Apoptosis Markers in Aggressive Periodontitis

Study Overview
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Brief Summary
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The aim of the present trial is to assess the gingival crevicular fluid level of caspase-3 and apoptosis inducing factor (AIF) in Generalized versus Molar-incisor grade C periodontitis. The present study will be carried out on patients selected from those attending on the outpatient clinics of Department of Oral Medicine, Periodontology, Oral Diagnosis and Dental Radiology, Faculty of Dental Medicine, Al-Azhar University, Assiut.

Detailed Description
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Periodontal diseases are inflammatory disorders that give rise to tissue damage and loss, as a result of complex interactions between pathogenic bacteria and host immune response. Gingivitis is generally regarded as a site-specific inflammatory condition initiated by dental biofilm accumulation and characterized by gingival redness and edema and the absence of periodontal attachment loss.(1,2) periodontitis, a disease characterized by gingival inflammation combined with connective tissue attachment and bone loss. Aggressive periodontitis is a form of periodontal disease characterized by rapid attachment loss, bone destruction, non-contributory medical history and family history of the cases Apoptosis is a fundamental and complex biological process that enables an organism to kill and remove unwanted cells during development, normal homeostasis and disease. Caspases are proteins implicated in the activation and execution of apoptosis was identified in humans as being crucial for this process. Caspases 3 appear to be activated in a protease cascade that leads to inappropriate activation or rapid disablement of key structural proteins and important signaling, homeostatic and repair enzymes and is essential for some of the characteristic changes in cell morphology and certain biochemical events associated with the execution and completion of apoptosis. However, the specific requirements of this caspase in apoptosis were until now largely unknown. Apoptosis-inducing factor (AIF) is a mitochondrion-localized flavoprotein with nicotinamide dinucleotide (NADH) oxidase activity is encoded by a nuclear gene. It has been shown to translocate from mitochondria to the cytosol as well as the nucleus when apoptosis is induced and mediate caspase-independent death because inhibition of caspase activation. Many authors view apoptosis as a process that is near-to-synonymous to massive caspase activation. The existence of a caspase-independent death effector thus does not fit the dominant scheme. During the last 2 years, several papers advocated the hypothesis that AIF is indeed a caspase-dependent death effector, meaning that the translocation of AIF from the mitochondrion (where it would be inert) to the nucleus (where it would induce apoptosis) would be caspase-dependent.

Official Title
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Assessment of Apoptosis Markers Caspase-3, Caspase-8, Caspase-9 and Apoptosis Inducing Factor (AIF) Levels in Generalized Versus Molar-incisor Grade C Periodontitis

Conditions
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Molar-incisor Pattern Grade C Periodontitis ( Localized Aggressive Periodontitis), Generalized Grade C Periodontitis (Generalized Aggressive Periodontitis)

Intervention / Treatment
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* Diagnostic Test: ELISA assessment of apoptosis markers in gingival crevicular fluid


Participation Criteria
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Eligibility Criteria
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inclusion criteria Patients must be free from any systemic diseases according to criteria of Modified Cornell Medical Index. Males and females aged ≥18 years old. All subjects should be cooperative. exclusion criteria females, who were lactating or pregnant, will be excluded. smoker's or former smokers also will be excluded . any other periodontal diseases, except generalized grade c periodontitis or molar-incisor grade c periodontitis Patients received antibiotics or non- steroidal anti-inflammatory drugs 3 months or periodontal treatment 6 months' before the beginning of the study

Ages Eligible for Study
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Minimum Age: 18 Years
Maximum Age: 75 Years

Sexes Eligible for Study
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All

Accepts Healthy Volunteers
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No

