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Inflammatory mechanisms in localized aggressive periodontitis

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Inflammatory mechanisms in localized aggressive periodontitis

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INFLAMMATORY MECHANISMS IN LOCALIZED AGGRESSIVE
PERIODONTITIS PATIENTS

by
Mahnaz Zandi

_____________________________________________________________________

A Thesis Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(CRANIOFACIAL BIOLOGY)

August 2009

Copyright 2009 Mahnaz Zandi
ii
Dedication

To my dear parents who were my first teachers, and my brothers for their support.

iii
Acknowledgments

I gratefully acknowledge Dr. Hessam Nowzari, who played a major role in my
development, both on a scientific and personal level.
I thank Dr. Sandra Rich for guidance and outstanding support and making this
thesis possible.
I acknowledge Dr. Mahvash Navazesh and Dr. Michael Paine for courage and
giving me the opportunity to improve my skills as a researcher.
I want to reserve special words to Dr. Vali Kermani for his input, concern and
opinion.
I would like to acknowledge Dr. Gewn Uman for suggestions regarding the
statistical analysis.
I thank Ursula Czoik for taking care of me in the graduation process.
iv
Table of Contents
Dedication ............................................................................................................... ii
Acknowledgments.................................................................................................. iii
List of Tables ......................................................................................................... vi
Abstract ................................................................................................................ viii
Chapter 1: Introduction ............................................................................................1
Microbiology of Periodontal Disease ................................................................1
Periodontitis as an infectious disease ...........................................................2
Biofilms and complex bacterial ecologies in periodontal disease ...............2
Figure 1 – Oral Microbiota Complexes .......................................................3
Current concepts about periodontal destruction ..........................................4
Viruses in periodontal disease .....................................................................4
Bio film formation in supragingival area .....................................................5
Subgingival bacterial configuration .............................................................6
Immunology of Periodontal Disease ..................................................................7
T cells and cytokines in periodontal disease ................................................8
Chemokines in periodontal disease ............................................................10
B cells and antibody regulation in periodontal disease ..............................12
T-cell receptor affinity ...............................................................................13
Regulatory T cells ......................................................................................14
Nuclear Factor Kappa Beta ........................................................................16
Innate immunity .........................................................................................17
Susceptibility to periodontal disease ..........................................................17
Localized Aggressive Periodontitis .................................................................19
Nomenclature: ............................................................................................19
Prevalence of Localized Aggressive Periodontitis ....................................22
Etiology of Localized Aggressive Periodontitis ........................................26
Genetic .................................................................................................26
Bacteriology .........................................................................................34
Immunology .........................................................................................44
Chapter 1 Endnotes ..........................................................................................60
Chapter 2: Methods and Materials .........................................................................82
Objective ..........................................................................................................82
Research Questions ..........................................................................................82
Hypothesis........................................................................................................82
Patients .............................................................................................................83
Sample Collection ............................................................................................84
Laboratory Procedure.......................................................................................84
v
Table of Contents (continued)

Bacterial culture .........................................................................................84
Real Time Polymerase Chain reaction (RT- PCR) ....................................85
Statistical methods ...........................................................................................86
Tables: Demographics .....................................................................................88
Tables: PCR Analyses......................................................................................90
Tables: Crosstabs .............................................................................................94
Tables: Microbiology (Bacterial Cultures) ....................................................104
Chapter 3: Results ................................................................................................111
Chapter 4: Discussion ..........................................................................................113
Chapter 4 Endnotes ........................................................................................122
Chapter 5: Conclusion..........................................................................................125
Bibliography ........................................................................................................126

vi
List of Tables
Table 1: Patients ID ...............................................................................................88
Table 2: Patients Sex ..............................................................................................88
Table 3: Patients Age .............................................................................................88
Table 4: Patients Race ............................................................................................89
Table 5: PCR Analysis for A.a ..............................................................................90
Table 6: PCR Analysis for CMV ...........................................................................90
Table 7: PCR Analysis for NFKb ..........................................................................91
Table 8: PCR Analysis for TNFa ...........................................................................92
Table 9: PCR Analysis for ICAM1 ........................................................................93
Table 10: Crosstab for A. a. ...................................................................................94
Table 11: Crosstab for CMV..................................................................................95
Table 12: Crosstab for NFKb .................................................................................96
Table 13: Crosstab for TNFa .................................................................................97
Table 14: Crosstab for ICAM1 ..............................................................................98
Table 15: Crosstab for A. a. ...................................................................................99
Table 16: Crosstab for CMV................................................................................100
Table 17: Crosstab for NFKb ...............................................................................101
Table 18: Crosstab for TNFa ...............................................................................102
Table 19: Crosstab for ICAM1 ............................................................................103
Table 20: Bacterial Culture for A. a.....................................................................104
Table 21: Bacterial Culture for P. gingivalis .......................................................104
Table 22: Bacterial Culture for P. intermedia ......................................................104
vii
List of Tables (continued)
Table 23: Bacterial Culture for T. forsythia.........................................................105
Table 24: Bacterial Culture for Campylobacter species ......................................105
Table 25: Bacterial Culture for Eubacterium Species ..........................................105
Table 26: Bacterial Culture for Fusobacterium species .......................................106
Table 27: Bacterial Culture for P. micros ............................................................106
Table 28: Bacterial Culture for Enteric gram negative rod ..................................106
Table 29: Bacterial Culture for Beta hemolytic streptococci...............................106
Table 30: Bacterial Culture for Yeast ..................................................................107
Table 31: Bacterial Culture for Eikenlla corrodens .............................................107
Table 32: Bacterial Culture for Staphylococcus species......................................107
Table 33: Bacterial Culture for D. pneumosintes ................................................107
Table 34: Ranks ...................................................................................................108
Table 35: Statistics Tests for Periodontal Pathogens ...........................................109
Table 36: Means for Periodontal Pathogens ........................................................110

