˚C for later bacterial microbiological analysis. All samples 
will be taken to the UNC Cytokine Analysis Lab for validation of collection and storage.  
 
We will evaluate the  microbial content determined by sequencing the V3-V4 variable 
region of 16S rRNA gene using next generation sequencing (50). The bacterial diversity 
and relative abundance (% of total reads) of each genus and taxon will be evaluated using 
the QIIME pipeline analysis on the 16S rRNA Illumina sequencing with references 
databases (SILVA, Greengenes and the Human Oral Microbiome Database). This is a 
high-throughput state-of-the art technology (50, 51). 
 

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Gingival Tissue/Biopsy: Biopsy samples of gingival tissues will be collected from all 
individuals at baseline and 21-days of biofilm-overgrowth. A trained dentist will use a 
scalpel blade to collect 2 biopsy samples per subject for a total of 128 samples.  Samples 
will be obtained using topical and local anesthesia. Each biopsy shall be ≤0.5 cm thick. 
Biopsies will only be collected from the papillae of posterior teeth. The scalpel blade shall 
be oriented in an inverse bevel direction to capture the base of the periodontal pocket or 
gingival sulcus.  Each biopsy will contain oral epithelium, gingival connective tissues and 
loosely adherent cells lining the periodontal pocket. Papillae height will be retained by 
using vertical mattress suture technique, minimizing facial reduction, and coronal 
reposition of the flap. The biopsy wound will be undermined with minimal flap reflection 
and closed with a suture. Post-operative instructions, including placing ice over the 
surgical area and avoiding hot liquids for the remainder of the day, will be given to the 
subject. 
Gingival 
biopsy 
samples 
will 
be 
bi-sectioned 
for 
immunohistochemistry/immunofluorescence imaging and mRNA expression.  
1. For RNA extraction, samples will be immediately placed in RNAlater® (Ambion 
Inc.) which is an aqueous, non-toxic tissue storage reagent that rapidly permeates 
tissue to stabilize and protect cellular RNA in situ in unfrozen specimens. Samples 
will be placed in a microfuge tube containing ~1ml of RNAlater and kept at 4°C 
overnight, then RNA later is removed. Samples will stored at -80º C until used for 
analysis.  
2. For Immunohistochemistry/Immunofluorescence, samples will be fixed in 4% 
paraformaldehyde and stored at -20°C for 24 hours and then placed in 70% 
ethanol for further analysis.  
7.3.2 
Specimen Preparation, Handling, and Storage 
All biological samples are collected into pre-labeled barcoded vials for laboratory 
processing. Clinical data are collected and stripped of all subject identifiers and provided 
a bar-coded ID that link the subject’s clinical data to the study visit, examiner, date and 
dataset. Biological samples are analyzed by the laboratory in a blinded manner and at 
the data closeout the ID links are established to connect the clinical data to the laboratory 
data. None of that data contains any patient identifiers. Identification design uses a 
barcode that includes the study ID, subject ID, visit ID and biological specimen ID.  
Example barcode:   
 
• “ITS” is the acronym that identifies the specific IRB approved research study.  
• “1001” is the subject identifier   
• “03” is the visit ID (visit 3) 

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• “904” is the specific biological sample ID  
7.3.3 
Specimen Shipment 
Biological specimens will not be shipped to an outside location for analysis, except for 
plaque samples. Specimens will be taken, by GO Health staff, to the principal 
investigator’s laboratory, 3412 Koury Oral Health Sciences Building and processed or 
stored for future analysis. Plaque samples will be stored at -80˚C for later bacterial 
microbiological analysis at the Microbiome core facility. Tissue samples will be stored at 
-80º C until use for RNA extraction and at 4˚C for histological processing. 
 
