References

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Carranza·s Clinical Periodontology; 10th ed. Elsevier publication. Textbook of Clinical Periodontology, Glickman; 6th ed. Oral microbiology ² Philip Marsh, Michael Martin ² 4th ed Oral microbiology & immunology ² Nisengard & Newman -2nd ed Immunology of Oral Diseases ² Thomas Lehner ² 3rd ed Essential microbiology for dental students ² Lakshman Samaranayake ² 3rd ed Anne. D. Haffajee & sigmund.S. Socransky. Microbial etiological agents of destructive periodontal diseases. Periodontology 2000, Vol. 5, 1994, 78-1 Tatsuj Nishihara & Takeyoshi koseki. Microbial etiology of Periodontitis. Periodontology 2000, Vol. 36, 2004, 14²26.

Terms used

      Aerobes Anaerobes Facultative anaerobes Obligatory Capnophilic Microaerophilic .

approximately 1014 microorganisms consisting of more than 400 different types of bacteria.  Within hours . After weaning (>2 years).  Within 2 weeks .MICROBIOLOGY OF ORAL CAVITY The colonization of the oral cavity starts about the time of birth.a nearly mature microbiota is established in the gut of the newborn. Body contains 10 times more bacteria than human cells.anaerobic bacteria can be detected in the infant·s edentulous mouth.colonized by low numbers of mainly facultative and aerobic bacteria. .  Second day .

. 51000 bacteria were isolated  509 distinct taxa were recognized  141 were detected only once.

Habitat  Teeth are the primary habitat for periopathogens.  Mucosal surfaces ² dorsum of tongue & tonsils. Therefore. . teeth can even be considered as a port of entry for periodontopathogens.

Dental plaque 

Dental plaque is defined as a specific but highly

variable structural entity, resulting from colonization of microorganisms on tooth surface, restorations and Other parts of the oral cavity which consists of salivary components like mucin, desquamated epithelial cells, debris and microorganisms all embedded in a gelatinous extracellular matrix.

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STRUCTURE AND COMPOSISTION OF DENTAL PLAQUE

Dental plaque is composed primarily of microorganisms. One gram of plaque (wet weight) contains approximately 1011 bacteria. In a periodontal pocket,  Healthy crevice - 103 bacteria. 

Deep pocket - 108 bacteria.
Nonbacterial microorganisms that are found in plaque include Mycoplasma species, yeasts, protozoa, and viruses.

Dental plaque is broadly classified based on its position on the tooth surface toward the gingival margin: * Supragingival plaque: * Marginal plaque: * Subgingival plaque: Subgingival microbiota differs in composition from the supragingival plaque, primarily  Availability of blood products and  Low oxidation reduction (redox) potential, GCF substances which acts as nutrients characterizes the anaerobic environment.

 Marginal plaque ² prime importance in the initiation and development of gingivitis. .  Tissue associated subgingival plaque ² important in the tissue destruction that characterizes different forms of periodontitis.  Supragingival plaque and tooth associated subgingival plaque ² critical in calculus formation and root caries.The site specificity of plaque is significantly associated with diseases of the periodontium.

and oxygen concentrations.  These microcolonies have micro environments with differing pH·s.  These chemical signals trigger the bacteria to produce potentially harmful proteins and enzymes.BASIC BIOFILM STRUCTURE  They are grouped in microcolonies surrounded by an enveloping intermicrobial matrix.  The matrix is penetrated by fluid channels that conduct the flow of nutrients. waste products.  The bacteria in a biofilm communicate with each other by sending out chemical signals. . metabolites. enzymes. and oxygen. nutrient availability.

.  Loose layer: on top of the lower layer. microbes are bound together in a polysaccharide matrix with other organic and inorganic materials. it can extend into the surrounding medium.  Fluid layer: bordering the biofilm has a rather stationary sublayer and a fluid layer in motion. Appears that is often irregular in appearance.LAYERS  Lower plaque layers: these are dense.  Nutrient components penetrate this fluid medium by molecular diffusion.