viii
Abstract

Introduction: Localized Aggressive Periodontitis (LAP) has a bacterial etiology,
with infection of the gingival tissues by several bacterial pathogens. However, in addition
to bacterial etiology, host immune and inflammatory factors are implicated in the
pathogenesis and progression of localized aggressive periodontitis. Objective: The
objective of this study is to explore underlying mechanisms involved in selective sites of
infection, and the subsequent inflammatory process in LAP. Methods and Materials: Ten
LAP patients participated in this study (3 male and 7 female), with an age range of from
13- 32 years. Samples were taken from one tooth with the deepest pocket for a test site and
one tooth with healthy periodontium for a control site from each individual. Laboratory Tests:
Bacterial culture was conducted to evaluate the fourteen periodontal pathogens. Also
Real-Time Polymerase Chain Reaction (RT PCR) was done to analyze the presence of
Aggrigatibacter actinomycetemcomitans (A.a.) RNA, Cytomegalovirus (CMV) RNA,
Nuclear factor kb (NFKb), Tumor Necrosis Factor alpha (TNFa), and Inter Cellular
Adhesion Molecule 1 (ICAM1). Results: No periodontal pathogens were present in the
control sites. All the test teeth revealed some periodontal pathogens. A.a. was not present
in any test samples. The mean percentages of other pathogens in test teeth were 5.28 for
Fusobacterium species, 4.90 for Campilobacter species, 3.93 for T. forsythia, 3.53 for P.
intermedia, 2.73 for P. gingivais, 1.40 for Enteric gram negative rod, 0.69 for Beta
hemolytic streptococci, 0.38 for D. pneumosintes. With PCR analysis CMV RNA and
A.a RNA was not present in any of the test or control samples. Measurement of NFkb
ix
and ICAM1 expression by RT- PCR showed there was marked increase in the affected
sites when compared with control sites. TNFa was not present in any of the test or control
samples. Conclusion: The preliminary findings of this study suggest upregulation of
active NFkb and ICAM1 play an important role in LAP.

1

Chapter 1: Introduction
Periodontal diseases are multifactorial infections elicited by a complex of
bacterial species that interact with host tissues and cells, causing the release of a broad
array of inflammatory cytokines, chemokines, and mediators, some of which lead to
destruction of the periodontal structures, including the tooth-supporting tissues, alveolar
bone, and periodontal ligament.
Microbiology of Periodontal Disease
Many life-threatening medical pathogens were discovered during the late 19th
century. The accumulation of knowledge of how to treat these medical pathogens has
saved many patients. At that time, three criteria known as Koch's postulate were
established to identify medically serious pathogens. In general, the postulate was aimed at
major pathogens, but not opportunistic pathogens or viral infections, and it is generally
accepted that some modifications of the postulate are necessary to cover opportunistic
pathogens, including periodontopathic or cariogenic bacteria in oral infectious diseases.
Socransky
1, 2
proposed the following modifications of Koch's postulate for the
determination of periodontopathogens:
• the presence of the putative pathogen in proximity to the periodontal lesions in
high number;
• patients infected with these periodontal pathogens often develop high levels of
antibodies in serum, saliva, and gingival crevicular fluid , and may also develop a
cell- mediated immune response;
• experimental implantation of the organism into an animal model should lead to at
least some characteristics of naturally occurring periodontal disease;
• clinical treatment that eliminates the bacteria from the periodontal lesions should
result in clinical improvement;
2

Loesche
3
described the non-specific plaque hypothesis (NSPH) and the specific
plaque hypothesis (SPH). According to the NSPH, caries and periodontal disease result
from the elaboration of noxious substance by entire plaque flora, while the SPH suggests
that only certain plaque cause infection because of presence of a pathogen(s) and/or a
relative increase in the levels of certain indigenous plaque organization.
Periodontitis as an infectious disease
The trigger for the initiation of disease is the presence of complex microbial
biofilms
4
that colonize the sulcular regions between the tooth surface and the gingival
margin through specific adherence interactions and accumulation due to architectural
changes in the sulcus (i.e., attachment loss and pocket formation). The characteristics of
microbiological progression from periodontal health to gingivitis (e.g., chronic
inflammation of the gingival tissue without tissue destruction), and eventually to
periodontal disease are vast and complicated
5
. It has been estimated that nearly 700
bacterial species, which show some structural organization in the biofilms
6
, can colonize
the oral cavity of humans, although it remains unclear how this multitude of bacteria
compete, coexist, and/or synergize to initiate this chronic disease process.
Biofilms and complex bacterial ecologies in periodontal disease
Studies of Socransky and Haffajee and their colleagues
7
have described improved
methods for examining the association of oral microbial communities with the change
from health to disease. These investigators stratified the microbiota into groups or
complexes, representing bacterial consortia that appear to occur together and that are
3

associated with the biofilms of gingival health, gingivitis and periodontitis
8, 9
.The
different microbial complexes have been associated with the sequence of colonization on
the tooth surface as well as with disease severity. Bacterial species contained in the
yellow, green, and purple complexes appear to colonize the subgingival sulcus first and
predominate in gingival health. In contrast, orange complex bacteria are associated with
gingivitis and gingival bleeding. Interestingly, bacteria of the orange complex may also
be associated with red complex microorganisms including Porphyromonas gingivalis,
Tannerella forsythensis (previous names Bacteroides forsythus or Tannerella
forsythensis), and Treponema denticola, organisms found in greater numbers in diseased
sites and in more advanced periodontal disease (Figure 1).

Figure 1 – Oral Microbiota Complexes
4

Current concepts about periodontal destruction
An early strategy for the identification of periodontopathic bacteria was to
compare the microbial compositions of subgingival plaque samples from healthy,
gingivitis, and periodontitis sites. Although the bacterial composition changes as plaque
matures, it is difficult to determine the disease-related difference between plaque samples
from gingivitis and periodontitis sites. In 1989, Socransky et al.
10, 11
proposed the 'Burst
theory,' in which periodontal destruction occurs in periodic, relatively short episodes, and
not in a time-dependent manner. This theory, based on epidemiologic studies of
subgingival pocket configuration, refers to the active and inactive plaque characteristics
associated with periodontal destruction. In the early 1990s, the microflora composition in
active sites that had recently progressed to periodontal breakdown was compared with
those of inactive sites
12, 13
.

Some resident periodontopathogens were identified in
subgingival pockets, and a carrier-state with a periodontopathogen was considered to be a
future risk indication of periodontal breakdown
14, 15
.
Viruses in periodontal disease
Various herpesviruses, including herpes simplex virus (HSV), human
cytomegalovirus (HCMV) and Epstein–Barr virus type-1 (EBV-1), have recently been
detected in crevicular samples from aggressive types of periodontitis
16
. Herpesviruses are
capable of infecting various types of cells, including polymorphonuclear leukocytes,
macrophages, and lymphocytes. Herpesvirus-infected inflammatory cells can reduce host
defense mechanisms, giving periodontopathic bacteria the opportunity to overgrow in the
subgingival area and to invade tissues and cells more efficiently
17
. Slots and coworkers
5