7.4 
Questionnaire Administration 
There are no questionnaires in place for this research study.  

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8 ASSESSMENT OF SAFETY 
The principal investigator is responsible for all aspects of the conduct of the study. There 
will be a primary study coordinator assigned to track patients, IRB compliance, adverse 
events and protocol adherence and study deviations.  The principal investigator will meet 
weekly with the study coordinator to track progress, enrollment and study adherence.  
8.1 
Specification of Safety Parameters 
Safety monitoring reporting for this study will focus on unanticipated problems involving 
risks to participants, including unanticipated problems that meet the definition of a serious 
adverse event. Any adverse experience that is determined to be reportable to the 
Regulatory Authorities will be promptly reported to the Institutional Review Board (IRB).  
8.1.1 
Unanticipated Problems  
The Office for Human Research Protections (OHRP) considers unanticipated problems 
involving risks to subjects or others to include, in general, any incident, experience, or 
outcome that meets all of the following criteria: 
• unexpected in terms of nature, severity, or frequency given (a) the research 
procedures that are described in the protocol-related documents, such as the IRB-
approved research protocol and informed consent document; and (b) the 
characteristics of the subject population being studied; 
• related or possibly related to participation in the research (possibly related means 
there is a reasonable possibility that the incident, experience, or outcome may 
have been caused by the procedures involved in the research); and 
• suggests that the research places subjects or others at a greater risk of harm 
(including physical, psychological, economic, or social harm) than was previously 
known or recognized. 
8.1.2 
Serious Adverse Events  
A serious adverse event (SAE) is one that meets one or more of the following criteria: 
• Results in death 
• Is life-threatening (places the subject at immediate risk of death from the event as 
it occurred) 
• Results in inpatient hospitalization or prolongation of existing hospitalization 
• Results in a persistent or significant disability or incapacity 
• Results in a congenital anomaly or birth defect 
 

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An important medical event that may not result in death, be life threatening, or require 
hospitalization may be considered an SAE when, based upon appropriate medical 
judgment, the event may jeopardize the subject and may require medical or surgical 
intervention to prevent one of the outcomes listed in this definition. 
8.2 
Reporting Procedures 
The principal investigator will be responsible to monitor the overall study that includes the 
research data as well as the clinical procedures. All adverse events will be reported by 
the PI to the IRBs and appropriate agencies. 
The Principal Investigator and study coordinator will review adverse events weekly and 
report them to the Institutional Review Board within 30 days.  All “unanticipated” adverse 
events will be reported to the IRBs within 2 week of the investigator becoming aware of 
the event. Any related and unexpected serious adverse events (SAE) will be reported to 
the IRB within 24 hours of the principal investigator becoming aware of the event. 
Unanticipated patient deaths will be immediately reported to the IRB’s and NIH.  A 
summarized copy of all adverse events will be submitted to the IRB’s annually or as 
requested. 
Incidents or events that meet the Office of Human Research Protections (OHRP) criteria 
for unanticipated problems require the creation and completion of an unanticipated 
problem report form.  OHRP recommends that investigators include the following 
information when reporting an adverse event, or any other incident, experience, or 
outcome as an unanticipated problem to the IRB: 
• appropriate identifying information for the research protocol, such as the title, 
investigator’s name, and the IRB project number; 
• a detailed description of the adverse event, incident, experience, or outcome;  
• an explanation of the basis for determining that the adverse event, incident, 
experience, or outcome represents an unanticipated problem;  
• a description of any changes to the protocol or other corrective actions that have 
been taken or are proposed in response to the unanticipated problem. 
 
Adverse Event (AE) 
An adverse event is any unfavorable and unintended diagnosis, sign (including an 
abnormal laboratory finding), symptom, or disease temporally associated with the study 
intervention, whether or not related to the intervention. Adverse events include new 
events not present during the pre-intervention period and pre-existing conditions that 
have increased in severity. 
 
 

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Expected clinical/dental Adverse Events (AE’s) 
•             Apthous and canker lesions 
•             Tooth sensitivity  
•             Cheek bite 
•             Linealba 
•             Gingival soreness 
•             Mild gingival inflammation 
•             Mild gingival bleeding 
•             Mild tooth mobility 
•             Mild gingival abrasion or irritation 
• 
TMJ pain and soreness 
 
 
Unexpected Adverse Event  
An unexpected adverse event is an adverse reaction, the nature or severity of which is 
not consistent with the clinical course of a subject receiving the study procedures. 
Expected events are untoward clinical occurrences that are perceived by the 
investigator to occur with reasonable frequency in the aforementioned study 
procedures. Examples of adverse events that are expected include apthous and canker 
lesions,tooth sensitivity, cheek bite, linealba, gingival soreness, mild gingival 
inflammation, mild gingival bleeding, mild tooth mobility, mild gingival abrasion or 
irritation, and TMJ pain and soreness. Such events, will not be considered reportable 
unless the event is considered by the investigator to be unexpectedly severe or frequent 
for an individual patient .If expectedness of an event is unclear, the study’s Principal 
Investigator will be consulted to make this determination.  
 