Glycoproteins . potassium. gingival crevicular fluid.Lipid material .Albumin . and fluoride.Polysaccharides-dextran.Predominantly calcium and phosphorous . . INORGANIC . and bacterial products.The intercellular matrix consists or organic and inorganic materials derived from saliva. of sodium. INTERCELLULAR MATRIX: ORGANIC CONSTITUENTS COMPONENTS .Trace amt.

. The source of inorganic constituents: Supragingival plaque .  The fluoride component of plaque is largely derived from external sources such as fluoridated toothpastes. rinses and fluoridated drinking water.primarily saliva Subgingival plaque ² Crevicular fluid (a serum transudate).

 Initial adhesion and attachment of bacteria.DENTAL BIOFILM FORMATION AT THE ULTRASTRUCTURAL LEVEL The process of plaque formation can be divided into three major phases:  The formation of the pellicle on the tooth surface.  Colonization and plaque maturation. .

a thin. covers the tooth surface. Glycoproteins (mucins) Proline-rich proteins Phosphoproteins (statherin) Histidine-rich proteins Enzymes (alpha amylase) Other molecules which acts as adhesion sites.FORMATION OF THE PELLICLE Within nanoseconds after a vigorously polishing the teeth. . This pellicle consists of. saliva-derived layer called the acquired pellicle.

(through Brownian motion. . from a certain distance (50 nm). . through long range and short range forces. Random contacts may occur. liquid flow or active bacterial movement). Phase 2: Initial adhesion.INITIAL ADHESION AND ATTACHMENT OF BACTERIA Phase 1: Transport to the surface. Initiated by the interaction between the bacterium and the surface.

 Phase 4: Colonization of the surface and biofilm formation.  A firm anchorage between bacterium and surface will be established by specific interactions (covalent. ionic or hydrogen bonding). .  Eg: A.Phase 3: Attachment. viscosus possesses fimbriae that contain adhesions that specifically bind to proline rich proteins of the dental pellicle.

Actinobacillus actinomycetemcomitans serotype a.are either independent or defined complexes. .Member of Yellow (Streptococcus spp.Members of Green complexes (Eikenella corrodens. Secondary colonizers: . 2. Early colonizers: .COLONIZATION AND PLAQUE MATURATION COMPLEXES OF PERIODONTAL MICROORGANISMS: 1. and Capnocytophaga species) and Red complexes. .) or purple complexes (Actinomyces odontolyticus).

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 First 2 to 8 hours ² Pioneering streptococci saturate the salivary pellicular binding sites and thus covering 3% to 30% of the enamel surface.  Next 20 hours ² a short period of rapid growth is observed. The further growth of the plaque mass occurs preferably by the multiplication of already adhering microorganisms rather than by new colonizers .GROWTH DYNAMICS OF DENTAL PLAQUE Ultrastructural Aspects:  Important changes within first 24 hours.  After 1 day ² the term ¶Biofilm· is fully deserved because organization takes place within it.

. then slows down.  After 4 days ² average of 30% of the total tooth crown area will be covered with plaque and no more increase substantially with time. CLINICAL ASPECTS Clinically it follows an exponential growth curve.  During next 3 days ² plaque growth increases at a rapid rate. There will be an ecological shift within of the biofilm there is a transition from the early aerobic environment to a highly oxygen-deprived environment. covering <3% of the vestibular tooth surface.  First 24 hours ² negligible plaque.DE NOVO SUPRAGINGIVAL PLAQUE FORMATION.

and have impaired host defences. The resident microflora also reduces the risk of infection by acting as a barrier to colonization by exogenous (and often pathogenic species (termed ¶colonization resistance·) . Play a critical role in the normal development of the physiology of the host.BENEFITS OF DENTAL PLAQUE: Dental plaque is part of the natural resident microflora of the human body. Germ-free animals have altered mucosal surfaces. poor nutrient absorption. suffer from nutrient deficiencies.

The absence.ASSOCIATION OF PLAQUE MICROORGANISMS WITH PERIODONTAL DISEASES The current concept on the etiology of periodontitis considers three groups of factors that determine whether active periodontitis will occur in a subject: A susceptible Host. . or a small proportion . The presence of pathogenic species. of beneficial bacteria.

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PATHOGENS STRENGTH OF RELATIONSHIP WITH DISEASE: .