described a model of periodontal pathology of activated and latent herpesviruses in
accordance with the segmental pattern of breakdown in most periodontitis patients
18
.
Bio film formation in supragingival area
For a better understanding of the microbial ecology, dental plaque can be divided
into supra and subgingival plaque. After subgingival plaque becomes established in the
periodontal pocket, supragingival plaque may not have any effect on the ecologic niches
of subgingival plaque
19
. However, the initial step of subgingival plaque formation is
supported by the presence of supragingival plaque
20
.
The frontier of supragingival plaque around the gingival sulcus provides much
information on the initiation of periodontal pocket formation, though there is still
controversy regarding the mechanism of periodontal pocket formation. Microbial biofilm
is the main player in the etiology of periodontitis at this stage. Supragingival plaque
contains many bioactive end products, such as fermented organic acid, sulfur components,
tissue-digesting enzymes, peptidoglycan, and lipopolysaccharide. These components are
diffused from supragingival plaque to the surface of gingival epithelium, and increase the
flow of gingival crevice fluid and inflammatory fluid from periodontal tissue. This new
nutritional supply, which is delivered from serum, changes the ecosystem of the plaque
adjacent to the inflamed gingiva. In this new environment, proteolytic bacteria in the
plaque expand their ecologic niche and produce proteases, which accelerate tissue
damage. These findings suggest that protease-producing bacteria, such as P. gingivalis,
B. forsythus, and T. denticola, may be involved as initiators of disease activity.
6

Subgingival bacterial configuration
Subgingival plaque can be divided into three kinds: attached, unattached, and
epithelium-associated.
21
Attached subgingival plaque is tooth-associated and is
predominantly composed of gram-positive rods and cocci.
22
These populations are
considered to survive under limited nutrition and strictly anaerobic conditions, and are
relatively stable in subgingival plaque. Attached plaque is also associated with the
deposition of subgingival calculus and root caries. Gram-negative and motile rods
dominate in unattached plaque, which extends to the frontier of apical plaque.
23, 24
This
appears to be the most bioactive area, because a large amount of inflammatory gingival
crevice fluid is excreted from periodontal tissue. P. gingivalis and T. denticola, the
dominant members of unattached plaque, are considered to induce and accelerate
inflammation as periodontopathogens. Epithelium-associated plaque is loosely attached
to the gingival epithelium, and consists of gram-negative and motile rods.
Immunostaining has revealed that Prevotella nigrescens/ Prevotella intermedia is one of
the main members of epithelium-associated plaque
25
. Epithelium-associated plaque is
considered to play an important role in periodontal pathogenesis, and is especially
involved in the bacterial invasion of connective tissue
26
. These three kinds of subgingival
plaque seem to be closely related and reflect the ecosystem of the subgingival microbial
community.
There is a plaque-free zone (PFZ) between the advancing apical plaque border
and epithelial attachments
27
. It is generally considered that few bacteria are present in the
PFZ; however, recent studies have found that small bacterial aggregates, which are called
7

islands, can be observed by scanning electron microscopy
28
. Scanning electron
microscope analysis has identified P. gingivalis, T. denticola, and Actinomyces viscosus
in more than half of the PFZ samples investigated as primary colonizers
29
. In addition,
the apical limit of subgingival plaque connects with some bacterial islands within the
PFZ. These findings suggest that bacteria in the PFZ may be critical periodontopathogens
in the frontier area of apical plaque.
It is now well-known that periodontitis is not associated with a single
microorganism, but is a consortium of bacteria participating in the initiation and
progression of periodontitis. For periodontopathic bacteria to cause periodontal diseases,
it is essential that they are able to colonize subgingival pockets and produce virulence
factors that directly damage host tissue. The results of current genome study projects of
several periodontopathogens will provide detailed information about the etiology of
periodontal diseases, and will likely show new possibilities for the treatment and
prevention of periodontal diseases.
Immunology of Periodontal Disease
Chronic inflammatory periodontal disease results from the inflammatory response
to bacteria in dental plaque and may either remain confined to the gingival tissues, or
progress leading to attachment loss. Disease progression is due to a combination of
factors, including the presence of periodontopathic bacteria, high levels of
proinflammatory cytokines, matrix metalloproteinases and prostaglandin E
2
(PGE
2
) and
low levels of inflammation inhibitory cytokines including interleukin (IL)-10,
transforming growth factor (TGF)-β and tissue inhibitors of metalloproteinase
30
. In some
8

individuals, neutrophils and cell mediated immunity may limit the extent of attachment
loss. However, in susceptible people as determined by genetic and environmental factors,
the presence of defined periodontopathic bacteria such as Porphyromonas gingivalis,
Aggregatibatcer actinomycetemcomitans (formely named Actinobacillus
actinomycetemcomitans) or Tannerella forsythia may limit clearance by neutrophils and
disease progression may occur. The adaptive immune response is under the control of T
cells which regulate B cell/ plasma cell differentiation and antibody production.
Clearance of bacteria by neutrophils may depend upon the presence of interferon (IFN)-γ
and may be further enhanced by protective antibodies which in turn are controlled by the
types of cytokines produced by T cells
30
.
T cells and cytokines in periodontal disease
The development and regulation of an immune response depends to a large extent
on the local production of a number of cytokines which can determine whether the
response will be a protective or non-protective one. The immune response to infection is
regulated by the balance between T helper (Th) 1 and Th2 cytokines. The net effect of the
Th1 cytokines IL-2 and IFN-γ is to enhance cell mediated responses, while that of the
Th2 cytokine IL-4 is to suppress cell mediated responses and hence enhance the
resistance associated with humoral immunity
31
.
It is evident that both T and B cells are present in periodontal disease tissues
32
, the
majority of T cells being activated memory/primed cells
33
. Both T and B cells extracted
from gingival tissues have been reported to be at a more advanced stage of the cell cycle
than peripheral blood T and B cells, indicative of activation within the tissues
34
or the
9

selective extravasation of activated cells. The infiltrate in the periodontal lesion consists
of lymphocytes and macrophages, and it has been hypothesized that T lymphocytes
predominate in the stable lesion, while the proportion of B cells and plasma cells is
increased in the progressive lesion
35, 36
. This has prompted the suggestion that T cells
with a Th1 cytokine profile may be the major mediator in the early/stable lesion. The
production of IFN-γ would enhance the phagocytic activity of both neutrophils and
macrophages and hence containment of the infection. However, the lesion persists due to
the continual formation of the plaque biofilm
37
. The dominance of B cells/plasma cells in
the advanced/progressive lesion would suggest a role for Th2 cells. If the innate response
is poor, low levels of IL-12 would be produced and a poor Th1 response may occur,
which may not then contain the infection. Mast cell stimulation and the subsequent
production of IL-4 would encourage a Th2 response, B-cell activation and antibody
production. If these antibodies are protective and clear the infection, the disease will not
progress, but if on the other hand they are non-protective, the lesion will persist and
continued B-cell activation would result in large amounts of IL-1 and hence tissue
destruction
37, 38
.
A role for IL-10 was suggested by another study which demonstrated two distinct
profiles of cytokine expression in CD4
+
gingival lymphocytes isolated from inflamed
periodontal tissues: in both, IFN-γ, IL-6 and IL-13 mRNA were present, but in only one
was IL-10 mRNA present. In most cases, IL-2, IL-4 and IL-5 mRNA were not detected
39
.
IL-10 has been demonstrated to inhibit lipopolysaccharide-induced B-cell proliferation in
the mouse
40
such that decreased IL-10 in periodontitis may possibly allow continued
10