Serious Adverse Event (SAE) 
A serious adverse event is any untoward medical occurrence that meets the definition of 
adverse event and results in death, is life-threatening, requires or prolongs 
hospitalization, causes persistent or significant disability/incapacity, results in congenital 
anomalies/birth defects or, in the opinion of the investigators, represents other 
significant hazards or potentially serious harm to research participants or others. 
 
Grading of Adverse Events 
The following scale will be used to grade the severity of adverse events noted during 
the study: 
Severity Criteria: 
1 = Mild adverse event – Did not interfere with normal activity 
2 = Moderate adverse event – Interfered with normal activity to some extent 
3 = Severe adverse event – incapacitating with inability to work or do usual activity 

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Serious Criteria:  
1 = Caused or prolonged hospitalization  
2 = Resulted in persistent or significant disability/incapacity  
3 = Congenital anomaly/birth defect 
4 = Life threatening 
5 = Fatal 
6 = Other event considered serious by investigator. Site investigators may use their 
discretion in classifying events as serious if they represent a significant hazard or 
potentially serious harm to research participants or others. 
 
Attribution of Adverse Events 
Site investigators will also evaluate the association of each AE with the periodontal 
intervention according to the following categories: 
a. Unrelated: Adverse event(s) clearly not be related to investigational 
procedure(s)/agent(s) or other intervention. 
b. Unlikely: Adverse event(s) doubtfully related to investigational procedure(s)/agent(s) 
or other intervention. 
c. Possibly: Adverse events(s) may be related to investigational procedure(s)/agent(s) 
or other intervention; known to occur but the temporal relationship is unclear; other 
causes are possible or biologically very plausible. 
d. Probably: Adverse event(s) likely associated with investigational 
procedure(s)/agent(s) or other intervention; known to occur and temporal relationship is 
appropriate or improvement is seen upon withdrawal of study drug; other causes are 
unlikely. 
e. Definitely: Adverse event(s) clearly associated with the investigational 
procedure(s)/agent(s) or other intervention; known to occur and clear temporal 
relationship is noted or improvement is seen upon withdrawal of study drug; other 
causes are very unlikely. If re-challenge is attempted, the adverse event must return. 
 
*An adverse event will be considered associated with the study procedures if in the 
estimation of the local investigator the event is at least possibly related to the study 
procedures procedures. 
 
Reporting Adverse Events 
 
All AEs and SAEs will be recorded on the electronic AE Eorm incorporated in the 
CDART “Dental Toolkit” Module. Adverse events that are (1) unexpected, (2) related or 
possibly related to participation in the research, and (3) serious or suggest that there 
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of Human Research Ethics (OHRE) within 7 calendar days of the investigator becoming 
aware of the information. The Principal Investigator will assure that documentation of 
each event is adequate to permit accurate inferences regarding causation (e.g., 
temporal associations, onset, course, response to patient or physician intervention, 
alternative etiologies) and severity. 
 
All unanticipated problems will be reported to NIDCR’s centralized reporting system 
via Rho Product Safety: 
• Product Safety Fax Line (US):  1-888-746-3293 
• Product Safety Fax Line (International):  919-287-3998 
• Product Safety Email:  rho_productsafety@rhoworld.com 
General questions about SAE reporting can be directed to the Rho Product Safety Help 
Line (available 8:00AM – 5:00PM Eastern Time):   
• US:  1-888-746-7231 
International: 919-595-6486 
 

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9 STUDY OVERSIGHT 
The investigator will be responsible for study oversight, including monitoring safety and 
ensuring that the study is conducted according to the protocol and ensuring data integrity.  
The principal investigator will review the data for safety concerns and data trends at 
regular intervals, and will promptly report to the IRB and NIDCR any Unanticipated 
Problem (UP), protocol deviation, or any other significant event that arises during the 
conduct of the study.  
 