MICROBIAL SPECIFICITY OF PERIODONTAL DISEASE NONSPECIFIC PLAQUE HYPOTHESIS: This hypothesis maintains that periodontal disease results from the ´elaboration of noxious products by the entire plaque floraµ SPECIFIC PLAQUE HYPOTHESIS: The specific plaque hypothesis states that only certain plaque is pathogenic. and its pathogenicity depends on the presence of or increase in specific microorganisms. ECOLOGICAL PLAQUE HYPOTHESIS: A change in the nutrient status of a pocket or chemical and physical changes to the habitat are thus considered the primary cause for the overgrowth by pathogens. .

. which has a significant impact on both its diagnosis and its treatment. Currently periodontitis is considered a mixed infection.COMPLICATING FACTORS The periodontal microbiota is a complex community of microorganisms. with alternating episodes of rapid tissue destruction and periods of remission. Recent microbiologic tests clearly indicate that the presence of periodontal pathogens by itself is not sufficient for the development of periodontitis. many of which are still difficult or impossible to isolate in the laboratory. Studies have established that disease progresses at different rates.

Veilonella parvula. and C. sanguis. including S. Protective or beneficial to the host. approximately 75% to 80% of the recoverable microflora is gram-positive with most of the remainder belonging to gramnegative species of the Veillonella and Fusobacterium genera.MICROORGANISMS ASSOCIATED WITH SPECIFIC PERIODONTAL DISEASES PERIODONTAL HEALTH  At periodontally healthy sites the microbial load is low with only 102 to 103 bacteria. ochraceus.  In health. .  Finally. the microflora in health is mostly of a nonmotile nature.

with approximately 104 to 106 bacteria. .GINGIVITIS  The microbial load at diseased sites is greater. Equal proportions of gram positive (56%) and gram negative (44%) species.  The microbiota of chronic gingivitis (plaque induced) consist of app. as well as facultative (59%) and anaerobic (41%) microorganisms. which uses the steroid as growth factors. intermedia.  Pregnancy associated gingivitis is accompanied by increases in steroid hormones in crevicular fluid and dramatic increases in levels of P.

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g. . gingivalis and Tannerella forsythensis). P.CHRONIC PERIODONTITIS Cultivation of plaque microorganisms from sites of chronic periodontitis reveals high percentages of anaerobic (90%) and gram negative (75%) bacterial species. Certain gram-negative bacteria with pronounced virulence properties have been strongly implicated as etiologic agents (e.

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C. rectus. and Veillonella parvula.LOCALIZED AGGRESSIVE PERIODONTITIS The microflora of subgingival biofilms from patients with LAP is similar to that of patients with chronic periodontitis and is predominantly composed of gram negative. Actinobacillus actinomycetemcomitans plays a causative role in LAP. However. some populations of patients with LAP do not harbor A. naeslundii. F. nucleatum. the most numerous isolates are several species from the genera Eubacterium. On a percentage basis. A. and anaerobic rods. . especially in cases in which patients harbor highly leukotoxic strains of the organism. and in still others P. actinomycetemcomitans. gingivalis may be etiologically more important. capnophilic.

. forsythensis A. The predominant subgingival bacteria in patients with generalized aggressive periodontitis are P. gingivalis. actinomycetemcomitans.GENERALIZED AGGRESSIVE PERIODONTITIS The subgingival flora in patients with generalized aggressive periodontitis resembles that in other forms of periodontitis. T. and Campylobacter species.

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forsythensis. T. Persistence of Streptococcus constellatus has also been reported . intermedia. S. In other patients. P. staphylococci. persistently high levels are found of one or more of the following bacteria: P. gingivalis. intermedius. and Eikenella corrodens. Peptostreptococcus micros. and Candida.REFRACTORY CHRONIC PERIODONTITIS The microflora taken from progressing sites in some of these patients is unusually diverse and may contain enteric rods.

respectively.NECROTIZING ULCERATIVE GINGIVITIS/PERIODONTITIS The majority of the spirochetes (treponemes) associated with necrotizing ulcerative gingivitis are uncultivable.4%. gingivalis and F. . but it is clear that they constitute a very large and diverse group. nucleatum accounting for 7-8% and 3. More than 50% of the isolated species were strict anaerobes with P.