polyclonal B-cell activation. Wassenaar et al.
41
showed functional differences in CD8
+
T-
cell clones. Those that produced high levels of IFN-γ but no IL-4 or IL-5 (Th1) mediated
cytolytic activity. Other CD8 clones produced high levels of IL-4 together with IL-5 and
displayed no cytotoxicity but could suppress the proliferative response of cytotoxic CD8
T-cell clones. It was concluded that CD8
+
T cells may participate in the local response by
suppressing IFN-γ producing cells and favoring humoral immune responses.
Chemokines in periodontal disease
The regulation of leukocyte migration into and through the tissues is determined
by the expression of adhesion molecules on endothelial cells and other cells such as
keratinocytes, which are induced by pro-inflammatory cytokines as well as to a group of
cytokines with chemotactic properties, the chemokines. Chemokines are responsible for
the recruitment and subsequent activation of particular leukocytes into inflamed tissues
42

and therefore play a central role in the final outcome of the immune response by
determining which subsets of leukocytes form the inflammatory infiltrate. Th1 and Th2
cells differ in their migratory properties and chemotactic responsiveness so that
chemokines may regulate local immune reactions by influencing the local balance of T-
cell subsets
43
.
Recently, MCP-1 (monocyte chemoattractant protein-1) production by monocytes
has been shown to be regulated by IL-10
44
. Reduced IL-10 in periodontitis could result in
reduced MCP-1 and cell-mediated responses.
However, another chemokine, MIP-1α (macrophage inflammatory protein-1α)
has been reported both to shift the immune response to a Th2-type response
45
and to
11

recruit Th1 cells. Immunohistochemistry has shown higher numbers of MIP-1α positive
leukocytes in periodontal disease tissues
46
. Microchemotaxis experiments have shown
MIP-1α to be a potent chemoattractant for B cells and cytotoxic T cells, although at
higher concentrations the migration of CD4 cells was enhanced
47
. Both MIP-1α
48, 49
and
MCP-1
50
have been shown to be involved in the recruitment of neutrophils.
The role played by RANTES in the migration of T-cell subsets and Th1/Th2 cells
is controversial. RANTES was recently detected in the gingival crevicular fluid of
patients with periodontitis but not subjects with clinically healthy gingival, and the
concentration was significantly higher in samples from active sites than in those from
inactive sites
51
. Furthermore, RANTES levels decreased after periodontal therapy
suggesting a relationship between this chemokine and periodontal disease status
52
. IP-10
on the other hand is specific for activated T cells. It was shown to target Th1 cells
selectively resulting in the upregulation of IFN-γ rather than IL-4 peripheral blood
producing T cells
53
. However, an immunohistological study reported no differences in IP-
10 or RANTES in the gingival tissues suggesting no predominant T-cell subset
recruitment by these chemokines in gingivitis or periodontitis.
54

Taubman and Kawai
55
have demonstrated that Th1-type T cells which
preferentially express CCR5 and CXCR3 are found predominantly in diseased gingival
tissues whereas little CCR5 expressed by Th2 cells could be found. It was also shown
that the chemokine ligands RANTES and MIP-1α (CCR5) and IP-10 (CXCR3) were
elevated in inflamed periodontal tissues. These authors cite these results as supporting
evidence for Th1 involvement in periodontal bone resorption.
12

B cells and antibody regulation in periodontal disease
B cells and plasma cells produce and secrete immunoglobulins which protect the
host by various methods including prevention of bacterial adherence, inactivation of
bacterial toxins and by acting as opsonins for phagocytosis by neutrophils. Antibodies
can downregulate or up-regulate the immune response. If the result of B cell
differentiation is protective antibody production, elimination of the causative organism
would ensue and periodontal disease progression would stop. Production of non-
protective antibodies in susceptible subjects could on the other hand result in continual
connective tissue breakdown. Periods of destruction would proceed.
56

The inability of specific antibodies to eliminate the causative organisms of
periodontal disease could be due to a number of factors, including poor antigenicity of
virulence determinants and elicitation of antibodies with poor anti-bacterial properties
57
.
The production of anti-P .gingivalis antibodies with different avidities in various forms of
periodontal disease have been suggested to reflect the quality of the humoral response
which may affect progression of the disease
58
. Non-protective low avidity anti-
P.gingivalis antibodies may be incapable of effectively mediating a variety of immune
responses.
59, 60

During the chronic phase of the disease, the antibody response has been suggested
to be generally protective, facilitating bacterial clearance and arresting disease
progression
61
. Anti-P.gingivalis protease antibodies which occur late in periodontitis
infections have been demonstrated to block the anti-opsonizing activity against C3 and
IgG
62
. An increased capacity of serum to opsonize P. gingivalis has been shown to be a
13

distinctive feature in patients with a past history of destructive periodontal disease
63
.
Opsonic IgG antibodies to A. actinomycetemcomitans which may facilitate neutrophil-
mediated phagocytosis and be protective against this periodontopathic organism have
also been demonstrated
64
. Repeated infection with A. actinomycetemcomitans has also
been shown to elicit an anti-leukotoxin antibody that protects neutrophils from the
leukocidal activity of the leukotoxin.
65