 
 

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10 CLINICAL SITE MONITORING 
Clinical site monitoring will not be done for this study; however, the NIDCR reserves the 
right to conduct independent audits or clinical monitoring as necessary. 
 

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11 STATISTICAL CONSIDERATIONS 
11.1 
Study Hypotheses 
The central hypothesis of this proposal is that IFI16 functions to attenuate the host 
response that drives periodontitis. We propose to investigate whether macrophage-
derived IFI16 expression modulates tissue inflammation via AIM2. Human tissues will be 
used for confirming results generated by the principal investigator from in vitro and in vivo 
models that show high expression of AIM2 and IFI16 under inflammatory conditions (4). 
Better characterization of the cellular and molecular biology of inflammasomes will help 
identify important potential therapeutic targets for the treatment and prevention of 
periodontal diseases. The following “hypothesis” corresponding to Study Objective 1 
(section 3.1) is: 
Primary hypotheses corresponding to the second study objective will be tested: 
1a. There will be a temporal change in expression of AIM2 (mRNA fold change and 
percentage of positive cells for histological analysis) induced by SIBO in healthy and 
severe periodontitis subjects 
1b. There will be a temporal change in expression of IFI16 (mRNA fold change and 
percentage of cells for histological analysis) induced by SIBO in healthy and severe 
periodontitis subjects 
1c. There will be differences in temporal changes in expression of AIM2 (mRNA fold 
change and percentage of cells for histological analysis) induced by SIBO between 
healthy and severe periodontitis subjects 
1d. There will be differences in temporal changes in expression of IFI16 (mRNA fold 
change and percentage of cells for histological analysis) induced by SIBO between 
healthy and severe periodontitis subjects 
Exploratory hypotheses corresponding to the second study objective will be assessed: 
2a. The stent-induced change in expression of protein AIM2 will correlate with 
differentially expressed (between healthy and severe periodontitis subjects) 
periodontal pathogens identified by 16S rDNA (% of total reads)  at baseline (visit 2) 
2b. The stent-induced change in expression of protein IFI16 will correlate with 
differentially expressed (between healthy and severe periodontitis subjects) 
periodontal pathogens identified by 16S rDNA (% of total reads)  at baseline (visit 2) 
2c. The stent-induced change in expression of protein AIM2 will correlate with the 
temporal change in periodontal pathogens identified by 16S rDNA (% of total reads)   
2d. The stent-induced change in expression of protein IFI16 will correlate with the 
temporal change in periodontal pathogens identified by 16S rDNA (% of total reads)   

