Transcript P. gingivalis

What happens at the interface between
plaque and subgingival tissue?
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Clinical observations: inflammation, apical
migration of junctional epithelium, tissue
destruction, bone resorption, etc.
Analysis of potential bacterial virulence factors:
characterize specific activity, assay behavior of
isogenic mutants.
Tissue culture models: mono- or multilayer cultured
cells challenged with bacteria or bacterial products.
Overview
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Periodontal disease in context: a common,
chronically progressive polymicrobial disease
whose progress is mediated by both bacterial
factors and host immune responses.
Inflammatory
mediators
(cytokines)
(prostaglandins)
Dys-regulation of
host proteinase
inhibitors
Evasion of host
defenses
Inflammatory
mediators
(cytokines)
Proteases
Proteases
Capsule
Leukotoxin
Leukotoxin
phagocytic
cell
Lysosomal
enzymes and free
radicals
Activation of
inflammatory
response
Hyaluronidase
Colllagenase
Arginine-specific
protease
bacterium
Surface-associated
material
LTA, LPS
Cytotoxins
TISSUE DAMAGE
BONE RESORPTION
from Marsh, 1999
Pattern recognition in
periodontal microbiology
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Approximately 15-20 cultivable species have been associated with
periodontal diseases.
Most (except A.a.) are:
 Anaerobic and
 Asaccharolytic
Recognizable pattern:
 Anaerobic, asaccharolytic bacterial species are associated with
periodontal disease
Tentative conclusion / hypothesis to be tested:
 Anaerobic, asaccharolytic bacteria cause most forms of
periodontal disease.
Suspected periodontal pathogens
Species
Clinical entitiy
Oxygen sensitivity
Porphyromonas gingivalis
AP, EOP, RP
Anaerobic
Bacteroides forsythus
AP, EOP, RP
Anaerobic
Treponema denticola
AP, EOP, RP
Anaerobic
Prevotella intermedia
AP, ANUG?
Anaerobic
Fusobacterium nucleatum
AP, ANUG?
Anaerobic
Eubacterium nodatum
AP
Anaerobic
Selenomonas noxia
AP
Anaerobic
Porphyromonas gracilis
AP
Anaerobic
AP, ANUG
Anaerobic
AP, RP
Anaerobic
Eubacteriumspp.
AP
Anaerobic
Selenomonas spp.
AP
Anaerobic
Streptococcus intermedius
AP
Anaerobic
A. actinomycetemcomitans
LJP, EOP?, RP?
Microaerophilic
Wolinella recta
AP
Microaerophilic
Eikenella corrodens
AP?
Microaerophilic
Treponema vincentii
Peptostreptococcus micros
Cluster analysis of 32 subgingival species from 13,261
samples. 5 clusters were formed with >60% similarity
and included 29 of the 32 species evaluated.
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(orange)
(yellow)
(purple)
(red)
Socransky et al. 1998
(S. Socransky)
(S. Socransky)
Periodontal Diseases:
Diagnostic tests and vaccine
development
Microbial considerations
Prospects
Limitations
Is Periodontal Disease due to:
The nonspecific overgrowth of bacteria, mostly anaerobes, on the
tooth surface?
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Dirty Mouth Syndrome
OR
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The overgrowth of certain bacteria, mostly anaerobes, on the
tooth surface?
Specific Infection
OR
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Genetic predisposition to periodontal disease?
The target population for diagnostic tests
Why target specific patients for treatment?
•You can not expect to treat everyone..
•You would like to treat patients by most appropriate means.
Populations you would like to treat:
•Those most at risk to develop disease.
•Those at most risk for having refractory disease.
The target population for diagnostic tests
3.3
Boyer et al., 1996
Relationship between trypsin-like enzyme
activity and abscess formation and lethality:
P. gingivalis injected subcutaneously in mice
Characteristics of
P. gingivalis strains
Lesions in mice
Abscess
lethality
++
+
+TLCK / PMSF
-
-
+TLCK
+
-
+DTT
++
++
W50
Trypsin +
Mutant strains
3079.03
Trypsin +
++
++
NG4819
Trypsin -
-
-
BE1
Trypsin +/-
+
-
SW5
Trypsin +/-
-
-
Adapted from Kesavalu et al, AADR 1992
Comparison of BANA test, DNA probes and
immunological probes in detection of
periodontal pathogens in plaque samples
Reference standard is DNA Probes for
P. gingivalis and T. denticola
Sensitivity
Accuracy
BANA Test
90%
83%
ELISA
94%
88%
Sensitivity
Accuracy
BANA Test
91%
85%
DNA Probe
93%
88%
Reference standard is antibodies to
P. gingivalis T. denticola and B. forsythus
There were no significant differences between the DNA probes,
antibodies and the BANA test.
Loesche et al., 1992, J.Clin.Micro.30:420
Accuracy of the BANA test
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The BANA test detects the presence of BANA-positive species in
plaque samples. Known BANA-positive species are T. denticola, P.
gingivalis, and B. forsythus, all of which are anaerobic species and
putative periodontal pathogens. The accuracy of the BANA test in
detecting these species in plaque samples is about 85%, which is
comparable to an 88% accuracy for DNA probes and 87% accuracy
for immunologic reagents. This information may aid in diagnosis if
clinical signs of periodontitis are present.
Clinicians make a diagnosis using clinical findings, a background
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
make a clinical diagnosis in the absence of the above information.
BANA test: not-quite current version
Diagnostic Tests to Detect
Genetic Susceptibility to Periodontitis
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Another strategy:
 Test
based on detection of a genetic polymorphism in the
gene encoding IL-1 that is associated with a higher than
normal level of IL-1 response and subsequent
inflammation.
 This
polymorphism is present in about 30% of the general
population.
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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 News
March 9, 1999
Medical Science Systems seeks patent on gene sequence
Medical Science Systems Inc. announced it has filed for patent
protection for a new gene sequence and its function. This
sequence is in the chromosome region that regulates interleukin-1
(IL-1) cytokine production and levels, which plays a major role in
the body's inflammatory response, immune response, and bone
and connective tissue metabolism.
