1

CONTENTS

PART I

• Introduction

• Definitions

• Historical background

• Microbiologic aspects of the

microbial-host interaction

Actinobacillus

Actinomycetum Comitans

Treponema Denticola

PART II

T. Forsythia

P.Gingivilis

Capnocytophaga species

• Immunologic aspects of the

microbial host interaction

• Connective tissue alterations:

tissue destruction in

periodontitis

PART III

• Collagen destruction in host

bacterial interaction

• Mechanism of alveolar bone

destruction in periodontitis.

• Toll like receptors in

microbial host interaction

• Conclusion

• References

3

INTRODUCTION

•The mouth, like all external surfaces of the body and the gut, has a

substantial microflora living in symbiosis with a healthy host.

•The microflora of the mouth contains hundreds of species of aerobic

and anaerobic bacteria.

• Cultural studies indicate that more than 500 distinct microbial

species can be found in dental plaque.

4

•Although bacteria are necessary for periodontal disease to take

place, a susceptible host is also needed.

•The immune-inflammatory response that develops in the gingival and

periodontal tissues in response to the chronic presence of plaque

bacteria results in destruction of structural components of the

periodontium leading, ultimately, to clinical signs of periodontitis.

5

•The host response is essentially protective, but both hypo-

responsiveness and hyper-responsiveness of certain pathways can

result in enhanced tissue destruction (Bruce l Pihlstrom 2005 ).

• Closer investigations of the destructive pathway of periodontal

disease began to focus on the relation-ship between bacteria and the

host response in the initiation and progression of periodontal disease..

6

•This shift in etiological theory produced a paradigm that called

attention to the fact that although microorganisms are the cause of

periodontitis, the clinical expression of the disease depends on how

the host responds to the extent and virulence of the microbial

burden.

• It was found that degradation of host tissue results from this

bacterial-host interaction. (Casey Hein 2004)

7

A dynamic equilibrium exists between dental plaque bacterium and innate

host defense system. Dental plaque bacteria have adapted survival

strategies favoring growth, while the host limits growth by combination of

innate and adaptive immune responses. This interaction represents a highly

evolved interaction between bacteria and host.

8

WHAT IS HOST ?

An organism which harbors the parasite .

9

WHAT IS BACTERIA?

• Extremely small—usually 0.3 to 2.0

micrometers in diameter—and relatively

simple microorganisms possessing the

prokaryotic type of cell construction.

10

INTERACTION

•The combined effect of two or more independent variables acting

simultaneously on a dependent variable.

11

•Symbiosis: It is an association in which both host and parasite are so

dependent upon each other that cannot live without the help of other

and none of them suffers any harm from the association.

• Commensal: Is a non disease forming organism; part of the resident

flora.

• Commensalisms: is an association in which parasite is deriving benefits

without causing injury to its host.

12

• Parasitism: is an association where parasites gets benefits and the

host gets harmful effects.

•Mutualism: is an association where parasite and host both are

benefited.

13

• Pathogen :is an organism that causes disease.

•Opportunistic pathogens: are normally not

pathogenic , but are able to become so if their

local environment is changed, they can overgrow and

the microbial load can cause disease.

14

•Virulence : ability of a microorganisms to cause the disease

•Toxins : Anything that is injurious, destructive, or fatal

or

A poisonous substance, especially a protein, that is produced by living

cells or organisms and is capable of causing disease when introduced

into the body tissues but is often also capable of inducing neutralizing

antibodies or antitoxins.

15

ETIOLOGY OF PERIODONTAL DISEASE PAST TO PRESENT

1960 Calculus Theory

1965 -1975 Nonspecific Plaque Theory

1975-1985 Specific Plaque Theory

1985- Today Host Bacterial Interaction Theory

16THE HOST-BACTERIAL INTERACTION THEORY AND THE RISK CONTINUUM: CASEY HEIN CONTEMPORARY ORAL HYGIENE DECEMBER 2004

BEFORE 1960 CALCULUS THEORY

17

Cascade equation : Calculus + tooth = disease

Etiological Factors : Calculus acted as mechanical irritant thought to be the sole cause

of disease.

Clinical implications : Prophylaxis every 6monthsto

remove calculus

1965-1975 NONSPECIFIC PLAQUE THEORY

Cascade equation: too much plaque + tooth = disease

Etiological factors : bacterial plaque caused gingivitis and, in great enough quantities, would cause gingivitis to

progress to periodontitis; all plaque was the same; too

much plaque caused disease.

Clinical implications : plaque control alone could prevent

or control disease; prophylaxis was recommended

18

1975-1985 SPECIFIC PLAQUE THEORY

Cascade equation : specific bacteria +tooth = disease

Etiological factors : microbial plaque became differentiated—

some bacterial species were identified as pathogenic and

specifically virulent in evading host defenses.

Clinical implications : discovery that healthy sites had different kinds of microbes than diseased sites; DNA probe and sensitivity

testing introduced to identify specific periodontal pathogens

responsible for disease progression; eradication of plaque still considered key.

19

1985-TODAY HOST-BACTERIAL INTERACTION THEORY

Cascade equation : mixed infection of bacteria + host

response to bacterial infection = disease

Etiological factors : it is the interaction of the host with

pathogenic bacteria that determines whether periodontal

disease initiated or whether disease progresses; recognition

of certain risk factors that make certain patients more susceptible to disease

Clinical implications : the introduction of periodontal disease risk assessment;

recognition of the need for a 2-pronged approach to treatment that includes the use of locally applied antimicrobials and host

modulatory strategies, ie, subdose doxycycline (periostat)

20

22

• Periodontal disease has been referred to as a 'mixed bacterial

infection' to denote that more than one microbial species contributes

to the development of disease.