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11.2 
Sample Size Considerations 
In order to evaluate the expression of IFI16 and AIM2 in subjects, 72 (36 subjects/group) 
will be recruited from the patients, students, staff and general population at the University 
of North Carolina-School of Dentistry. We will enroll 72 subjects to accommodate a 10% 
drop-out rate. Individuals with healthy tissues and severe periodontitis will be enrolled 
based on Morelli et al 2017 (1). Sample size calculations are based, in part, on preliminary 
cross-sectional (non-SIBO experiment) data on expression levels of Aim2 from 
immunohistochemistry reported in Xue et al. (2015; Table 3) who reported statistically 
significant differences in healthy (n=10; mean=0.50; s.d.=0.14) and severe periodontitis 
(n=10; 
0.97; 
s.d.=0.27) 
subjects 
with 
respect 
to 
AIM2 
expression 
from 
immunohistochemical analyses.  For the first study objective (section 3.1), there will be 
99% power to detect differences in AIM2 and IFI16 expressions at Visit 2 between healthy 
and severe periodontitis at these levels using two-sided t-tests at the 0.05 significance 
level.  When comparing change in AIM2 expression levels from visit 2 to visit 5, 
assumptions of a common standard deviation of 0.25 and an intra-subject correlation of 
0.40 across the two time points indicate that power will be at least 80% to determine 
differences in change between healthy and severe periodontitis subjects that are as small 
as 0.40; in a worst case (but not realistic) scenario, an intra-subject correlation of 0 would 
give 80% power to detect differences in temporal change that are as small as 0.50. Xue 
et al (2015; Figure 1c) found no statistically significant cross-sectional differences 
between healthy and severe periodontitis subjects with respect to mRNA expression of 
AIM2; they did not report this data numerically precluding power analysis.  
11.3 
 Final Analysis Plan 
1) (Primary) compare the expression (fold-change relative to baseline) of AIM2 and IFI16 
between healthy (PPC-A, PPC-B) and severe periodontitis (PPC-E, PPC-G) subjects 
during an acute inflammatory response using the stent-induced biofilm overgrowth model 
3) (Exploratory) correlate the expression of these proteins with periodontal pathogens 
For the primary hypotheses involving gingival tissue samples, univariate and multivariate 
nonparametric testing procedures for SIBO experiments (Preisser, Sen and Offenbacher, 
2011) will be utilized.  In particular, a multivariate Wilcoxon Signed Rank test will use all 
available data to jointly test for changes in AIM2 and IFI16 expression levels over time 
(visit 2 to visit 5). If the overall multivariate test is statistically significant at the 0.05 level, 
then univariate Wilcoxon Signed Rank tests will be applied to AIM2 and IFI16 proteins 
individually. Similar procedures will be applied for expression levels from mRNA and from 
immunohistochemistry. Additionally, multivariate and univariate Wilcoxon Rank Sum 
Tests will be applied at the 0.05 significance level to compare SIBO-induced temporal 
change in Aim2 and IFI16 expression levels between healthy and severe periodontitis 
subjects.  

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Parametric statistical procedures will be used to summarize and test periodontal clinical 
measurements related to secondary hypotheses. Means and their 95% confidence 
intervals will be used to summarize measurements at each time point in healthy and 
severe periodontitis subjects.  As these measurements will be collected at visits 2, 3 4 
and 5, linear mixed models will be used to assess change over time within groups and 
differences in change over time between healthy and severe periodontitis groups. 
Using 16s rRNA sequencing, expression counts for a large number (~20,000) of 
Operational Taxonomic Units (OTUs) with known taxonomy will be obtained from 72 
samples at baseline and among them approximately 64 samples at Day 21 of SIBO.  A 
few hundred taxa will be identified by collapsing expression data within genera 
corresponding to candidate periodontal pathogens.  Change in pathogen levels from 
baseline to Day 21 among severe periodontitis subjects will be assessed with univariate 
Wilcoxon Signed Rank tests applied to each OTU (hypothesis 2f) or Wilcoxon Rank 
Sum Tests for comparison of Day 21 minus baseline differences in expression counts 
between healthy and severe periodontitis groups (hypothesis 2h). False discovery rate 
(FDR; Benjamini and Hochberg, 1995) and family-wise error rate (FWER; Hochberg, 
1988) methods, e.g., as described in Preisser et al. (2011), will be applied to resulting  
p-values for exploratory and confirmatory testing, respectively, to identify statistically 
significant taxa.  
Temporal changes in periodontal pathogen levels that are statistically significant 
controlling for multiple testing as described above will be correlated with stent-induced 
changes in AIM2 and IFI16 proteins using Spearman rank correlation coefficients and 
corresponding 95% confidence intervals.  
Statistical computation for analysis of the data will be done my Mr. Moss in consultation 
with Dr. Marchesan and Dr. Preisser. 
All hypothesis tests that are observed to be NOT statistically significant will be reported 
as being inconclusive. 
 
  
 