The B-cell response requires T-cell help in the form of cell–cell contact as well as
cytokines, which are responsible for the expansion and differentiation of B cells into
plasma cells and in class switching
66
. T cells are necessary for both specific antibody
production and polyclonal B-cell activation
67
and as they are the dominant cell type in the
cell-mediated (macrophage/lymphocyte) response, T-cell determination of the resulting
antibody response must play a fundamental role in the pathogenesis of periodontal
disease.
T-cell receptor affinity
Differentiation of Th1 and Th2, T-cell subsets is determined during priming and
is influenced by a number of factors, including the cytokine environment, the antigen
itself, antigen dose, the route of administration, the nature of the antigen presenting cell
and costimulatory molecules. Recent studies have shown that the affinity of the major
histocompatibility complex/peptide/T-cell receptor interaction determines the
differentiation of CD4
+
cells into either Th1 or Th2 cells
68
. In the presence of IL-12, a
short T-cell receptor stimulation has been shown to induce Th1 polarization, IL-12
exerting its effect during and after T-cell receptor signaling. Th2 polarization, on the
14

other hand, was found to require prolonged T-cell receptor signaling and IL-4 was
effective only when present during T-cell receptor triggering
69
. These authors concluded
that the duration of T-cell receptor stimulation was crucial in influencing Th1/Th2
polarization. Furthermore, Busch & Palmer
70
showed that in vivo expansion of T cells
after bacterial infection is accompanied by an increase in the T-cell affinity for antigen. T
cells which have undergone a number of rounds of in vivo expansion have been
demonstrated to express a narrower range of T-cell receptors and to bind major
histocompatibility complex/peptide complexes with greater affinity. The strength of the
T-cell receptor signal has also been found to determine the involvement of CD28
costimulation in CD4 T-cell differentiation. In this study, IL-4-producing Th2 cells were
generated after priming with a weak T-cell receptor signal but not with a strong signal,
and this was dependent on CD28/B7 interactions
71
. It was concluded that a more
sustained engagement of the T-cell receptor by major histocompatibility complex
polarize T-cell receptor transgenic splenocytes to a Th2 profile.
Regulatory T cells
While Th1 and Th2 subpopulations of T cells determine the response to infection
based on the cytokine pattern induced, distinct so-called regulatory T cells with
immunosuppressive function and different cytokine profiles have been described that
may prevent infection-induced immunopathology or prevent pathogen elimination by
suppressing protective Th1 responses
72
. Three distinct regulatory T cell (Tr) subsets have
been described. Tr1 CD4 cells secrete high levels of IL-10 and low to moderate levels of
transforming growth factor (TGF)-β, and have been shown to prevent the development of
15

Th1-mediated autoimmune diseases and suppress immune responses to pathogens,
tumors and alloantigen
73
. The suppressive effects of Tr1 clones are reversed by IL-10
neutralization, suggesting that, regardless of T-cell antigen-specificity, Tr1 suppression is
an effect mediated by IL-10. Th1 and Th2 cells reciprocally regulate the other
subpopulation by the secretion of IFN-γ and IL-4 and also possibly by IL-10 via a
negative feedback loop. Tr1 cells which secrete high levels of IL-10 can suppress Th1
responses to an infectious pathogen
74
.
The second subset of regulatory T cells is Th3 CD4 cells, which primarily secrete
TGF-β. As this cytokine is secreted by many cell types, Th3 cells may have a major role
in immune regulation. TGF-β is an important anti-inflammatory agent and IL-1
inhibitor
75
.
CD4
+
CD25
+
T cells make up approximately 5–10% of the peripheral blood T-
cell pool and immunosuppression occurs by inhibition of IL-2 production, via a
mechanism dependent on cell–cell contact as well as the expression of CTLA-4, which is
a CD28 homolog and negative regulator of T-cell activity
76
.
The development and persistence of chronic infections have been postulated to be
due to an imbalance of either a Th1 or a Th2 profile, although suppression of a protective
immune response by regulatory T cells may be a major factor
72
. Pathogen-stimulated IL-
10 or TGF-β by innate cells including macrophages and dendritic cells may suppress the
immune response early in infection, this suppression being maintained by the induction
of Tr1 or Th3 cells. Pathogens which cause chronic infections may export Tr cells to
counteract protective Th1 responses, which in turn will prolong survival
72
.
16

Nuclear Factor Kappa Beta
NFKB (nuclear factor kappa beta) is a transcription factor that plays important
roles in the immune system
77, 78
. NFKB regulates the expression of cytokines, inducible
nitric oxide synthase (NOS), cyclo-oxgenase 2 (COX-2), growth factors, inhibitors of
apoptosis and effectors enzymes in response to ligation of many receptors involved in
immunity including T-cell receptors (TCRs), B-cell receptors (BCRs) and members of
the Toll-like receptor/IL-1 receptor super family. NFKB also plays a role in the
development and the activity of a number of tissues including the central nervous
system
79
. Moreover, pathological dysregulation of NFKB is linked to inflammatory and
autoimmune diseases as well as cancer. In mammals, the NFKB family is composed of
five related transcription factors: p50, p52, ReiA (p65), c-Rel and RelB
80
. There are two
signaling pathways leading to the activation of NFKb known as the canonical pathway
(or classical) and the non-canonical pathway (or alternative pathway)
81
. The common
regulatory step in both of these cascades is activation of an IkB.
Recent advances in bone cell biology demonstrated the crucial role of the receptor
activator of nuclear factor- KB (RANK) ligand (RANKL)/osstoprotegrin (OPG) system
in osteoclast differentiation and function. RANKL is a cytokine that belongs to the TNF
family and is essential for the induction of the osteoclastogenesis. OPG is also a cytokine
that belongs to TNF family that inhibit osteoclastogensis by being involved in
competitive binding of RANK with RANKL. Several studies demonstrated the functional
association of RNKL and OPG with periodontal disease. In animal experiments, CD4
+

Tcell stimulated by Actinobacillus actinomycetemcomitans induce RANKL production
82,
17

83
and participate in alveolar bone destruction. Two other studies that used samples
obtained from human with periodontal disease also demonstrated an elevated level of
RANKL expression and a reduced level of OPG expression in the affected tissues when
compared with those in healthy gingival tissue
84, 85
.
Innate immunity
Phagocytic cells such as neutrophils and macrophages constitute the first line of
defense against bacterial infection. Neutrophils can be found within the gingival sulcus
and migrate through the junctional epithelium in all stages of periodontal disease. In the
sulcus, neutrophils form a barrier between the epithelium and plaque
86
which in most
cases prevents bacterial invasion of the epithelium and underlying connective tissue
87
.
Macrophages are important mediators of inflammation. On exposure to antigen,
macrophages both initiate and enhance the immune response by the secretion of a number
of proinflammatory cytokines such as IL-1 and IL-6, T-cell regulating cytokines
including IL-10 and IL-12 and a number of chemokines which influence the recruitment
of additional monocytes, neutrophils and lymphocytes into the gingival tissues
88
.
Macrophages also act as antigen-presenting cells in the initial stages of the immune
response and play a vital role in the effector stages as microbicidal cells
89
.
Susceptibility to periodontal disease
Innate susceptibility to periodontal disease is influenced by host genotype
90
.
Genetic polymorphisms in Fc receptors on phagocytic cells may be significant in
determining susceptibility to bacterial infection
91
. Individuals with low affinity Fc
18