• In general, gram-negative facultative or anaerobic bacteria appear to

represent the predominant bacterial species that have been

implicated in the disease process include Porphyromonas gingivalis,

A cetemcomitans, Treponema denticola, Bacteroides forsythus,

fusobacterium nucleatum, Prevotella intermedia, Campylobacter

rectus, Peptostreptococcusmicros and Eikenella corrodens.

23

Formation of plaque

Host tissue invasion

Bacterial evasion of

host defense

mechanism

Virulence factors of different

micro-organisms

24

FORMATION OF PLAQUE Acquisition

Adherence or retentation

Initial survival

Longer term survivial

Avoidance of elimination

Multiplication

Elaboration of virulence

factor

25

1. Invasion of body tissue

2. Bacterial endotoxins

3. Bacterial enzymes

4. Exotoxins

HOST TISSUE INVASION

26

Mode of entry of bacteria:

• Through the ulceration in the epithelium

• Through gingival sulcus

• Through periodontal pockets

• Direct penetration of microorganisms

Organisms capable of invading tissues directly:(AAP 1996)

• Actinibacillus actinomycetum comitans

• P. gingivilis

• Fusobacterium nucleatum

• Trepanoma denticola

Advantages of tissue invasion:

• Tissues can provide reservoir for colonization

• Can not be eliminated easily by mechanical methods.

• Systemic antibodies are required to eliminate bacteria

• The presence of bacteria within the tissue makes periodontitis more resistance to the treatment

27

BACTERIAL EVASION OF HOST DEFENSE MECHANISM

Direct damage to polymorph nuclear leucocytes and macrophages

Reduced PMN chemotaxis

Degradation of immunoglobulins

Degradation of fibrin Altered lymphocyte function

Damage to crevicular epithelium

Production of volatile sulphur compounds

Degradation of periodontal tissues by bacterial enzyme .

28

VIRULENCE FACTORS OF DIFFERENT MICRO-ORGANISMS

Actinobacillus Actinomycetum Comitans

Treponema Denticola

T. Forsythia

P. Gingivilis

Capnocytophaga Species

29

ACTINOBACILLUS ACTINOMYCETUM COMITANS

•Actinomycetemcomitans is a gram-negative, non-spore

forming, non-motile, capnophilic , facultative anaerobic

coccobacillus .

•A. Actinomycetemcomitans can be classified into six

distinct serotypes (a–f) based on surface polysaccharides

located on the O side chains of lipopolysaccharide.

30

•The presence of A. Actinomycetemcomitans in subgingival

plaque has been associated with aggressive periodontitis

with serotype b twice as prevalent as serotype a .

•Animal studies have shown that serotype b induces the pro-

inflammatory cytokine interleukin-6 (IL-6) and IL-1

(another pro-inflammatory cytokine) from thymocytes than

serotype a or c.

31

ANTIGENS OF ACTINOBACILLUS ACTINOMYCETUMCOMITANS

Polysaccharide and

lipopolysaccharide

Leukotoxin

Extracellular proteolytic

enzymes

GroEL heat shock

protein

Fimbriae

32

LIPOPOLYSACHRIDES OF AA

• It induce bone resorption by promoting differentiation of osteoclast

precursor cells and by activating osteoclast cells.

• Ito et al have reported that lipopolysaccharide from

A. Actinomycetemcomitans promotes differentiation of osteoclasts

in vitro in the presence of 1,25-dihydroxyvitamin D3 and

dexamethasone

33

LEUKOTOXIN OF AA

•Actinomycetemcomitans produces a 116 kDa immunomodulating

protein antigen, termed leukotoxin.

•The leukotoxin is a pore-forming protein and is a member of the

repeats-in-toxin (rtx) family of bacterial toxins.

34

• In a study by Zambon Et Al. Fifty-five percent of localized aggressive

periodontitis patients were shown to be infected with A.

Actinomycetemcomitans strains that produced a leukotoxin that was

able to lyse human peripheral blood polymorphonuclear leukocytes and

HL-60 cells (a promyelocytic cell line) in vitro.

35

At high concentrations the toxin binds non-specifically to cellmembranes forming large pores allowing the rapid influx of ca2+ andloss of ATP resulting in necrosis.

At low concentrations the toxin binds to specific cell surface proteinson susceptible cells and form small diameter pores allowing theuncontrolled influx of na+ and the activation of apoptosis

36

•Thus the A. Actinomycetemcomitans leukotoxin is a potent cytotoxic

antigen able to kill immune cells, resulting in the dysregulation of the

host immune response and the release of a variety of enzymes and

reactive molecules from phagocytic cells that may result in tissue

damage and further inflammation .

37

EXTRACELLULAR PROTEOLYTIC ENZYMES OF AA

• Proteolytic activity in subgingival plaque, in particular trypsin-like

proteolytic activity, has been highly correlated with clinical

measurements of periodontitis.

38

proteolytic enzymes in A. actinomycetemcomitans culture supernatants have been reported to degrade

IgG (all four subclasses)

Serum (but not secretory) IgA

IgM but not IgD or IgE in vitro.

Thus the proteolytic activity of A. actinomycetemcomitans may be an important virulence factor involved in dysregulation of the host's immune response.

39

GROEL HEAT SHOCK PROTEIN OF AA

•A.Actinomycetemcomitans

produces an antigenic 64 kDa

GroEL protein that is equally

expressed on the cell surface of

all serotypes a–e.