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12 SOURCE DOCUMENTS AND ACCESS TO SOURCE DATA/DOCUMENTS 
As part of the responsibilities assumed by initiating and conducting the study, the principal 
investigator will maintain adequate case histories for the subjects recruited under this 
research protocol. These case histories include source documentation, hard copy case 
report forms and electronic files.  
To ensure compliance with the Health Insurance Portability and Accountability Act 
(HIPAA) guidelines, all research data entered into the database will be de-identified. 
Accordingly, there will be no identification of subjects by name, geographic residence 
(smaller than a state), date, telephone or fax number, electronic mail address, social 
security number, medical record number, health plan beneficiary number, account 
number, certificate or license number, vehicle identifiers and serial numbers (including 
license plate number), device identifiers and serial numbers, web universal resource 
locators (URLs), internet protocol (IP) address numbers, biometric identifiers (including 
finger and voice prints) full face photographic images or any comparable images, or any 
other number, characteristic or code derived from actual identifiers. 
Data will be collected on desktop and/or laptop computers using the web based data entry 
program module, Dental Toolkit. This system resides in a secure server environment 
within a hardened data center on the UNC campus and is governed by standard UNC 
and UNC Schools of Medicine and Dentistry information security guidelines. 
Source documentation for this study will consist of a combination of the dental eCRF 
(electronic case report form) containing medical histories, demography, vital signs, 
height and weight, body mass index, oral assessments and indices, sample tracking, 
AE’s, protocol violations, subject disposition and paper based CRF’s including IRB 
approved consent forms. All final data recorded in the dental eCRF will be copied onto 
CDs and kept with paper CRF’s at the clinical site. 
Computers and hard copies of the data sheets are kept in a locked office. Research 
samples will be identified only by unique numbers assigned to the participants. 
Biological specimens will also be coded. Linkage to these codes will be retained by the 
study coordinator and kept in locked workrooms accessible only by study personnel. 
Data will not be shared outside the immediate research team. 
 
Study staff will maintain appropriate medical and research records for this study, in 
compliance with ICH E6, Section 4.9 and regulatory and institutional requirements for the 
protection of confidentiality of subjects. Study staff will permit authorized representatives 
of NIDCR and regulatory agencies to examine (and when required by applicable law, to 
copy) research records for the purposes of quality assurance reviews, audits, and 
evaluation of the study safety, progress and data validity.    
 

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13 QUALITY CONTROL AND QUALITY ASSURANCE 
Clinical examiners will be calibrated prior to commencement of the study for training of 
study procedures and for documentation of acceptable intra- and inter-examiner 
measurement reliability. All study examiners will have participated in an examiner 
calibration session conducted at the University of North Carolina, School of Dentistry in 
the GO Health Center prior to becoming a certified study examiner. Examiner calibration 
is done annually at the General and Oral Health Center as a standard protocol. 
Trained quality control personnel will ensure that the research study is conducted, 
recorded and reported in accordance with the protocol, Standard Operating Procedures 
(SOPs), Good Clinical Practice (GCP) and the applicable regulatory requirements.  
A data quality assurance program will be established that will consist of 1) real-time 
detection and correction of errors within the Dental Toolkit, and 2) periodic data review by 
study personnel.  At the time of data entry, the Dental Toolkit alerts the user to missing, 
out-of-range, and inconsistent values and provides the user the opportunity to correct 
errors in real time.  In accordance with federal regulations, the system records all 
elements of data entry (i.e., time, date, verbatim text, and the name of the person 
performing the data entry) in an electronic audit trail.  For data not directly entered into 
the Dental Toolkit, study personnel will verify key outcome measurements by comparing 
data in the system with source documentation.  Other data will be entered directly into the 
Dental Toolkit without source documentation.  The frequency and the number of records 
reviewed are described in the Clinical Quality Management Plan. Missing values will be 
documented.  
This study coordinator will monitor source documentation daily to ensure that data fields 
are complete and to verify that any transcribed data are accurately reflected on the study 
electronic case report forms. All study staff will receive training on the electronic data 
capturing tool, Dental Toolkit, prior to first subject being enrolled.  
 