receptor for IgG2 (Fc RIIa) have reduced IGg2-mediated phagocytosis of encapsulated
bacteria
92
.
Cytokine polymorphisms have been reported to influence immune response in
periodontal disease. IL1 polymorphisms have been claimed to be risk factor for severe
periodontal disease with genotype positive individual
93
.
Neuroendocrine regulation may be significant in periodontal disease susceptibility.
Leukocytes express receptors to neurotransmitors, especially noreadrenalin, as well as
receptors for several hormones produced by endocrine glands, including corticosteroid,
growth hormone, thyroid hormone, substance P and ACTH. On the other hand, many
cytokines produced by leukocytes can act on neurons and endocrine gland including IL-1,
Il-6 and TNF-a
94
.
Environmental risk factors such as tobacco smoking, psychological stress, and
systemic disease such as diabetes modify the host response and may be major
determinants of susceptibility to periodontal disease
95
.
T cell response to P.gingivalis and in particular CD4 response may be depending
on major histocompatibility complex genes. The specific antibody response to
P.gingivalis requires the presence of T cells
96
, and T cell response has been shown to
influence periodontal bone destruction
97
.
It is clear that the immunoregulatory profile is fundamental in determining the
ultimate outcome of periodontal disease.
19

Localized Aggressive Periodontitis
Nomenclature:
Localized Aggressive Periodontits (LAP) occurs in young and medically healthy
patients. It is characterized by a rapid and extensive periodontal destruction that is
localized to interproximal areas of permanent first molars and incisors.
Gottlieb
98
(1928) was the first to describe the disease. He called it “Deep
Cementopathia”, because he believed that the original defect was in the cementum. In
1938, Wannenmacher
99
called the disease “Parodontitis Marginalis Progressiva”. He
stated that the disease has inflammatory entity and that the bone resorption appeared
mostly in the incisor and first molar areas. In fact, Wannenmacher should be considered
the first to have described LAP.
In 1940, Thoma and Goldman
100
used the term of “Paradontosis. They believed
pocket formation is due to the breakdown of the principal fibers of the periodontal
ligaments due to resorption of the alveolar bone. Then connective tissue will proliferate
and replaces the resorbed bone. This proliferation displaces the tooth, and occlusion will
influence the direction of tooth migration.
The English term “Periodontosis” was coined by Orban and Weinmann
101
in 1942.
They divided the disease into three stages. In the first two stages there is no inflammation
or pocket formation.
The Nomenclature Committee of the American Academy of periodontology
(1950) also referred to periodontosis as a degenerative non-inflammatory destruction
originating in one or more of periodontal structures characterized by migration and
20

loosening of the teeth in the presence or absence of secondary epithelial proliferation and
pocket formation or secondary gingival disease.
McCall
102
(1951) commented that, because the incisors and first molars are the
first teeth to erupt, and thus have been exposed to occlusal stress for the longest time,
they show the first signs of alveolar bone loss.
Seidler
103
and coworkers (1950) described the pattern of bone loss. They stated
that in the older age groups the bone destruction affected almost all the teeth but in
younger individuals only the first molars and incisors were affected.
Kaslick and Chasens
104
(1968) used the new term “peridontosis with
periodontitis.” They studied 27 U.S. army recruits having this disease. They found that
the pattern of connective tissue loss on opposite sides of the mouth was mirror image.
They concluded that this finding was supportive evidence for the idea that periodontosis
could be a hereditary or developmental defect.
Bear
105
(1971) gave his definition of the periodontosis as a disease of
periodontium occurring in otherwise healthy adolescents, which was characterized by a
rapid loss of the alveolar bone about more than one tooth of the permanent dentition.
There are two basic forms in which it occurs. In Localized form the first molars and
incisors and in generalized form, most of the dentition can be affected. The amount of
destruction manifested is not commensurate with the amounts of local irritants present.
Also Baer
105
(1971) said that in extracted teeth affected by periodontosis microscopic
examination revealed plaque adhering to the root surfaces, suggesting that the local
irritants were present.
21

Borring, Moller and Ferandsen
106
(1978) performed third molar transplantations
into sackets of freshly extracted first molars of eight patients with juvenile periodontitis.
No pocket depth over 3 mm was neither found, nor any abnormal mobility of the teeth
after bone healing. This study makes doubtful any primary role of the bone in this disease.
The term of juvenile periodontisis was introduced by Buttler
107
(1969).
Wearhaug
108
(1976) showed that there is always a thin layer of sub gingival plaque which
grows epically at maximum rate of 5 m per day, i.e., 1.8 mm per year. He concluded that
rather than a degenerative process, there must be some deficiency in the host defense
mechanisms which allows the exaggerated destructiveness, and claimed that the disease
should actually be called destructive juvenile peritonitis.
In 1997, the committee of Nomenclature of the American Academy of
Periodontology published a Glossary of Terms recommended for use, which labeled this
disease as juvenile periodontitis. Since1999, the classification system for periodontal
disease has been changed. The term juvenile periodontitis has been changed to
Aggressive Periodontitis, and Adult Periodontitis has been changed to chronic
periodontitis. The distinction between chronic and aggressive forms of periodontitis is
based on (a) the amount and pattern of periodontal destruction and (b) the patient’s age
and medical status. If the patients are young, medically healthy, and present with
extensive periodontal destruction they might have a form of aggressive periodontisis. If
periodontal destruction is localized to interproximal areas of the permanent first molars
and incisors, the diagnosis is Localized Aggressive Priodontitis (LAP). In most cases of
LAP the amounts of microbial deposits are inconsistent with the severity of periodontal
22

tissue destruction.
109
If the destruction is found around at least three permanent teeth
other than the first molars and incisors, the diagnosis of Generalized Aggressive
Periodontitis (GAP) is usually made.
Both forms of aggressive periodontitis are plaque-induced infections and host
responses to plaque bacteria that are responsible for most of the tissue destruction.
However, the plaque biofilms are often clinically thinner than in cases of chronic
periodontitis. Based on several specific clinical and host-response differences between
LAP and GAP, it is clear that LAP is not merely a localized from of GAP (Armitage,
2002)
109
.
LAP generally has a circumpubertal onset or is first detected and diagnosed
during puberty, whereas GAP is usually detected and diagnosed in people less than 30
years of age. However, some patients with Gap may be older than 30 years of age. It has
been suggested by Lang et al., that patients with LAP usually mount a robust serum
antibody response to periodontal pathogens, whereas patients with GAP exhibit poor
antibody response to infecting agents
110
.
Prevalence of Localized Aggressive Periodontitis
Estimates of prevalence of localized Aggressive Periodontitis (LAP) vary greatly.
Russell
111
(1957) studied urban children in the United States and found advanced
periodontal disease in 3% of white and 3.2% of black children. He
112
also studied the
prevalence of periodontal disease in different population. He found advanced periodontal
disease in 5% of Lebanese children aged 10 to 14 years, and in 10% of Palestinian
children in refugee camps in Lebanon.
23