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PERIODONTAL ABSCESSES:

The bacteria isolated from abscesses are similar to those associated with chronic and aggressive forms of periodontitis. An average of approximately 70% of the cultivable flora in exudates from periodontal abscesses are gram-negative and about 50% are anaerobic rods. Periodontal abscesses revealed a high prevalence of the following putative pathogens: F. nucleatum (70.8%), P. micros (70.6%), P. intermedia (62.5%), P. gingivalis (50.0%), and T. forsythensis (47.1%). Enteric bacteria, coagulase-negative staphylococci, and Candida albicans have also been detected.

PARVOBACTERIA 

Actinobacilli  Only bacilli isolated from oral cavityActinobacillus actinomycetem comitans  Habitat : sub gingival  0.4 -1 um  Straight or curved rods with rounded ends.  Grow as white, translucent. Smooth & nonhemolytic colonies on blood agar  Growth is promoted by CO2.

ACTINOBACILLUS ACTINOMYCETEMCOMITANS

Aclinobaciltus" refers to the internal star-shaped morphology of its bacterial colonies on solid media and to the short rod or bacillary shape of individual cells selective media: tryptone-soy-serum-bacitracin-vanomycin agar (white translucent colonies with * shaped internal structure) This species was first recognized as a possible periodontal pathogen in lesions of localized juvenile periodontitis.

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 Culture :grows slowly under anaerobic condition (blood/serum glucose agar at 37 deg C)  Produces small. A georgiae  Gm + filamentous branching rod. A naeslundii. A odontolyticus. A myeri. non.soil organism  Found in dental plaque.sporing.ACTINOMYCETES  True bacteria with long branching filament. non-acid fast. creamy white adherent colonies on blood agar.  Species : A israelli.  Important genera: actinomyces (microaerophilic / anaerobic) Nocardia (aerobic) Actinomyces .  Non-motile. .

A israelii .

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non motile.PORPHYROMONAS GINGIVALIS It is a Gram negative. asaccharolytic rods non-motile that usually exhibit coccal to short rod morphologies. Culture: grow anaerobically with dark pigmentation on media containing lysed blood.  P. gingivalis is a member of the much investigated ´black pigmented Bacteroidesµ group. melaninogenicus . Does not ferment carbohydrate. B. Initially grouped into a single species.  Second consensus periodontal pathogen. Found in sub gingival sulcus. anaerobic.

P gingivalis .

TANNERELLA FORSYTHIA Third consensus periodontal pathogen. B. The organism is a Gram negative. forsythus was detected more frequently and in higher numbers in active periodontal lesions than inactive lesions. anaerobic. B. forsythus. was first described in 1979 as a ´fusiformµ Bacteroides. highly pleomorphic rod. spindle shaped. This species has been shown to produce trypsin like proteolytic activity and induce apoptotic cell death .

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At least 15 species of subgingival spirochetes have been described. a spirochete has been implicated as the likely etiologic agent of acute necrotizing ulcerative gingivitis  The organism has been considered as possible periodontal pathogens since the late 1800s and in the 1980s.  Difficulty in distinguishing individual species. helical shaped. anaerobic. highly motile microorganisms that are common in many periodontal pockets.  Clearly. .SPIROCHETES These are Gram negative.

denticola was found to be more common in periodontally diseased than healthy sites These ´were the most frequently detected spirochetes in supra and subgingival plaques of periodontitis patients. Pathogen related oral spirochetesµ (PROS) were also shown to have the ability to penetrate a tissue barrier in in vitro systems .T.

round-ended anaerobic rod have been shown to be particularly elevated in acute necrotizing ulcerative gingivitis and also in certain forms of periodontitis . intermedia is the second black pigmented Bacteroides to receive considerable interest in pathogenesis of chronic periodontitis. It is a Gram negative.PREVOTELLA INTERMEDIA/PREVOTELLA NIGRESCENS P. short.

intermedia and P. gingivalis and was shown to induce mixed infections. intermedia that show identical phenotypic traits have been separated into two species. . nigrescens. It has also been shown to invade oral epithelial cells in vitro Strains of P. P.This species appears to have a number of virulence properties exhibited by P.

and bacterion. a spindle. a small spindleshaped rod. polymorphum. and animalis. . fusiforme. nucleatum is the type species of the genus Fusobacterium. Gharbia and Shah (1990) divided Fusobacterium species into four subspecies: subspecies nucleatum. which belongs to the family Bacteroidaceae.FUSOBACTERIUM NUCLEATUM: F. a small rod: thus. The name Fusobacterium has its origin in fusus.