40

•Heat shock proteins have been suggested to be associated with the

etiology and pathogenesis of both experimental and naturally occurring

autoimmune diseases such as juvenile chronic arthritis, rheumatoid

arthritis and atherosclerosis.

41

• Kirby et al. Reported the potent osteolytic active component in the

surface-associated material was identified as Groel, as a monoclonal

antibody inhibited A. Actinomycetemcomitans induced bone

resorption. GroEl stimulates the recruitment and activation of

osteoclasts in a dose dependent manner.

42

FIMBRIAE OF AA

•Actinomycetemcomitans produces highly

antigenic bundle-forming fimbriae that

are 5 nm in diameter and several μm in

length.

43

•The fimbriae are suggested to play an important role in colonization

as fimbriated A. Actinomycetemcomitans strains have greater

affinity for epithelial cells and saliva-coated hydroxyapatite than

non-fimbriated strains.

• Fimbriae may also have a role in A. Actinomycetemcomitans invasion

of epithelial cells by receptor-mediated endocytosis.

44

TREPONEMA DENTICOLA•T. Denticola is a gram-negative, motile,

asaccharolytic, anaerobic spirochete with

typical helical morphology.

•Ultrastructure of the outer membrane sheath of T. Denticola is similar to

the outer membrane of other gram-negative bacteria. However, the lipid

composition of the outer sheath is similar to lipoteichoic acid of the cell

surface of gram-positive bacteria.

45

ANTIGENS OF TREPONEMA DENTICOLA

Lipopolysachrides

Major sheath protein

Flagellum

Extracellular

proteolytic enzymes

Other antigenic proteins

46

LIPOPOLYSACHRIDES OF T DENTICOLA

• Lipoprotein preparations of T. Denticola have been reported to induce

the inflammatory mediators, nitric oxide, tnf-α and IL-1 from

macrophages, in a dose dependent manner

•Gopalsami et al have reported that the outer membrane lipid of

T. Denticola was able to induce bone resorption .

47

MAJOR SHEATH PROTEIN OF TD

•A major surface protein and antigen of T. Denticola is the major

sheath protein (msp)

• It binds to host matrix proteins and form pores on gingival

fibroblasts, epithelial cells, lymphocytes and erythrocytes

48

•The 53-kda protein also enhances the inflammatory response by

triggering degranulation of polymorphonuclear cells and the specific

release of collagenase, gelatinase and matrix metalloproteinases mmp-

8 and mmp-9.

49

Flagellum of T d

•T. Denticola, like all spirochetes, result in a cork screw-like

locomotion, which aids movement in highly viscous environments like

gingival crevicular fluid and the penetration of gingival epithelial and

connective tissue.

50

•The flagellar filament consists of three core proteins flaB1 (35 kda),

flaB2 (35 kda) and flaB3 (34 kda) and a major sheath protein flaA

(38 kda).

•Although each of the filament proteins are antigenic, flaA and flaB3

have been shown to be the immunodominant antigens.

• FlaA has been suggested to have a role in adherence of T. Denticola

to host cells as it binds fibronectin .

51

EXTRACELLULAR PROTEOLYTIC ENZYMES OF TD

• Important extracellular protein antigens of T. Denticola are its

proteolytic enzymes, reviewed by Potempa Et Al.

•The prolylphenylalanine specific, chymotrypsin-like, serine proteinase

known as dentilisin or trepolisin, is the best characterized

T. Denticola protease. This enzyme occurs on the cell surface .

52

The proteinase has shown to degrade :

Bradykinin,

Substance P

Angiotensin I And II

Host Protease Inhibitors Α1-antitrypsin,

Antichymotrypsin,

А2-macroglobulin,

AntithrombinIII,

Antiplasmin

CystatinC.

53

•Dentilisin degrade pro-Il1β into its bioactive forms and thus stimulate

the inflammatory response

•The ability to degrade host matrix proteins, inflammatory and

protease regulatory proteins and peptides may contribute to the

uncontrolled degradation of periodontal tissues and enhance disease

progression.

54

Ishihara et al found that dentilisin plays a significant role in virulence.

• Helps t. Denticola to bind to the ground substanceglycosaminoglycan, hyaluronate

• Induce apoptosis in epithelial cells.

• Dysregulate the inflammatory response by degrading vasoactivepeptides, hormones and neuropeptide

55

OTHER ANTIGENIC PROTEINS OF TD

A putative hemolysin, a 46-kDa antigenic protein named CYSTALYSINis suggested to play a central role in

• Iron acquisition by T. Denticola as it is able to agglutinate and lyseerythrocytes

• Cause the oxidation and sulphuration of hemoglobin

56

57

58

REFERENCES

The host-bacterial interaction theory and the risk continuum: casey hein, contemporary oral hygiene december 2004

Text book of periodontology: carranza 10 th edn

Text book of microbiology: ananthnarayana 7th edn

Modulation of the host response in periodontal therapy. J periodontol. 2002;73(4): 460-470

Role of antigen in periodontal disease;periodontol 2000;2004;101

59

60

WHAT IS HOMEOSTASIS ?

•Homeostasis, is the property of a system in which variables are

regulated so that internal conditions remain stable and relatively

constant. Examples of homeostasis include the regulation of

temperature and the balance between acidity and alkalinity (ph).

•The concept was described by Claude Bernard in 1865 and the word

was coined by Walter Bradford Cannon in 1926

61

BENEFICIAL MICROBES TO MAINTAIN HOMEOSTASIS

•Amongst the bacteria of more than 700 species now identified within

the human oral microbiota, it is the streptococci that are numerically

predominant.