Study Plan
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How is the study designed?
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Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Generalized grade C periodontitis<br>generalized aggressive periodontitis ( generalized Grade C ,as in new classification) patients characterized with Bone loss/age index more than 1 and extent more 30 % | Diagnostic Test: ELISA assessment of apoptosis markers in gingival crevicular fluid<br>* Gingival crevicular fluid (GCF) samples will be collected to assess the level of caspase-3,8,9 and apoptosis inducing factor (Aif)<br>|
| Molar-incisor grade C periodontitis<br>localized aggressive periodontitis ( Molar-incisor pattern Grade C ,as in new classification) patients characterized with limited affection on molars and incisors and their Bone loss/age index more than 1. | Diagnostic Test: ELISA assessment of apoptosis markers in gingival crevicular fluid<br>* Gingival crevicular fluid (GCF) samples will be collected to assess the level of caspase-3,8,9 and apoptosis inducing factor (Aif)<br>|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| caspase 3 , picograms/millimeter (pg/ml) in gingival crevicular fluid | GCF from the mesio-buccal site of each tooth was collected using paper strips held in place for 30 seconds, and then removed. GCF volume (ml) was measured using a calibrated machine. After volume determination, strips were stored in sterile collection tubes that were labeled and then stored at - 80C. Enzyme-linked immunosorbent assays were used to quantify caspase 3 amount in picograms and its concentration (pg/ml) estimated according to the following formula= total caspase 3 amount (pg) / volume GCF (ml). | through diagnosis completion, an average of 6 months | 
| caspase 8 , picograms/millimeter (pg/ml) in gingival crevicular fluid | GCF from the mesio-buccal site of each tooth was collected using paper strips held in place for 30 seconds, and then removed. GCF volume (ml) was measured using a calibrated machine. After volume determination, strips were stored in sterile collection tubes that were labeled and then stored at - 80C. Enzyme-linked immunosorbent assays were used to quantify caspase 8 amount in picograms and its concentration (pg/ml) estimated according to the following formula= total caspase 8 amount (pg) / volume GCF (ml). | through diagnosis completion, an average of 6 months | 
| caspase 9 , picograms/millimeter (pg/ml) in gingival crevicular fluid | GCF from the mesio-buccal site of each tooth was collected using paper strips held in place for 30 seconds, and then removed. GCF volume (ml) was measured using a calibrated machine. After volume determination, strips were stored in sterile collection tubes that were labeled and then stored at - 80C. Enzyme-linked immunosorbent assays were used to quantify caspase 9 amount in picograms and its concentration (pg/ml) estimated according to the following formula= total caspase 9 amount (pg) / volume GCF (ml). | through diagnosis completion, an average of 6 months | 
| Apoptosis Inducing Factor(AIF), picograms/millimeter (pg/ml) in gingival crevicular fluid | GCF from the mesio-buccal site of each tooth was collected using paper strips held in place for 30 seconds, and then removed. GCF volume (ml) was measured using a calibrated machine. After volume determination, strips were stored in sterile collection tubes that were labeled and then stored at - 80C. Enzyme-linked immunosorbent assays were used to quantify AIF amount in picograms and its concentration (pg/ml) estimated according to the following formula= total AIF amount (pg) / volume GCF (ml). | through diagnosis completion, an average of 6 months | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Gingival Index | gingival index used to measure gingival inflammation. The degree of gingival inflammation was assessed as follow: 0 = Normal gingiva. Mild inflammation, slight change in color, slight edema and no bleeding on probing. Moderate inflammation, redness, edema and bleeding on probing. Sever inflammation, marked redness, edema and tendency to spontaneous bleeding. | through diagnosis completion, an average of 6 months | 
| Plaque Index | Plaque index measured to assess plaque accumulation around gingival margin. The degree of plaque accumulation was assessed as follows: 0 = no plaque around the gingival margin. = A thin film of plaque around the gingival margin. The plaque may be recognized only by running a probe across the tooth surface. = moderate accumulation of soft deposits on the gingival margin and/or adjacent tooth surface, which can be seen by naked eye. = Abundance plaque accumulation within the gingival pocket and/or on the gingival margin and adjacent tooth surface and hard deposits on the tooth surface are seen. | through diagnosis completion, an average of 6 months | 
| Attachment level | Attachment level by mm was measured using color -coded probe. The distance from the cemento-enamal junction to the free gingival margin and the distance from the free gingival margin to the base of the pocket where properly measured. The difference between the two measurements yields the attachment level in millimeter (mm). | baseline, immediately after complete diagnosis and before any intervention or therapy | 
| Bleeding on probing (BOP) | Bleeding on probing (BOP) at a force of 0.3N.with a manual pressure of sensitive probe, recorded on distal, facial, mesial, gingival surfaces. Calculation = (Number of bleeding surfaces / total number of tooth surface) *100.Expressed in percentage (%) of gingival units bleeding on probing FMBS. In the rating system, 0 indicates absence of bleeding on probing, with 15%, 20% and >20% indicating an increased percentage of inflammation/infection. | through diagnosis completion, an average of 6 months | 
| radiographic evaluation at molar-incisor area | Marginal bone loss around all central incisors and first molars was evaluated using periapical radiographs. The distance from cemento-enamel junction to the crest of alveolar bone will represent bone lost by mm | through diagnosis completion, an average of 6 months |