What happens at the interface between What happens at the interface between plaque and subgingival tissue?plaque and subgingival tissue?

Clinical observationsClinical observations:: inflammation, apical inflammation, apical migration of junctional epithelium, tissue migration of junctional epithelium, tissue destruction, bone resorption, etc.destruction, bone resorption, etc.

Analysis of potential bacterial virulence factorsAnalysis of potential bacterial virulence factors:: characterize specific activity, assay behavior of characterize specific activity, assay behavior of isogenic mutants.isogenic mutants.

Tissue culture modelsTissue culture models:: mono- or multilayer cultured mono- or multilayer cultured cells challenged with bacteria or bacterial products.cells challenged with bacteria or bacterial products.

OverviewOverview

Periodontal disease in contextPeriodontal disease in context: a common, : a common, chronically progressive polymicrobial disease chronically progressive polymicrobial disease whose progress is mediated by both bacterial whose progress is mediated by both bacterial factors and host immune responses.factors and host immune responses.

bacteriumphagocytic

cell

TISSUE DAMAGETISSUE DAMAGE BONE RESORPTIONBONE RESORPTION

Inflammatory Inflammatory mediators mediators (cytokines) (cytokines)

(prostaglandins)(prostaglandins)

Dys-regulation of Dys-regulation of host proteinase host proteinase

inhibitorsinhibitors

ProteasesProteases

LeukotoxinLeukotoxin

Evasion of host Evasion of host defensesdefenses

Inflammatory Inflammatory mediators mediators (cytokines)(cytokines)

Proteases Proteases Capsule Capsule

LeukotoxinLeukotoxin

Activation of Activation of inflammatory inflammatory

responseresponse

Lysosomal Lysosomal enzymes and free enzymes and free

radicalsradicals

from Marsh, 1999

Surface-associated Surface-associated materialmaterialLTA, LPSLTA, LPSHyaluronidase Hyaluronidase

Colllagenase Colllagenase Arginine-specific Arginine-specific

proteaseprotease

CytotoxinsCytotoxins

Pattern recognition in Pattern recognition in periodontal microbiologyperiodontal microbiology

Approximately 15-20 cultivable species have been associated with Approximately 15-20 cultivable species have been associated with periodontal diseases.periodontal diseases.

Most (except Most (except A.a.A.a.) are:) are: Anaerobic Anaerobic andand Asaccharolytic Asaccharolytic

Recognizable patternRecognizable pattern:: Anaerobic, asaccharolytic bacterial species are associated with Anaerobic, asaccharolytic bacterial species are associated with

periodontal diseaseperiodontal disease Tentative conclusion / hypothesis to be testedTentative conclusion / hypothesis to be tested::

Anaerobic, asaccharolytic bacteria cause most forms of Anaerobic, asaccharolytic bacteria cause most forms of periodontal disease.periodontal disease.

Suspected periodontal pathogensSuspected periodontal pathogens

Species Clinical entitiy Oxygen sensitivityPorphyromonas gingivalisPorphyromonas gingivalis AP, EOP, RPAP, EOP, RP AnaerobicAnaerobic

Bacteroides forsythusBacteroides forsythus AP, EOP, RPAP, EOP, RP AnaerobicAnaerobic

Treponema denticolaTreponema denticola AP, EOP, RPAP, EOP, RP AnaerobicAnaerobic

Prevotella intermediaPrevotella intermedia AP, ANUG?AP, ANUG? AnaerobicAnaerobic

Fusobacterium nucleatumFusobacterium nucleatum AP, ANUG?AP, ANUG? AnaerobicAnaerobic

Eubacterium nodatumEubacterium nodatum APAP AnaerobicAnaerobic

Selenomonas noxiaSelenomonas noxia APAP AnaerobicAnaerobic

Porphyromonas gracilisPorphyromonas gracilis APAP AnaerobicAnaerobic

Treponema vincentiiTreponema vincentii AP, ANUGAP, ANUG AnaerobicAnaerobic

Peptostreptococcus microsPeptostreptococcus micros AP, RPAP, RP AnaerobicAnaerobic

EubacteriumEubacteriumspp.spp. APAP AnaerobicAnaerobic

Selenomonas Selenomonas spp.spp. APAP AnaerobicAnaerobic

Streptococcus intermediusStreptococcus intermedius APAP AnaerobicAnaerobic

A. actinomycetemcomitansA. actinomycetemcomitans LJP, EOP?, RP?LJP, EOP?, RP? MicroaerophilicMicroaerophilic

Wolinella rectaWolinella recta APAP MicroaerophilicMicroaerophilic

Eikenella corrodensEikenella corrodens AP?AP? MicroaerophilicMicroaerophilic

Cluster analysis of 32 subgingival species from 13,261 Cluster analysis of 32 subgingival species from 13,261 samples. 5 clusters were formed with >60% similarity samples. 5 clusters were formed with >60% similarity

and included 29 of the 32 species evaluated. and included 29 of the 32 species evaluated.