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spindle shaped (cigar shaped) rod that has been recognized as part of the subgingival microbiota for over 100 years. anaerobic. Acts as ´microbial bridgeµ facilitating coaggregation between early and Late colonizers.F. The species can induce apoptotic cell death in mononuclear and polymorphonuclear cells. This species is the most common isolate found in cultural studies of subgingival plaque samples comprising app. nucleatum is a Gram negative. 7-10% of total isolates. .

constellatus and S.THE MILLERI STREPTOCOCCI: Cultural studies of the last two decades have also suggested the possibility that some of the streptococcal species are associated with and may contribute to disease progression. These species was found to be elevated at sites which demonstrated recent disease progression. intermidius might contribute to disease progression in subsets of periodontal patients. evidence suggests that the milleri streptococci. S. Streptococcus anginosus. At this time. .

Emphasis have been placed on enteric organisms. particularly in individuals who have responded poorly to periodontal therapy. K. . Interest has grown in groups of species not commonly found in the subgingival plaque as initiators or possibly contributors to the pathogenesis of periodontal disease.OTHER SPECIES Obviously all periodontal pathogens have not yet been identified. pneumoniae. Slots et al (1990) found Enterobacter cloaceae. staphylococcal species as well as other unusual mouth inhabitants. Klebsiella oxytoca and Enterobacter agglomerans which constitute more than 50% of strains isolated from Chronic periodontitis patients. Pseudomonas aeruginosa.

VIRUSES More recently. possibly by changing the host response to the local subgingival microbiota HSV-1 and EBV are significantly associated with destructive periodontal disease including chronic and aggressive periodontitis. Epstein Barr. HSV-1 detected sites is associated with severity and progression of destructive periodontal disease . viruses including cytomegalo. Papilloma and herpes simplex have been proposed to play a role in the etiology of periodontal diseases.

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Dogan B. albicans serotypes and genotypes and suggested geographic clustering of C. Reynaud AH (2001) found a weak correlation between yeasts in periodontal pockets and E. albicans clones in Subgingival samples of Chronic Periodontitis patients.FUNGI Hannula J. Slots (2001) showed geographical differences in the subgingival distribution of C. . saburreum.

The presence of two pathogens at a site could have no effect or diminish the potential pathogenicity of one or other of the species.MIXED INFECTIONS: At the pathogenic end of the spectrum. Alternatively. it is conceivable that different relationships exist between pathogens. pathogenicity could be enhanced either in an additive or synergistic fashion. It is not clear whether the combinations suggested in the experimental abscess studies are pertinent to human periodontal diseases .

MICROBIAL DIAGNOSTIC TESTING .

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IMMUNOLOGY .

Local immunopathology Histopathological & ultra-structural changes in experimental gingivitis & periodontal diseases have suggested 4 stages of development:     Initial Early Established Advanced lesion .

 In relatively plaque free subjects small No of leucocytes migrate through JE towards gingival crevice.Initial lesion:  May be in response to generation of chemotactic substances by bacterial plaque Ags  Difficult to differentiate b/w normal & pathological tissue reaction. .

 Bld vsls dilate with exudation of fluid with Igs (IgG). . fibrin & PMNs  Few lymphocytes & macrophages ² in JE & adjacent CT. complement.Local immunopathology  Develops within 2 ² 4 days of plaque accumulation.  Lesion is localized to gingival sulcus & JE & CT.

Systemic immunity  Serum ABs forms Variety of immune complexes activates Classical C pathway generate C3a & C5a induce Increased vascular permeability & are chemotactic for PMNs .