•Streptococcus salivarius as an oral probiotic. (Burton JP et al benef

microbes. 2011 jun;2(2):93-101 )

•S. Sanguinis, S. Oralis, actinomyces naeslundii, neisseria subflava, and

veillonella dispar.(The pros and cons of oral bacteria by Rosemary )

62

PART II

63

CONTENT

PART II

T. Forsythia

P.Gingivilis

Capnocytophaga species

• Immunologic aspects of the microbial host interaction

• Connective tissue alterations: tissue destruction in periodontitis

PART III

• Collagen destruction in host bacterial interaction

• Mechanism of alveolar bone destruction in

periodontitis.

• Toll like receptors in microbial host interaction

• Conclusion

• References

64

P. GINGIVALIS

P. Gingivalis is a gram-negative, non-spore forming, non-motile,

asaccharolytic, obligate anaerobic coccobacillus.

65

P. GINGIVALIS ANTIGENS

Capsule

Lipopolysaccharide

Fimbriae

Extracellular

proteolytic enzymes

Heat shok protein

Haemagglutinins

Antigenic outer

membrane proteins

66

CAPSULE OF PG

•One major surface antigen of P. Gingivalis is the capsule, a

polysaccharide hetero polymer up to 15 nm thick, that surrounds the

outer membrane.

• P. Gingivalis capsule also inhibits the attachment of periodontal

ligament fibroblasts to the tooth root surface and long-term

exposure to capsular-polysaccharide alters the properties of the

tooth root surface decreasing the ability of fibroblasts to attach.

67

• Encapsulated strains of P. Gingivalis are also more resistant to

phagocytosis by polymorphonuclear cells.

• Can multiply intracellularly, preventing neutrophil migration to the

site of inflammation, block the key step of inflammation , it also

decrease the ability of other MO to stimulate E- selectin on

endothelial cell.

68

The decreased ability to activate the alternative complement pathway and the increase in cellular hydrophilicity due to the capsular polysaccharide are proposed mechanisms by which encapsulated P. Gingivalis is resistant to phagocytosis.

69

LIPOPOLYSACCHARIDE OF PG

• P. Gingivalis lipopolysaccharide induces a variety of cytokines from a

number of different cells. Human gingival fibroblasts secrete the

pro-inflammatory cytokines Il-1β, IL-6 and IL-8 in response to

incubation with P. Gingivalis lipopolysaccharide .

70

• P. Gingivalis lipopolysaccharide also induces the secretion of nitric

oxide, Tnf-α, PGE-2 and IL-1 from human and murine macrophages .

•Stimulates the secretion of a higher amount of IL-6 via toll-like

receptors.

•Wendell and stein have shown that a combination of p. Gingivalis

lipopolysaccharide and nicotine had a synergistic effect upregulating

the expression of IL-6 and IL-8 in human gingival fibroblasts

71

• P. Gingivalis lipopolysaccharide is a potent inducer of bone resorption

by the stimulation of local Il-1α and Il-1β.

• Bomvan Noorloos Et Al have shown that other heat-sensitive

P. Gingivalis components have a greater role in activating osteoclasts

and inducing bone resorption.

• Isogai et al have suggested that P. Gingivalis lipopolysaccharide

induces apoptosis in t-cells.

72

FIMBRIAE OF PG

•The fimbriae are curly, single-stranded filaments, 5 nm in diameter

and are composed of fimbrillin .

• P. Gingivalis fimbriae are thought to be important for the adhesion of

the bacterium to host tissues.

• P. Gingivalis strains with type II fimbriae had a greater ability to

invade epithelial cells (types I–V) .

73

•Active invasion of endothelial cells by P. Gingivalis mediated via

fimbriae stimulates the expression of the surface cell adhesion

molecules ICAM-1, VCAM-1, P and E-selectins, thus recruiting

leukocytes to the site and enhancing the inflammatory response

• P. Gingivalis fimbriae have also been shown to stimulate the release of

Il-1α, Il-1β Tnf-α, neutrophil chemotactic factor, and Tnf-α, IL-6 and

IL-8 from polymorphonuclear cells

74

EXTRACELLULAR PROTEOLYTIC ENZYMES OF PG

• Pick et al. (1994) separated the trypsin-like activity in P. Gingivalis

culture supernatants called' gingipain' and found that there were two

separate cysteine proteinase activities, one with arginine and one with

lysine specificity.

75

HEAT SHOCK PROTEIN OF PG

•The P. Gingivalis heat shock protein GroEl is highly antigenic and anti-

p. Gingivalis GroEl antibodies have been detected in gingival tissue

extracts.

• P. Gingivalis GroEl may lead to an autoimmune reaction

76

HAEMAGGLUTININS

•The cell surface Haemagglutinating adhesin, HA-AG2, is a surface

antigen of P. Gingivalis all of the sera from chronic periodontitis

patients reacted with two HA-AG2 proteins with masses of 43 and 49

kDa .

•Specific antibodies to HA-AG2 have been shown to inhibit

haemagglutination and binding of P. Gingivalis to epithelial cells.

77

ANTIGENIC OUTER MEMBRANE PROTEINS

• Curtis and co-workers, have identified a 55 kDa major antigenic outer

membrane protein of P. Gingivalis .

•A number of studies have investigated the antigenic profile of the

outer membrane of P. gingivalis and have identified a range of

immunodominant proteins by their molecular mass.

78

•The 46 kDa antigen was suggested to be the predominant

immunoreactive protein with sera from patients with severe

periodontitis

79

T. FORSYTHIA

•T. Forsythia is a gram-negative, anaerobic, saccharolytic fusiform

bacterium.