Socransky et al. 1998

(green)

(orange)

(yellow)

(red)

(purple)

(S. Socransky)(S. Socransky)

(S. Socransky)(S. Socransky)

Periodontal Diseases:Periodontal Diseases:

Diagnostic tests and vaccine Diagnostic tests and vaccine developmentdevelopment

Microbial considerationsMicrobial considerations

ProspectsProspects

LimitationsLimitations

Is Periodontal Disease due to:Is Periodontal Disease due to:

The nonspecific overgrowth of bacteria, mostly anaerobes, on The nonspecific overgrowth of bacteria, mostly anaerobes, on the tooth surface?the tooth surface? Dirty Mouth SyndromeDirty Mouth Syndrome

OROR The overgrowth of certain bacteria, mostly anaerobes, on the The overgrowth of certain bacteria, mostly anaerobes, on the

tooth surface?tooth surface?Specific InfectionSpecific Infection

OROR Genetic predisposition to periodontal disease?Genetic predisposition to periodontal disease?

The target population for diagnostic testsThe target population for diagnostic tests

Why target specific patients for treatmentWhy target specific patients for treatment??

•You can not expect to treat everyone..You can not expect to treat everyone..•You would like to treat patients by most appropriate means.You would like to treat patients by most appropriate means.

Populations you would like to treatPopulations you would like to treat::

•Those most at risk to develop disease. Those most at risk to develop disease. •Those at most risk for having refractory disease.Those at most risk for having refractory disease.

The target population for diagnostic testsThe target population for diagnostic tests

Boyer et al., 1996Boyer et al., 1996

3.3

Relationship between trypsin-like enzyme Relationship between trypsin-like enzyme activity and abscess formation and lethality: activity and abscess formation and lethality: P. gingivalisP. gingivalis injected subcutaneously in mice injected subcutaneously in mice

Characteristics of Characteristics of

P. gingivalis strainsP. gingivalis strains

Lesions in miceLesions in mice

AbscessAbscess lethalitylethality

W50W50 Trypsin +Trypsin + ++++ ++

+TLCK / PMSF+TLCK / PMSF -- --

+TLCK+TLCK ++ --

+DTT+DTT ++++ ++++

Mutant strainsMutant strains

3079.033079.03 Trypsin +Trypsin + ++++ ++++

NG4819NG4819 Trypsin -Trypsin - -- --

BE1BE1 Trypsin +/-Trypsin +/- ++ --

SW5SW5 Trypsin +/-Trypsin +/- -- --Adapted from Kesavalu et al, AADR 1992Adapted from Kesavalu et al, AADR 1992

Comparison of BANA test, DNA probes and Comparison of BANA test, DNA probes and immunological probes in detection of immunological probes in detection of

periodontal pathogens in plaque samplesperiodontal pathogens in plaque samples

Reference standard is DNA Probes for Reference standard is DNA Probes for

P. gingivalisP. gingivalis and and T. denticolaT. denticola SensitivitySensitivity AccuracyAccuracy

BANA TestBANA Test 90%90% 83%83%

ELISAELISA 94%94% 88%88%

Reference standard is antibodies to Reference standard is antibodies to

P. gingivalisP. gingivalis T. denticolaT. denticola and and B. forsythusB. forsythus SensitivitySensitivity AccuracyAccuracy

BANA TestBANA Test 91%91% 85%85%

DNA ProbeDNA Probe 93%93% 88%88%

There were no significant differences between the DNA probes, There were no significant differences between the DNA probes, antibodies and the BANA test.antibodies and the BANA test. Loesche et al., 1992, J.Clin.Micro.30:420 Loesche et al., 1992, J.Clin.Micro.30:420

Accuracy of the BANA testAccuracy of the BANA test

The BANA test detects the presence of BANA-positive species in The BANA test detects the presence of BANA-positive species in plaque samples. Known BANA-positive species are plaque samples. Known BANA-positive species are T. denticolaT. denticola, , P. P. gingivalisgingivalis, and , and B. forsythusB. forsythus, all of which are anaerobic species and , all of which are anaerobic species and putative periodontal pathogens. The accuracy of the BANA test in putative periodontal pathogens. The accuracy of the BANA test in detecting these species in plaque samples is about 85%, which is detecting these species in plaque samples is about 85%, which is comparable to an 88% accuracy for DNA probes and 87% accuracy comparable to an 88% accuracy for DNA probes and 87% accuracy for immunologic reagents. This information may aid in diagnosis if for immunologic reagents. This information may aid in diagnosis if clinical signs of periodontitis are present.clinical signs of periodontitis are present.