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14 ETHICS/PROTECTION OF HUMAN SUBJECTS 
14.1 
Ethical Standard 
The investigator will ensure that this study is conducted in full conformity with the 
principles set forth in The Belmont Report: Ethical Principles and Guidelines for the 
Protection of Human Subjects of Research, as drafted by the US National Commission 
for the Protection of Human Subjects of Biomedical and Behavioral Research (April 18, 
1979) and codified in 45 CFR Part 46 and/or the ICH E6.   
14.2 
Institutional Review Board 
The protocol, informed consent form(s), recruitment materials and all participant materials 
will be submitted to the IRB for review and approval.  Approval of both the protocol and 
the consent form must be obtained before any participant is enrolled.  Any amendment to 
the protocol will require review and approval by the IRB before the changes are 
implemented in the study.  
UNC IRB and Office of Research Ethics 
Medical School Bldg. 52 
105 Mason Farm Road 
Chapel Hill, NC 27599-7097 
(919) 966-3113 
(919) 966-7879 (f) 
FWA#4801 
14.3 
Informed Consent Process 
Informed consent is a process that is initiated prior to the individual agreeing to participate 
in the study and continues throughout study participation.  Extensive discussion of risks 
and possible benefits of study participation will occur with each potential participant.   
Before any study procedures or assessments are performed, an IRB-approved consent 
form describing in detail the study procedures and risks of the study, will be given to each 
subject to read. Once trained study staff is assured that an individual understands the 
implications of participating in the study, the subject will be asked to give consent to 
participate in the study by signing the informed consent form. Consent forms will be IRB-
approved, and the participant is required to read and review the document or have the 
document read to him or her.  The investigator or designee will explain the research study 
to the participant and answer any questions that may arise. The participant will sign the 
informed consent document.   
Participants will be given the opportunity to discuss the study with their surrogates or think 
about it prior to agreeing to participate.  They may withdraw consent at any time 
throughout the course of the study.  A copy of the signed informed consent document will 
be offered to each participants for their records.  The rights and welfare of the participants 

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will be protected by emphasizing to them that the quality of their clinical care will not be 
adversely affected if they decline to participate in this study. 
The consent process will be documented in the clinical or research record. 
14.4 
Exclusion of Women, Minorities, and Children (Special Populations) 
There is no planned involvement of special classes of subjects, such as fetuses, 
neonates, women, children, prisoners, institutionalized individuals. Periodontal disease is 
a chronic condition that affects individuals over 30. Children will be excluded from this 
study, because we are seeking to understand the expression of IFI16 and AIM2 in severe 
periodontitis.  
14.5 
Participant Confidentiality 
The study protocol, documentation, data, and all other information generated will be held 
in strict confidence.  No information concerning the study or the data will be released to 
any unauthorized third party without prior written approval. 
Authorized representatives may inspect all study documents and records required to be 
maintained by the investigator, including but not limited to, medical records (office, clinic, 
or hospital) for the study participants.  The clinical study site will permit access to such 
records. Participant confidentiality is strictly held in trust by the investigators, study staff, 
and the sponsor(s) and their agents. This confidentiality is extended to cover testing of 
biological samples and genetic tests in addition to any study information relating to 
participants. 
14.6 
Future Use of Stored Specimens and Other Identifiable Data 
Plaque samples and gingival biopsies that are not utilized for the current proposal will 
be stored for later analysis. Subjects will be consented using a separate biorepository 
consent form approved by UNC IRB (#15-1814) for stored specimens. 
 
 

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15 DATA HANDLING AND RECORD KEEPING 
15.1 
Data Management Responsibilities 
The investigator is responsible for ensuring the accuracy, completeness, legibility, and 
timeliness of the data reported. Data collection and accurate documentation are the 
responsibility of the study staff under the supervision of the investigator. The investigators 
will maintain adequate case histories of study participants, including accurate case report 
forms (CRFs), and source documentation.  All source documents and laboratory reports 
must be reviewed by the study team and data entry staff, who will ensure that they are 
accurate and complete.  Unanticipated problems must be reviewed by the investigator or 
designee. 
15.2 
Data Capture Methods 
Data will be collected on desktop and/or laptop computers within the UNC School of 
Dentistry, GO Health Center. Research staff that have specifically been trained on the 
software data collection tools will be performing data entry. Other bar-coded data will be 
actually scanned into the software via desktop scanners. These computers are stored in 
locked offices, and are password protected. Data backup will be performed on password 
protected network. 
15.3 
Types of Data 
Data captured will include subject’s demographics, medical and dental histories, vital 
signs, body mass index, clinical oral exams and indices, adverse events, protocol 
deviations, clinical and laboratory notes and tracking of biological specimens. Reports 
may include any captured data retained on this module. 
15.4 
Schedule and Content of Reports 
Customized reports will be generated from the data capturing module. Reports may 
include any captured data retained on this module. Data will not be shared with anyone 
outside those listed as IRB approved personal or investigators. Biannual progress reports 
will be provided to the NIDCR and/or the medical monitor. Report may include data 
regarding enrollment and retention, unanticipated problems and protocol deviations, 
disposition of biological specimens, outcome measures, quality management findings 
and other relevant parameters. 
15.5 
Study Records Retention 
Study records will be maintained for at least three years from the date that the grant 
federal financial report (FFR) is submitted to the NIH. 
15.6 
Protocol Deviations 
A protocol deviation is any noncompliance with the clinical study protocol, Good Clinical 
Practice, or Manual of Procedures requirements.  The noncompliance may be on the part 