In a study of young adults in the army, Kaslickan and Chasens
113
(1968) found
that the prevalence of Localized Juvenile Periodontitis (LJP) in 3,777 young adults was
0.15%. In another study by Lacy and Brasher
114
(1977), the prevalence of LGP in a group
of 3,235 persons from a military population was 0.4%. Saxby
115
(1984) reported that
0.2% of Asians and 0.8% of black adolescents in England had localized juvenile
periodontitis. Harley and Floyd
116
(1988) examined a group of 12-19 year old school
children in Nigeria and found that 0.8% of these had radiographic and clinical features
characteristic of localized and generalized EOP. A hospital-based retrospective study in
17-34 years old Nigerians estimated the prevalence rate at 1.6% (Arowojolu & Nwokorie,
1997)
117
, whereas a study in a group of 18-26 years old Kenyans found a prevalence of
0.3% subjects with EOP (Wagaiyu & Wagaiyu
118
).
Loe and Brown
119
(1991) conducted a national survey of the oral health of U.S.
children aged 5 to 17 during the 1986-1987 school year. Eleven thousand and seven
adolescents aged 14 to 17 years received a periodontal assessment. Approximately 0.53%
of adolescents nationwide were estimated to have Localized Juvenile Periodontitis (GJP),
and 1.61% to have incidental loss of attachment (greater than or equal to 3 mm on one or
more teeth.). Blacks were at much greater risk for all forms of early onset periodontitis
than whites. Males were clearly more likely (4.3 to 1) to have GJP than females when
other variables were statically controlled. Gender association was more complicated for
LJP because gender interacted with race. Black males were 2.9 times likely to have LJP
as black females. In contrast, white females were more likely than white males to have
disease by about the same odds.
24

In a retrospective study over a three years period, 23 cases of LJP were diagnosed
from 5,480 Saudi subjects with different forms of periodontal disease. The overall
prevalence was 0.42%. The female to male ratio was 1.88:1. The difference in the sex
ratio was statistically significant. No statistically significant difference in the sex ratio
association regarding the site afflicted by Actinobacillus actinomycetemcomitans was
found (Nassar, M.M. et al, 1994)
120
.
Albander et al.
121
(1997) studied a sample of about 14,000 children representative
of 13-17 years old adolescents attending secondary schools in the United States, and
reported that 1.3% of whites, 5% Hispanics and 10% of blacks had clinical attachment
loss indicative of early onset periodontitis.
In a study by Stabholz et al. (1998)
122
of a unique population in Israel, they found
a high prevalence of LJP. This study had been done among adolescents 12-20 years of
age from a group of nuclear families living and functioning in a closed community. The
survey was carried out on a population of teenagers that had attended the same school
and their siblings. The population was 88 individuals from 30 families. Of the 86
individuals examined, 33 individual from 15 families were diagnosed as having LJP
(38.4%). Except for two pairs of families with genetic ties, no familial connections could
be traced between the individuals with LGP. Their findings strongly suggested an
environmental influence in the etiology at the disease.
The prevalence of aggressive periodontitis in adolescents and young adults from
Valedo Parabia in Brazil has been evaluated by Cortelli et al.
123
(2002). In a population of
young adult patients, aging between 15 and 25 years, from the Department of Dentistry,
25

University of Taubate, six hounded patients were included in their study. Ten subjects
(1.66%) were diagnosed as having localized aggressive periodontitis (2 male and 3
females), and 22 (3.66%) patients presented with generalized aggressive periodontitis (5
male and 16 females). In this study there was a positive correlation between the female
gender and occurrence of periodontal disease.
Albander et al.
124
(2002) studied the prevalence and severity of Early Onset
Periodontitis (EOP) among students attending secondary school in two regions of Uganda.
690 students aged 12-25 years were included in their study. Results showed that 199
(28.8%) study subjects exhibited clinical features of EOP, of which 16 (2.3%) subjects
showed generalized EOP, 29 (4.2%) localized EOP, and 154 (22.3%) indicated EOP. The
percentage of EOP-affected males was significantly higher than females (33.8% vs.
22.2%). No association was found between EOP prevalence and socioeconomic status.
Prevalence of Aggressive Periodontitis among Israeli army personal has been
evaluated by Levin et al. (2005)
125
. Aggressive periodontitis was found in 5.9% of the
subjects (4.3% localized and 1.6% generalized). At least one site with a probing depth >
or = 5mm was found in 20.1% of the subjects. Current smokers (39.9%) and subjects of
North African origin correlated with a high prevalence of aggressive periodontitis.
A review of the published literature suggests that the prevalence of LAP may
vary significantly between different countries. Low prevalence rates ranging between
0.1% and 0.2% have been reported in Europe (Hansen et al
126
. 1984, Saxby
115
1984,
Kronauer
127
et al., 1986), whereas high prevalence rates have been reported in Africa
26

(Harley & Floyd 1988
116
), Brazil (Gjermo et al
128
, 1984), Iraq (Albander et al.
129
, 1989),
and the United States
121
(Albander et al., 1997).
Etiology of Localized Aggressive Periodontitis
The etiology of Localized Aggressive Periodontitis (LAP) remains an interesting
research topic. LAP has a bacterial etiology, with infection of the gingival tissues by
several bacterial pathogens. However, in addition to bacterial etiology, host immune and
inflammatory factors are implicated in the pathogenesis and progression of localized
aggressive periodontitis. It is now generally accepted that LAP is an inflammatory
disease with a multifactorial nature. The risk factors can be classified into three major
categories:
1) Genetic Basis
2) Bacterial
3) Immunological (Host)
Genetic
Localized Aggressive Periodontitis has a primarily bacterial etiology but there is
increasing evidence that besides certain environmental factors, the onset and progression
of periodontitis can be influenced by different genetic factors. It has been noted that LAP
tends to cluster within families, thus favoring the theory that susceptibility to this disease
may be an inherited trait. To date, however, efforts to identify the genetic basis for
susceptibility to LAP have provided ambiguous results.
Heredity could be an etiologic factor for Localized Aggressive Periodontitis
(LAP). It has been pointed out that there are several inherited syndromes in which there is
27

a concomitant juvenile periodontitits such as Papillon-Lefevre syndrome,
Hypophosphatasia, Cyclic Neutropenia, Down’s syndrome, etc. (Fourel
130
1972,
Jorgenson et al., 1975
131
). Newman
132
(1976) suggested that the familial pattern of LAP
may result from (a) genetic predispositions to specific groups of bacteria, (b) a
genetically determined immunodeficiency, and (c) faulty or impaired formation and
maintenance of periodontal tissue integrity. Bear et al.
133
(1963) examined the karyotype
of the patients with juvenile peridontitis and found it normal. Also Kaslick et al.
134
(1971)
analyzed the ABO Blood groups. A large number of LJP patients were of blood group B
and a smaller number were of blood group O. They concluded that genetic factors must
play an important role in the etiology of periodontitis. In contrast, in his study Malena
135