Clinicians make a diagnosis using clinical findings, a background Clinicians make a diagnosis using clinical findings, a background history, and interpretation of tests such as the BANA test. It is history, and interpretation of tests such as the BANA test. It is incorrect to think that the BANA test, or any other test, can be used to incorrect to think that the BANA test, or any other test, can be used to make a clinical diagnosis in the absence of the above information.make a clinical diagnosis in the absence of the above information.

BANA test: not-quite current versionBANA test: not-quite current version

Diagnostic Tests to Detect Diagnostic Tests to Detect Genetic SusceptibilityGenetic Susceptibility to Periodontitis to Periodontitis

Another strategy:Another strategy:

Test based on detection of a genetic polymorphism in the Test based on detection of a genetic polymorphism in the gene encoding IL-1 that is associated with a higher than gene encoding IL-1 that is associated with a higher than normal level of IL-1 response and subsequent normal level of IL-1 response and subsequent inflammation.inflammation.

This polymorphism is present in about 30% of the general This polymorphism is present in about 30% of the general population.population.

PST (Medical Science Systems,Inc., San Antonio, Texas). This is the only test to reveal a specific genetic markerthat identifies individuals at high risk for severe periodontitis. A sample of the individual's DNA is collected from a finger stick blood sample in a dentist's office. The sample is then sent to Medical Science Systems, Inc. (MSS) and analyzed in a licensed genetic laboratory using advanced technology to determine if the individual isgenotype-positive or genotype-negative.

The evidence supports that when genotype-positive individuals are bacterially challenged, they are at least six times more likely to develop severe generalized periodontitis. Theymay therefore need to be managed even more aggressively to keep plaque levels under control.

Breaking NewsBreaking NewsMarch 9, 1999 March 9, 1999

Medical Science Systems seeks patent on gene sequenceMedical Science Systems seeks patent on gene sequence

Medical Science Systems Inc. announced it has filed for patent Medical Science Systems Inc. announced it has filed for patent protection for a new gene sequence and its function. This protection for a new gene sequence and its function. This sequence is in the chromosome region that regulates interleukin-1 sequence is in the chromosome region that regulates interleukin-1 (IL-1) cytokine production and levels, which plays a major role in (IL-1) cytokine production and levels, which plays a major role in the body's inflammatory response, immune response, and bone the body's inflammatory response, immune response, and bone and connective tissue metabolism. and connective tissue metabolism.

CURRENT (?) AND FUTURE (?) CURRENT (?) AND FUTURE (?) COMMERCIAL TESTSCOMMERCIAL TESTS

Evalusite (available in Europe and Canada). Evalusite (available in Europe and Canada). Omnigene (OmniGene Laboratory Services, Omnigene (OmniGene Laboratory Services,

Cambridge, MA). Cambridge, MA). MicroProbe (under development).MicroProbe (under development). PerioScan (BANA Test)(Oral B Laboratories)PerioScan (BANA Test)(Oral B Laboratories) PST (Medical Science Systems,Inc., San PST (Medical Science Systems,Inc., San

Antonio, Texas). Antonio, Texas).

CURRENT (?) AND FUTURE (?) CURRENT (?) AND FUTURE (?) COMMERCIAL TESTSCOMMERCIAL TESTS

Evalusite (available in Europe and Canada). This chair side immunoassay detects A. actinomycetemcomitans, P. gingivalis, and P. intermedia by complexing antibody to specific antigens on these bacteria. The plaque sample is mixed with a detergent and placed on a membrane with antibodies to the targeted bacteria. When the antibody-antigen complex forms, a colored enzyme substrate is added to the mixture. A positive test will form a blue dot on the reagent pad. This test takes about 10 minutes.

Omnigene (OmniGene Laboratory Services, Cambridge, MA, currently available). This DNA probe tests for A. actinomycetemcomitans, P. gingivalis, P. intermedia, E. corrodens, F. nucleatum, and C. rectus. Nonviable organisms can be detected. Results are given as negative of low, moderate, or high presence of the targeted bacteria.