80

T. FORSYTHEA ANTIGENS

Lipopolysachrides

Extracellular proteolytic enzymes

Other antigenic

outer membrane proteins

81

LIPOPOLYSACHRIDES OF T. FORSYTHIA

Very little is known about the structure and chemical composition of

T. Forsythia lipopolysaccharide, however a study by Vasel Et Al

indicated that T. Forsythia lipopolysaccharide may be similar to

P. Gingivalis lipopolysaccharide.

82

EXTRACELLULAR PROTEOLYTIC ENZYMES OF T F

•Several studies have shown that T. Forsythia produces cell surface

proteolytic enzymes. These include the trypsin-like serine proteases

and hemolytic cysteine–protease

•T. Forsythia trypsin-like activity, together with trypsin-like activities

of T. Denticola and P. Gingivalis in subgingival plaque samples have

been correlated with clinical parameters of periodontitis

83

•The trypsin-like activity has been suggested to play a role in binding

of T. Forsythia to erythrocytes, polymorphonucleocytes and

fibroblasts .

•Using PCR Tan Et Al. Showed that T. Forsythia occurred in 91% of

subgingival plaque samples from chronic periodontitis patients

whereas only 9% of subgingival plaque samples from healthy patients

contained this genotype.

84

OTHER ANTIGENIC OUTER MEMBRANE PROTEINS

•T. Forsythia produces a protein that has been suggested to induce

apoptosis especially in lymphocytes by the formation of membrane

pores.

•A GroEL-like protein with a molecular mass of 58 kDa is also produced

by T. Forsythia which may have important implications for the immune

response

85

•A major surface antigen of T. Forsythia is BspA, a 98-kDa protein

sera from chronic periodontitis patients, but not from healthy

individuals, recognized recombinant BspA in a western blot analysis.

• BspA also has been shown to stimulate proinflammatory cytokine

production in THP-1 mononuclear cells via interaction with CD14 and

Toll-like receptor

86

CAPNOCYTOPHAGA SPECIES

• Capnocytophaga species is a group of facultative gram-negative

fusiform rods.

•They require co2 for growth in culture, hence their name.

•The Capnocytophaga species have been associated with localized

early-onset periodontitis Eikenella Corrodens

87

• Capnocytophaga ochracea has been correlated with the presence of

disease in localized juvenile periodontitis patients and was not found

in healthy subjects in a family study by Williams Et Al.

88PERIODONTOLOGY 2000, VOL. 26, 2001

• Innate factors such as complement, resident leukocytes and

especially mast cells play an important role in signaling endothelium,

thus initiating inflammation.

• Acute inflammatory cells (i.e. Neutrophils) protect local tissues by

controlling the periodontal mcirobiota within the gingival crevice and

junctional epithelium .

90

• Chronic inflammatory cells, macrophages and lymphocytes protect the

entire host from within the subjacent connective tissue and do all

that is necessary to prevent a local infection from becoming systemic

and life threatening.

91

Acute bacterial challenge phase

Acute inflammatory

response

Immune response phase

92

ACUTE BACTERIAL CHALLENGE PHASE

There is a very high turnover of epithelium -that permits rapid replacement of cells and tissue components damaged by microbial challenge.

The gingival crevicular fluid flushes the sulcus or pocket and delivers complement proteins and specific antibodies.

Salivary secretions provides continuous flushing and supply of agglutinins and specific antibodies.

The intact epithelial barrier of the gingival, sulcular and junctional epithelium prevents bacterial invasion.

The epithelial and vascular elements response to bacterial challenge

93

ACUTE INFLAMMATORY RESPONSE

Vascular leakage enhances the localized response:

Leukocytes selectively emigrate from the vessels to alter the immuno-inflammatory cell populations in the gingival tissues

Neutrophil migration into the gingival sulcus:

The inflammatory infiltrate within the tissues:

• Opsonization

• Phagocytosis

94

VASCULAR LEAKAGE ENHANCES THE LOCALIZED RESPONSE

• In the presence of lipopolysaccharide or cytokines, the endothelial cells

of the microcirculation become activated. The vessels of the

microcirculation become inflamed, dilated and engorged with blood, and

the blood flow slows.

•The endothelial cell junctions open and protein-rich fluid leaves the

vessels at the site of the post-capillary venules /and accumulates in the

extra cellular matrix.

95

LEUKOCYTES SELECTIVELY EMIGRATE FROM THE VESSELS TO ALTER THE IMMUNE INFLAMMATORY CELL POPULATIONS

IN THE GINGIVAL TISSUES

•Neutrophils are believed to play an important role in controlling the

periodontal mcirobiota. They are the first leukocytes to arrive at the

site of inflammation and are always the dominant cell type within the

junctional epithelium and the gingival crevice

96

•Neutrophils exit the inflamed vessel of the' microcirculation and

migrate through the connective tissues and junctional epithelium, to

form a barrier between the subgingval microbial plaque and the

gingival tissue

•There is specific interactions among vascular cell adhesion molecules

and leukocyte integrins and thus the chance of inducing leukocyte

extravasation by diapedesis into the extravascular spaces.

97

NEUTROPHIL MIGRATION INTO THE GINGIVAL SULCUS

• Following extravasation, neutrophils seem to gain access to the more

coronal portion of the junctional epithelium and to selectively migrate

through this multilayered epithelium to gain access to the bacterial

flora.

98

Two mechanisms of possible importance in the regulation of neutrophilmigration towards the gingival sulcus or the periodontal pocket followingneutrophil extravasation:

The expression of leukocyte adhesion molecules such as the intercellularadhesion molecule I, in epithelial cells.