.Early lesion  Develops within 4 ² 7 days.  Dense lymphoid cell infiltration.  Often seen in normal gingiva ² when plaque control is not practiced efficiently.

 Exudation of serum Igs.Local immunopathology  Lymphocytes constitute 75%.  Most of them ² T-cell series with small No of B-cells. C.  Only few plasma cells.  Some macrophages  Fibroblasts adj to lymphocytes show degenerative changes  Localized loss of collagen fibers. . fibrinogen & leucocytes is increases.  GCF & leucocytes reach maximum.

Systemic immunity Lymphoid cells ² seeded into gingival focus of inflm releases Leukocyte migration inhibition augment Localization of leucocytes & proliferation of lymphocytes .

 Prominent plasma unfiltration. .  It is probable that B lymphocytes found in early Lesion have been stimulated by plaque Ags to differentiate into plasma cells.Established lesion  Develops within 2 ² 3 weeks.

some IgA & few IgM.  JE & oral epithelium proliferate apically into CT & there is loss of collagen. .  These produce IgG.Local immunopathology  Still confined to small site adj to gingival sulcus but consists of numerous plasma cells.  Gingival sulcus deepen & JE is converted into pathological pocket.  However they are also found among bld vsls & collagen fibers.  There are also some T lymphocytes.

viscosus)  Both T & B lymphocytes stimulate blast cells which are seeded into inflm foci & there will be continuous supply of these cells. IgG or IgM can be detected.  A slight increase in salivary IgA & serum IgA.  Potent activator ² LPS.Systemic immunity  Proliferative response of lymphocytes become evident at 14 ² 21 days of plaque accumulation. . dextran & levan as well as some bacteria ( A.

AA and some spirochetes ² potentially significant organisms.  Bacteroides gingivalis. .Advanced lesion  Established lesion persists for years & change into advanced lesion marks the transition from a chronic & successful defense reaction to destructive immunopathological mechanism  There are 2 principal schools of thought: ‡ That the host immune responses may be involved ‡ That some specific microorganism in dental plaque may be responsible for development of the advanced lesion.

Local immunopathology  This stage recognized clinically as periodontitis with pocket formn.permits direct access of plaque Ags & metabolites. destrun of collagenous PDL & bone resorpn. . lymphocytes & macrophages.  Breakdown of epithelial barrier --.  Which will lead to tooth mobility & eventually loss of tooth. ulcern of pocket epim.  The pathological changes extend apically & laterally with dense infiltration of plasma cells.

IgM & C as well as predominantly PMNL infiltration.  Gingival fluid contains high conc if IgG. IgA. Essential feature ² irreversible loss of PDL & bone with progressive increase in pocket formation. .

 Imp role in phagocytosis & bacterial killing.Systemic immunity  Role of macrophages  Antigen processing.  IL-1 production which activates .  Release lysosomal enzymes which enhances local damage. .Bone resorption. .Collagenase production by fibroblasts. .  Release PGs ² which affects immune responses.T cells to proliferation.

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Type IV HSR  Stimulates T& B cells to proliferate. .  This leads to expansion of cells involved in delayed HSR (not adequately investigated)  Dental plaque as well as microorganisms induce lymphoproliferative responses & release cytokines.

 There is decrease in lymphoproliferative responses to stimulation with oral microorganisms (Actinomyces.  Furthermore both CD4 & CD8 appear to be activated at the site of the lesion. Veilonella or Bacteroides) is due to the activity of CD8suppressor T cells& their soluble products.class 2 Ag. .proportion of CD4 (helper induced) to CD8 (cytotoxic-suppressor) cell subsets (2:1 to 1:1) in periodontitis.Immunopathology & mechanism of adult periodontitis  Immunocytochemical investigation from tissue from adult periodontitis ---. & some of them express HLA.

 Hence in adult periodontitis whilst quantitatively B cell activity may predominate. . the immunological changes are under T cell control.  Indeed it is possible that the lesion develops as a result of imbalance in T cell immunoregulation.  However the regulatory function of T cells extends to Polyclonal B cell activation.  This accounts for large Nos of B cells & plasma cells.Significance of polyclonal B cell activators  Elicit non-specific B cell proliferation & AB production.

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