CURRENT (?) AND FUTURE (?) CURRENT (?) AND FUTURE (?) COMMERCIAL TESTSCOMMERCIAL TESTS

MicroProbe (under development). This DNA probe system can be completed in the dental office in 1 hour.Oligonucleotide-coated beads specific for A actinomycetemcomitans, P gingivalis, and P intermedia arecontained on a small plastic card. The patient's plaque sample is dispersed, and the DNA is extracted with reagentscontained in the kit. The card with the beads is moved through the solutions of the kit, and a positive test resultswhen the white bead changes to blue. This reaction is precipitated by the antigen-antibody complex formation ofthe targeted bacteria.

PerioScan (BANA Test)(Oral B Laboratories, Edwood City, CA, currently available).N-benzoyl-DL-arginine-2-napthylamide (BANA) is a colorless substrate that is degradated by trypsin-like enzymesproduced by B. forsythus, P. gingivalis, and T. denticola. A blue-black color on a reagent card will formif the patient's plaque sample contains one or more of these organisms. The test can be done chairside, takes 15minutes, and is relatively inexpensive. The BANA test can not detect nonviable organisms, and it detects groups of bacteria, not individual species.

Progress Toward a Vaccine Progress Toward a Vaccine Against Periodontal Against Periodontal

DiseasesDiseases

Factors that make a disease/infection Factors that make a disease/infection amenable to vaccine development:amenable to vaccine development:

Factors that make a disease/infection Factors that make a disease/infection recalcitrant to vaccine development:recalcitrant to vaccine development:

Factors that make a Factors that make a disease/infection amenable disease/infection amenable

to vaccine development:to vaccine development: Limited number of pathogenic agents. Limited number of pathogenic agents.

The pathogen has a single major virulence factor.The pathogen has a single major virulence factor.

The pathogen appears in the blood.The pathogen appears in the blood.

Pathology of the disease is not caused by immune Pathology of the disease is not caused by immune mechanisms. mechanisms.

Factors that make a Factors that make a disease/infection recalcitrant disease/infection recalcitrant

to vaccine developmentto vaccine development A variety of pathogens or serotypes causes the A variety of pathogens or serotypes causes the

disease. disease.

The pathogen(s) possess a variety of virulence The pathogen(s) possess a variety of virulence factors. factors.

The pathogen is restricted to mucus membranes The pathogen is restricted to mucus membranes or is intracellular. or is intracellular.

Immunopathology can occur. Immunopathology can occur.

Ebersole et al., 2001Ebersole et al., 2001

Progress toward vaccination Progress toward vaccination against periodontal diseasesagainst periodontal diseases

For a non-life threatening disease a vaccine must be entirely For a non-life threatening disease a vaccine must be entirely safe and cause minimal or no side-effects.safe and cause minimal or no side-effects.

Caries and periodontal disease are localized to the tooth and Caries and periodontal disease are localized to the tooth and gingival area thus a successful vaccine must induce gingival area thus a successful vaccine must induce immunity at these sites. immunity at these sites.

Antibodies that occur in these area are mainly IgA in saliva Antibodies that occur in these area are mainly IgA in saliva although some IgG is present from gingival crevicular fluid. although some IgG is present from gingival crevicular fluid.

IgA is only a weak activator of complement and is poorly IgA is only a weak activator of complement and is poorly opsonic. The function of IgA seems to be limited to inhibition opsonic. The function of IgA seems to be limited to inhibition of attachment and neutralization of enzymes or toxins. of attachment and neutralization of enzymes or toxins.

Salivary IgA can be stimulated via the GALT thus oral Salivary IgA can be stimulated via the GALT thus oral vaccines for caries or periodontal disease may be feasible.vaccines for caries or periodontal disease may be feasible.

Periodontal disease vaccine Periodontal disease vaccine approaches:approaches:

Active Active immunizationimmunization : : Whole cells Whole cells Cellular components Cellular components

fimbriae or other antigens of fimbriae or other antigens of Porphyromonas Porphyromonas gingivalisgingivalis

Passive immunizationPassive immunization ((with specific antibodieswith specific antibodies):): Monoclonal antibodies to Monoclonal antibodies to P. gingivalis P. gingivalis antigensantigens Immune bovine milk Immune bovine milk ““ Egg yolk antibodyEgg yolk antibody ““

Periodontal disease vaccine Periodontal disease vaccine approaches:approaches:

Possible future approachesPossible future approaches:: Replacement therapy with non-pathogenic Replacement therapy with non-pathogenic

mutants.mutants.

Expression of vaccine proteins in bacteria Expression of vaccine proteins in bacteria that colonize the gut such as that colonize the gut such as S. lactisS. lactis or or Salmonella.Salmonella.

Gene therapy, eg. expressing the gene for the Gene therapy, eg. expressing the gene for the P. gingivalisP. gingivalis fimbriae in salivary gland cells. fimbriae in salivary gland cells.