Cytokines with potent and cell type-specific leukocyte chemotacticproperties: the chemokines and the neutrophil-selective interleukin 8, inparticular.

99

THE INFLAMMATORY INFILTRATE WITHIN THE TISSUES:

Opsonization Phagocytosis

100

OPSONIZATION

•Opsonization- refers to the coating of particles, such as bacteria,

with host proteins that facilitate phagocytosis.

•Specific antibody of the IGg isotype also facilitates phagocytosis

directly by binding with the neutrophil Fc receptor and appears to be

essential for phagocytosis of certain periodontal pathogens.

101

Patients with periodontitis often exhibit very high serum titers of IGg

to specific periodontal pathogens

102

PHAGOCYTOSIS

•Once the bacterial cell is bound to the neutrophil , ingestion

(phagocytosis) results in entrapment of the bacterial cell into the

membrane delimited structure known as the phagosome.

• Bacteria within the phagosome and phagolysosome may be killed by

oxidative or nonoxidative mechanisms

103

IMMUNE RESPONSE PHASE

•The bacterial products and epithelial derived cytokines activate the

local tissue mononuclear cells that shape the local immune response.

• Macrophages have been reported to be few in healthy gingiva.

Although increased in gingivitis, periodontitis, macrophage density

remain in low proportions relative to other cell types.

104

105

FACTORS CAUSING TISSUE DESTRUCTION

Bacterial products

Proteinases

Cytokines

Prostagland-ins

Reactive oxygen species

107

BACTERIAL PRODUCTS

•Degrade basement membrane and extracellular matrix proteins

including collagen, proteoglycans, and glycoproteins. This would

destroy periodontal connective tissue and facilitates bacterial

invasion.

• Interferes with tissue repair by inhibiting clot formation or lysing

the fibrin matrix in periodontal lesions.

108

•Activates latent host tissue collagenase which would enhance host-

tissue enzyme mediated tissue destruction.

• Inactivates proteins important in host defense.

109

PROTEINASES

•Matrix metalloproteinases (mmps) are considered to be primary

proteinases involved in periodontal tissue destruction by degradation

of extracellualr matrix molecules.

•They degrade extracellular matrix molecules, such as collagen, gelatin

and elastin.

110

•MMP-8 and MMP-l both collagenase are elevated in tissues and GCF

associated with periodontitis

• Elastase is capable of degrading wide range of molecules including

elastin, collagen, and fibronectin.

111

CYTOKINES

• Both IL-I and TNF-a induce production of proteinases in

mesenchymal cells, including MMPs, which may contribute to

connective tissue destruction.

• Il-I and TNF-a found in significant concentrations in GCF from

periodontally diseased sites.

112

• Increasing severity of periodontitis is associated with increased

concentrations' of IL-I and decreasing concentrations of IL-Ira .

• In a primate model of experimental periodontitis, application of

antagonists to IL-1 and TNF resulted in an 80% reduction in

recruitment of inflammatory cells in proximity to the alveolar bone

and a 60%reduction in bone loss.

113

PROSTAGLANDINS• Prostaglandins are arachidonic acid metabolites generated by

cyclooxygenases (COX-I, COX-2).

• Cox-2 is upregulated by IL-Ib , TNF-a and bacterial LPS and appears to be

responsible for generating the prostaglandin PGE2 that is associated with

inflammation.

• PGE2 is increased in periodontal sites demonstrating inflammation and

attachment loss.114

Elevated prostaglandin E2 levels are detected in thegingiva and gingival crevicular fluid of patients withperiodontal diseases, compared to periodontally healthysubjects in 1974, Goodson Et Al. reported a 10-foldincrease of prostaglandin E2 levels in inflamed gingivaltissue, compared with healthy gingival tissue.

115

REACTIVE OXYGEN SPECIES

•Any species capable of independent existence that contain one or more

unpaired electrons.

• Inflammatory cells and in particular PMN once stimulated produce

reactive oxygen species via metabolic pathway of the respiratory burst,

which occurs in the process of phagocytosis.

116

•These includes superoxide anion, hydrogen peroxide, hydroxyl radicle

and hyperchlorus acid.

117

ROS can cause

Protein damage

Lipid Peroxidation

DNA damage

118

119

120

ROLE OF GINGIPAIN

• Pick et al. (1994) separated the trypsin-like activity in P. Gingivalis

culture supernatants called' gingipain' and found that there were two

separate cysteine proteinase activities, one with arginine and one with

lysine specificity

121

Arg-gingipain have been found potently to enhance vascular permeability.

These enzymes increase gingival crevicular fluid production and thus providea continuous supply of nutrients for the bacterium, enhancing its growth andvirulence.

Arg-gingipain has also been found to be a very efficient enzyme for theproduction of the potent chemotactic factor c5a by directly cleaving the C5component of complement.

These enzyme also degrades c3,and in this way eliminates the creation ofc3-derived opsonins. This render sp. Gingivalis more resistant tophagocytosis by neutrophils

122

Fibrinogen is a major target for lys-gingipain and it thus increases thelocal clotting time, leading to gingival bleeding.

The bleeding of periodontal sites is of primary importance for the growthof P. Gingivalis, since it ensures the rich source of haem and iron that itrequires for survival.

Gingipains act as adhesins and have a strong binding affinity forfibrinogen , fibronectin and laminin. It inhibits haemagglutination.

Since these complexes are present on the surfaces of both the vesiclesand membranes of P. Gingivalis , they may play an important role in theattachment of this bacterium to host cells.