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of the subject, the investigator, or study staff.  As a result of deviations, corrective actions 
are to be developed by the study staff and implemented promptly. 
These practices are consistent with investigator and sponsor obligations in ICH E6: 
• Compliance with Protocol, Sections 4.5.1, 4.5.2, 4.5.3, and 4.5.4. 
• Quality Assurance and Quality Control, Section 5.1.1 
• Noncompliance, Sections 5.20.1 and 5.20.2. 
All deviations from the protocol must be addressed in study subject source documents 
and promptly reported to NIDCR and the local IRB, according to their requirements.   

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16 PUBLICATION/DATA SHARING POLICY 
This study will comply with the NIH Public Access Policy, which ensures that the public has 
access to the published results of NIH funded research. It requires scientists to submit 
final peer-reviewed journal manuscripts that arise from NIH funds to the digital archive 
PubMed Central upon acceptance for publication.   
 

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SUPPLEMENTAL MATERIALS 
These documents are relevant to the protocol, but they are not considered part of the 
protocol.  They are stored and modified separately. As such, modifications to these 
documents do not require protocol amendments. 
 
 
 

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Appendix I:  Schedule of Events 
Procedures 
Visit 1 
Screenin
g/Enroll
ment 
Day -28 
to ±2 
Visit 2 
Baseline 
Day 0 
±5 days 
Visit 3 
Day 7 
±5 days 
Visit 4 
Day 14 
±5 days 
Visit 5 
Day 21 
±5 days 
Visit 6 
Day 28 
±5 days 
Visit 7 
Day 35 
±5 days 
Visit 8 
Day 42 
±5 days 
Consenting 
X 
 
 
 
 
 
 
 
Medical 
Hx/ 
Update 
X 
X 
X 
X 
X 
X 
X 
X 
Oral Exam 
X 
X 
X 
X 
X 
X 
X 
X 
Pregnancy 
testing 
X 
 
 
 
 
 
 
 
Stent impression 
X 
 
 
 
 
 
 
 
Deliver stent 
 
X 
 
 
 
 
 
 
Discontinue 
stent 
 
 
 
 
X 
 
 
 
Adverse Events 
 
 
X 
X 
X 
X 
X 
X 
Height /Weight 
X 
 
 
 
 
 
 
 
Demographics 
X 
 
 
 
 
 
 
 
Plaque 
Collection 
 
X 
 
 
X 
 
 
 
Biopsy 
Collection 
 
X 
 
 
X 
 
 
 
PI, GI, PD, CAL, 
BOP 
X 
X 
X 
X 
X 
X 
 
 
Adult 
Prophylaxis/SRP  
 
 
 
 
X 
X 
X 
X 
 
 
 

IFI16 is a periodontitis modulating protein 
Version 8.0 
Protocol 
March 6, 2022 
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Based on NIDCR Clinical Study (Observational) Protocol Template v4.0 - 20140106 
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Appendix II:  Gingival biopsy 
 
Lingual 
3 
2 
 Site: A 
Site: B 
Mesial 
Example shown for biopsy collection of interproximal 
biopsy tissue sites between teeth #2 & 3. 
 
Strategy: 
During visit  (baseline, 0 day), one biopsy sample will be collected from the posterior 
interproximal unit determined by the dental provider. Sites will not be standardized since 
dentition will vary from subject to subject. 
 
During visit 5 (day 21 of SIBO), one biopsy sample will be collected from the posterior 
interproximal unit in SIBO, determined by the dental provider. Sites will not be 
standardized since dentition will vary from subject to subject.