Examples of anti-Examples of anti-P. gingivalisP. gingivalis vaccines under developmentvaccines under development

Feasibility of an HA2 Domain-Feasibility of an HA2 Domain-Based Periodontitis VaccineBased Periodontitis Vaccine

In a rat periodontitis model, preinoculation with the In a rat periodontitis model, preinoculation with the Porphyromonas gingivalisPorphyromonas gingivalis HA2 binding domain for HA2 binding domain for hemoglobin provided protection from disease. hemoglobin provided protection from disease.

Protection was associated with induced anti-HA2 IgG Protection was associated with induced anti-HA2 IgG humoral antibodies. The IgG subclass ratios suggested humoral antibodies. The IgG subclass ratios suggested that relatively lower Th2/Th1-driven responses were that relatively lower Th2/Th1-driven responses were directly associated with protection when rHA2 was directly associated with protection when rHA2 was administered in saline.administered in saline.

DeCarlo et al., 2003DeCarlo et al., 2003

Domain structure and homologies between Domain structure and homologies between the the P. gingivalis P. gingivalis proteases RGP-1 and KGPproteases RGP-1 and KGP

DeCarlo et al., 2003DeCarlo et al., 2003

conserved hemagglutinin domain conserved hemagglutinin domain involved in involved in P.g. P.g. adherenceadherence

Feasibility of an HA2 Domain-Feasibility of an HA2 Domain-Based Periodontitis VaccineBased Periodontitis Vaccine

Higher anti-HA2 IgG levels Higher anti-HA2 IgG levels resulted in less bone loss in resulted in less bone loss in the rat periodontitis model.the rat periodontitis model.

DeCarlo et al., 2003DeCarlo et al., 2003

Identification of vaccine candidate Identification of vaccine candidate antigens from a genomic analysis of antigens from a genomic analysis of

Porphyromonas gingivalisPorphyromonas gingivalis

Using bioinformatics methods, analyze the full Using bioinformatics methods, analyze the full P. gingivalisP. gingivalis genome sequence for genes encoding likely surface-genome sequence for genes encoding likely surface-expressed proteins.expressed proteins.

Clone these genes, express as recombinant Clone these genes, express as recombinant E. coliE. coli proteins. proteins.

Screen the cloned proteins to see if they are recognized by Screen the cloned proteins to see if they are recognized by anti-anti-P. gingivalisP. gingivalis antibodies. antibodies.

Test the recombinant proteins for ability protect against Test the recombinant proteins for ability protect against infection with infection with P. gingivalisP. gingivalis in an animal model. in an animal model.

Ross et al., 2001Ross et al., 2001

Identification of vaccine candidate Identification of vaccine candidate antigens from a genomic analysis of antigens from a genomic analysis of

Porphyromonas gingivalisPorphyromonas gingivalis

Ross et al., 2001Ross et al., 2001

Factors that make a Factors that make a disease/infection amenable disease/infection amenable

to vaccine development:to vaccine development: Limited number of pathogenic agents. Limited number of pathogenic agents.

The pathogen has a single major virulence factor.The pathogen has a single major virulence factor.

The pathogen appears in the blood.The pathogen appears in the blood.

Pathology of the disease is not caused by immune Pathology of the disease is not caused by immune mechanisms. mechanisms.

Factors that make a Factors that make a disease/infection recalcitrant disease/infection recalcitrant

to vaccine developmentto vaccine development A variety of pathogens or serotypes causes the A variety of pathogens or serotypes causes the

disease. disease.

The pathogen(s) possess a variety of virulence The pathogen(s) possess a variety of virulence factors. factors.

The pathogen is restricted to mucus The pathogen is restricted to mucus membranes or is intracellular. membranes or is intracellular.

Immunopathology can occur. Immunopathology can occur.

Antibiotics in treatment of Antibiotics in treatment of periodontal diseasesperiodontal diseases

Antibiotics used for their antimicrobial effects:Antibiotics used for their antimicrobial effects: MetronidazoleMetronidazole AmoxicillinAmoxicillin

Antibiotics used for their anti-inflammatory effects:Antibiotics used for their anti-inflammatory effects: Tetracyline and derivatives: low-dose doxycycline (Tetracyline and derivatives: low-dose doxycycline (PeriostatPeriostat): ):

these compounds are used at doses these compounds are used at doses belowbelow which antimicrobial effects which antimicrobial effects are seen.are seen.

at these levels, tetracycline derivatives inhibit activation of host at these levels, tetracycline derivatives inhibit activation of host metalloproteinases involved in tissue remodelling (collagenases).metalloproteinases involved in tissue remodelling (collagenases).