123

RESPIRATORY BURST

•Respiratory burst (sometimes called oxidative burst) is the rapidrelease of reactive oxygen species (superoxide radical and hydrogenperoxide) from different types of cells.

•Usually it denotes the release of these chemicals from immune cells,e.G., Neutrophils and monocytes, as they come into contact withdifferent bacteria or fungi .

124

• Respiratory burst plays an important role in the immune system. It is

a crucial reaction that occurs in phagocytes to degrade internalized

particles and bacteria.

•NADPH oxidase, an enzyme family in the vasculature (in particular,

in vascular disease), produces superoxide, which spontaneously

recombines with other molecules to produce reactive free radicals.

125

•The superoxide reacts with NO, resulting in the formation

of peroxynitrite, reducing the bioactive NO needed to dilate

terminal arterioles and feed arteries and resistance arteries.

•Superoxide anion, peroxynitrite, and other reactive oxygen species

also lead to pathology via peroxidation of proteins and lipids, and via

activation of redox-sensitive signaling cascades and protein

nitrosylation

126

•Myeloperoxidase uses the reactive oxygen species hydrogen

peroxide to produce hypochlorous acid. Many vascular stimuli,

including all those known to lead to insulin resistance, activate NADPH

oxidase via both increased gene expression and complex activation

mechanisms.

•To combat infections, immune cells use NADPH oxidase to reduce

o2 to oxygen free radical and then H2O2.

127

•Neutrophils and monocytes utilize myeloperoxidase to further

combine H2O2 with cl- to produce hypochlorite, which plays a role in

destroying bacteria. Absence of NADPH oxidase will prevent the

formation of reactive oxygen species.

128

ZIPPER MECHANISM The zipper mechanism, ingestion occurs by sequential engagement of a

phagocyte's membrane against the particle surface, and pseudopod

advance proceeds no further than receptor-ligand interactions permit.

129

SPECIFIC RESPONSE OF CAPSULE TOWARDS HOST

•The capsule of porphyromonas gingivalis leads to a reduction in the

host inflammatory response .

•A study done by Tiana Wyant et al in 2011 reported that the higher

virulence potentials of encapsulated strains than of non encapsulated

ones, evaluated using a mouse abscess model.

130

• Encapsulated P. Gingivalis strains trigger different host responses

than nonencapsulated mutant strains , thus indicating that the

presence of a capsule indeed has immunomodulating properties.

•The capsular polysaccharide locus has been identified by Aduse-opoku

et al. , which allowed for the generation of isogenic capsule-deficient

mutants.

131

•The analysis of such a mutant by Brunner Et Al. has shown that

encapsulation reduced the production of cytokines interleukin-1 (IL-

1), IL-6, and IL-8 by fibroblasts in response to P. Gingivalis infection ,

thus further demonstrating that the capsule modulates the host

response to bacteria

132

PART III

133

CONTENTS

• Collagen destruction in host bacterial interaction

• Mechanism of alveolar bone destruction in periodontitis.

• Toll like receptors in microbial host interaction

• Conclusion

• References

134

•Tissue remodeling is usually tightly regulated by a complex interplay

of cell-cell and cell-matrix interactions involving the production of

enzymes, activators, inhibitors and regulatory molecules such as

cytokines and growth factors.

•The accelerated breakdown of connective tissues occurring in

pathological situations, such as periodontal diseases.

136

•The endopeptidases (or proteinases) are key enzymes in tissue

degradative processes, since the protein components of most

matrices are the predominant determinants of tissue structure and

function.

137

• Collagenase :

Collagenase 1 or MMP 1 or fibroblast collagenase can hydrolyses

collagen type 1,2,3,6,8,and 10

Collagenase 11 or MMP 8 or PMN’s collagenase can hydrolyze collagen

type 1 and 3

•Gelatinase :

MMP 2 and MMP 9 can degrade collagen type 4, 7, 10 and 11 and

Elastin.

138

Stromeolysin :

•Stromeolysin 1

•Stromeolysin 2

•Stromeolysin 3

Degrade proteoglycans, basement membrane , laminin and fibronectin.

139

•Higher levels of antibodies to collagen type I were found in the

peripheral blood of patients with periodontitis than in healthy

controls.

• In addition to antibodies to collagen type-1 there are several other

types of auto reactive components. Increased reactions of IgG auto

antibodies to desmosomal proteins were observed in sera from

patients with periodontitis in comparison to sera from controls.

140

•Two regulators of MMP expression are IL-1 AND TGF-β

These substance are present in inflamed tissue (Sodek And Overall

1992)

• In macrophages MMP production is stimulated by LPS and inhibited by

INF-γ, IL-4 and IL-10.

141

142

MEDIATORS AFFECTING COLLAGEN

143

Increase in collagen synthesis:

• PDGF

• TGFβ

• FGF

• IGF

Decrease In Collagen Synthesis:

• Cytokines

• IL-1αβ

• INFγ

• TNFα

• Hormones like Glucocorticoids

• Others like PGE2

144

Bacterial factors

• Capsular and surface associated material

• LPS

• Lipoteichoic acid

• Peptodoglycans

• Muramyl dipeptide

• Lipoprotein

Host factors

• Inflammatory mediators

• PGE2

• Leukotrienes

• 12- HETE

• Heparin

• Thrombin

• Bradykinin

• Cytokines

• IL-1

• IL-6

• TNF

• TGF β

• PGDF

146

Bone loss in periodontal disease are listed by Haussmann in 1974

•Direct action of plaque products on bone progenitor cells induce their

differentiation into osteoclasts.

• Plaque products acts directly on bone destroying it through a non-

cellular mechanism.