CURRENT (?) AND FUTURE (?)
COMMERCIAL TESTS
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Evalusite (available in Europe and Canada).
Omnigene (OmniGene Laboratory Services,
Cambridge, MA).
MicroProbe (under development).
PerioScan (BANA Test)(Oral B Laboratories)
PST (Medical Science Systems,Inc., San
Antonio, Texas).
CURRENT (?) AND FUTURE (?)
COMMERCIAL TESTS
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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 antibodyantigen 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 (?)
COMMERCIAL TESTS
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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
Against Periodontal Diseases
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Factors that make a disease/infection amenable to
vaccine development:
Factors that make a disease/infection recalcitrant to
vaccine development:
Factors that make a
disease/infection amenable to
vaccine development:
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Limited number of pathogenic agents.
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The pathogen has a single major virulence factor.
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The pathogen appears in the blood.
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Pathology of the disease is not caused by immune
mechanisms.
Factors that make a
disease/infection recalcitrant to
vaccine development
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A variety of pathogens or serotypes causes the disease.
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The pathogen(s) possess a variety of virulence factors.
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The pathogen is restricted to mucus membranes or is
intracellular.
Immunopathology can occur.
Ebersole et al., 2001
Progress toward vaccination
against periodontal diseases
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For a non-life threatening disease a vaccine must be entirely safe
and cause minimal or no side-effects.
Caries and periodontal disease are localized to the tooth and
gingival area thus a successful vaccine must induce immunity at
these sites.
Antibodies that occur in these area are mainly IgA in saliva
although some IgG is present from gingival crevicular fluid.
IgA is only a weak activator of complement and is poorly opsonic.
The function of IgA seems to be limited to inhibition of attachment
and neutralization of enzymes or toxins.
Salivary IgA can be stimulated via the GALT thus oral vaccines for
caries or periodontal disease may be feasible.
Periodontal disease vaccine
approaches:
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Active immunization :
 Whole
cells
 Cellular components
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fimbriae or other antigens of Porphyromonas
gingivalis
Passive immunization (with specific antibodies):
 Monoclonal
antibodies to P. gingivalis antigens
 Immune bovine milk
“
 Egg
yolk antibody
“
Periodontal disease vaccine
approaches:
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Possible future approaches:
 Replacement
therapy with non-pathogenic mutants.
 Expression
of vaccine proteins in bacteria that
colonize the gut such as S. lactis or Salmonella.
 Gene
therapy, eg. expressing the gene for the P.
gingivalis fimbriae in salivary gland cells.
Examples of anti-P. gingivalis
vaccines under development
Feasibility of an HA2 Domain-Based
Periodontitis Vaccine
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In a rat periodontitis model, preinoculation with the
Porphyromonas gingivalis HA2 binding domain for
hemoglobin provided protection from disease.
Protection was associated with induced anti-HA2 IgG
humoral antibodies. The IgG subclass ratios suggested
that relatively lower Th2/Th1-driven responses were
directly associated with protection when rHA2 was
administered in saline.
DeCarlo et al., 2003
Domain structure and homologies between
the P. gingivalis proteases RGP-1 and KGP
conserved hemagglutinin domain
involved in P.g. adherence
DeCarlo et al., 2003
Feasibility of an HA2 Domain-Based
Periodontitis Vaccine
Higher anti-HA2 IgG levels
resulted in less bone loss in
the rat periodontitis model.
DeCarlo et al., 2003
Identification of vaccine candidate
antigens from a genomic analysis of
Porphyromonas gingivalis
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Using bioinformatics methods, analyze the full P. gingivalis
genome sequence for genes encoding likely surfaceexpressed proteins.
Clone these genes, express as recombinant E. coli proteins.
Screen the cloned proteins to see if they are recognized by
anti-P. gingivalis antibodies.
Test the recombinant proteins for ability protect against
infection with P. gingivalis in an animal model.
Ross et al., 2001
Identification of vaccine candidate
antigens from a genomic analysis of
Porphyromonas gingivalis
Ross et al., 2001
Factors that make a
disease/infection amenable to
vaccine development:

Limited number of pathogenic agents.

The pathogen has a single major virulence factor.

The pathogen appears in the blood.

Pathology of the disease is not caused by immune
mechanisms.
Factors that make a
disease/infection recalcitrant to
vaccine development
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
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A variety of pathogens or serotypes causes the
disease.
The pathogen(s) possess a variety of virulence factors.
The pathogen is restricted to mucus membranes or is
intracellular.
Immunopathology can occur.
Antibiotics in treatment of
periodontal diseases
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Antibiotics used for their antimicrobial effects:
Metronidazole
 Amoxicillin
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Antibiotics used for their anti-inflammatory effects:
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Tetracyline and derivatives: low-dose doxycycline (Periostat):
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these compounds are used at doses below which antimicrobial effects
are seen.
at these levels, tetracycline derivatives inhibit activation of host
metalloproteinases involved in tissue remodelling (collagenases).