147

• Plaque products stimulate gingival cells, causing them to release

mediators which in turn trigger bone progenitor cells to differentiate

into osteoclasts.

• Plaque products cause gingival cells to release agents that can act as

co-factor in bone resorption.

• Plaque products can cause gingival cells to release agents that destroy

bone by direct chemical action without osteoclasts.

148

MECHANISM OF BONE RESORPTION (BERKOVITZ)

Osteoblast induced

Osteoclast induced

149

OSTEOBLAST INDUCED

•All systemic and local bone resorbing factors exert their influence

by stimulating osteoblasts.

150

•Osteoblasts are involved in the regulation of osteoclasts function

because they have receptors for systemic factors such as

parathromone and 125-OH2 and locally produced factors such as

prostaglandins, leukotrienes and cytokines which effects local

changes and exert their influence by stimulating osteoblasts (Meikle

et. Al 1986)

151

•Stimulated osteoblasts stimulate osteoclast formation by secretion

of cytokines, growth factors in particular granulocyte macrophage

colony stimulating factors and macrophage colony stimulating factor

and interleukin-6.

152

• IL-6 secretion is stimulated by IL-1 attachment to osteoblasts

receptor. All these secreted cytokine in presence of IL-3 can

stimulate the development of precursors cells in marrow , these

precursors cells are stimulated by IL-6 to become osteoclasts

153

• Bone resorbing agent such as PTH hormone stimulates osteoblast to

synthesize and collagenase and plasminogen activator which digests

the osteoid exposing the mineralizing matrix which may be

chemotactic to osteoclast

•Osteoblasts release short range soluble activators for osteoclasts

(clinical oral science Harris and Edgar )

154

OSTEOCLAST MEDIATED

Osteoclastic resorption involves

Solubilization of mineral phase

Dissolution of organic matrix

155

•The resorption area is defined beneath the ruffled border of

osteoclast. This is highly specialized region of cytoplasmic in

folding of plasma membrane, this contains podosomes which are

specialized protrusion of the ventral surface of osteoclasts which

adhere directly to the bone surface being broken down.

•Osteoclasts also produce ROS which may play a role in

pathological demineralization of bone during disease (Gaarrett Et

Al in 1990)

156

•Osteoclasts : osteoclastogensis is enhanced by PTH, calcitriol,PGE2, thyroxin and IL-11.

• Formation of active osteoclasts require MCSF and involve cell tocell contact between precursors of monocyte-macrophage lineageand osteoblasts, stromal cells, these cells express receptorsactivator of NF-kb ligand (RANKL).

• RANKL attaches to RANK, A receptor on the cell surface ofosteoclasts and osteoclasts precursors to stimulate proliferationand differentiation of cells to form osteoclast phenotype

157

• In the presence of

periodontopathic organisms

CD4+ T cells presents

increased expression of

RANKL, triggering the

activation of osteoclasts and

causing bone loss.

158

ROLE OF NO IN BONE RESORPTION

There is good evidence to suggest that NO(nitric oxide) has biphasic effects on osteoclastic bone resorption.

Low concentrations of no have been shown to potentiate IL-1 induced bone resorption, based on the observation that NO inhibitors inhibit IL-1 induced bone resorption in vitro. Constitutive production of NO within osteoclasts has been suggested to be essential for normal osteoclast function.

159

160

•Toll gene products were first

discovered in 1985 and were described

as being critical for the embryonic

development of dorsal–ventral polarity

in the fruit fly, drosophila.

•They contain common extracellular

leucine-rich domain and a conserved

intracellular domain.

162

•They are critical for

recognition of microbes by the

innate immune system and for

bridging the innate and

acquired immune system.

163

•Toll-like receptors are predominantly expressed on cells of the innate

immune system, including neutrophils, dendritic cells and

monocytes/macrophages.

•These cells express different toll-like receptors, allowing them to

induce a wide variety of immune responses to specific pathogens.

165

•Neutrophils, the predominant innate immune cells in blood, express

toll-like receptor 1, 2, and 4 and TLR 10.

•They play a key role in host defense by recognizing, engulfing, and

killing microorganisms.

166

•Monocytes/macrophages express toll-like receptor 1, 2, toll-like

receptor 4, and 8.

• Recent report shows that activation of toll-like receptor 1 ,2 on

monocytes leads to their differentiation into macrophages rather

than dendritic cells.

167

SUMMARY

168

CONCLUSION

•The host-bacterial interaction theory may explain why otherwise

healthy individuals with moderate levels of plaque do not exhibit loss

of periodontal support. In these individuals, PMNS are effective in

blocking invading pathogens without destroying the collagen content

of the periodontium in the process.

169

• If these same pathogens attempted to invade the periodontium of

people predisposed to periodontal disease, it appears that impaired

chemotaxis and phagocytosis of defense cells may put these

individuals at significantly greater risk for progressive periodontal

destruction.

170

REFERENCES

The host-bacterial interaction theory and the risk continuum: casey hein,: contemporary oral hygiene December 2004

Text book of periodontology: Carranza 10 edn

Text book of microbiology: Ananthnarayana 7 edn

Mechanisms tissue matrix of connective destruction in periodontitis; john j. Reynolds & murray c. Meikle; periodantology 2000, vol. 14, 1997,144-157

171

Oringer rj. American academy of periodontology, research, science, and therapy committee. Modulation of the host response in periodontal therapy. J periodontol. 2002;73(4): 460-470.

The role of reactive oxygen and antioxidant species in periodontal tissue destruction periodontology 2000;2007;vol43

Toll-like receptors and their role in periodontal health and disease. Periodontology 2000;2007;vol43;41-50

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