(1972) found that the blood-phonotype A
1
was more susceptible to the disease than
phenotypes A
2
, B, AB, and O.
The association between periodontal disease and HLA-A
2
antigen has been
investigated, and it was found that only 25.5% of patients with juvenile periodontitis
were HLA-A
2
antigen positive whereas 61% of the normal controls were positive
(Kaslick et al
136
. 1975). Reinholdt and coworkers
137
(1977) also found a similar low
frequency of HLA-A
2
antigen in these patients. The author stated that tissue type
specification HLA-A9, and HLA –A28 had significantly higher frequency in juvenile
periodontitis patients.
Association of a Vitamin D Receptor (VDR) gene polymorphism with localized
Early-Onset Periodontal (EOP) disease has been investigated by Hening and coworkers
138

(2002). Genetic polymorphisms in the VDR gene were associated with the parameters of
28

bone homeostasis and disease with bone loss in particular osteoporosis. The data from
this study indicated that carriages of the less frequent allele of the Taq1 restriction
fragment length polymorphism in the VDR gene significantly increase the risk of
developing unequivocal evidence of localized disease. However, VDR gene type may not
affect the incidence of all cases of EOP. These findings support genetic basis for
periodontal disease and may help define sub-groups of this disease which share common
pathogenic factors.
Receptors for the Fc fragment of Immunoglobulin G (Fc gamma Rs) play a
crucial role in host defense against bacterial infection by linking humeral and cell-
mediated immune responses. Fc gamma Rs has different biologic activity. Genes
encoding allotypes with diminished activity have been suggested as potential risk factors
for infectious disease. These molecules, designated FcgRs, are involved in multiple
aspects of the host response to bacterial infection, including antibody-dependent cell-
mediated cytotoxicity, endocytosis, phagocytosis, release of inflammatory mediators, and
enhancement of antigen presenation.
139
Three classes of FcgRs are currently recognized
and are designated FcgRI (CD64), FcgRII (CD32), and FcgRIII (CD16).
140
The
neutrophil specific low affinity FcgRIIIb also exhibits two allelic forms designated NA1
and NA2. In a study by Fuy and Coworker (2003)
141
it has been shown that individuals
expressing Fc gamma RIIIb NA2/NA2 gene type have a greater risk for developing LAP.
A major portion of antibody to periodontitis-associated bacteria is the IgG2
subclass, which is reactive with serotype-specific carbohydrate antigens (Wilson &
Hamilton, 1992
142
; Lu et al., 1994
143
). Individuals affected by localized AgP have
29

elevated serum IgG2 levels (Zhang et al., 1996
144
). High levels of IgG2 reactive with
serotype-specific antigen of A.actinomycetemcomitans correlate with less severe disease
in generalized AgP (Califano et al., 1996
145
). These findings suggest that genetic
differences in AgP susceptibility may be mediated by antibody responses. A segregation
analysis of IgG2 in families with one or more AgP-affected members suggested that IgG2
levels are controlled by a single gene (Marazita et al., 1996
146
). However, studies of other
antibodies, such as IgE, indicated that the response is controlled by multiple genes
(Mathias et al., 2001
147
). Dihel and his coworkers
148
estimated heritability of IgG2 levels
to be 38% in sixty families with aggressive periodontitis.
Liy and coworkers (2003)
148
performed genetic linkage analysis with four
multigenerational families exhibiting the LAP phonotype. They found that the LAP
phonotype is linked to DNA marker, DIS 492, with LOD score 3.48. The haplotype
analysis showed that LAP locus is located between DIS492 and DIS533 on chromosome
1, covering about 26 million DNA base pairs.
Cytokines play an important role in inflammation.
150
Periodontal tissue cells
stimulated by periodontal pathogens secret proinflammatory cytokines, such as
interleukin (IL)-1a, IL-b, IL-6, IL-8, and tumor necrosis factors (TNF)-a, which play key
roles in the pathogenis of periodontal disease. TNFa and IL-1 are considered major
mediators of periodontal inflammation. IL-1, which has two main forms, IL-1a and IL-1B, is
the prototypic "multifunctional" cytokine. IL-1 affects nearly every cell type, often in concert
with other cytokines or small mediator molecules. The varied biologic properties of IL-1
result from its effects on the expression of various genes that regulate the production of
30

cytokines such as TNF-a, IL-2, IL-3, and IL-6.
151
IL-1 has been implicated in the pathogenesis
and clinical course of periodontal diseases because of its multiple proinflammatory
properties.
152, 153
It is a key mediator of inflammation and modulates extracellular matrix
components, enhances bone resorption in the periodontal tissues, stimulates fibroblasts
and other nucleated cells to produce matrix metalloproteinase, activates plasminogen, and
triggers prostaglandin synthesis. IL-1 also strongly stimulates connective tissue catabolism,
activates immunocytes, and regulates adhesion molecules that facilitate migration of
leukocytes into tissues.
154

IL-1 family genes are located in a cluster on human chromosome 2ql3
155
. A specific
genotype in the IL-1 cluster that includes a specific locus is associated with increased IL-1
production and increased susceptibility to severe periodontitis
155
. TNFa is another potent
immunomodulator and proinflammatory cytokine that has been implicated in the
pathogenesis of autoimmune and infectious disease. TNFa induces the secretion of
collagenase by fibroblasts, stimulates resorption of cartilage and bone, and has been
implicated in the destruction of periodontal tissue in periodontitis
156
. The TNFa gene lies
within the class III region of the major histocompatibility complex on the short arm of
human chromosome 6. There is a base-transition polymorphism at the -308 position of the
TNF-a promoter region
156
.
Higher production of IL-1 and TNF has been associated with enhanced response to
infection, in which local induction of these cytokines facilitates elimination of the microbial
invasion. TNF-a acts synergistlcally with IL-1.
31

Genetic markers have come to attention because of the genetic nature of AgP. In 1997,
Kornman et al. noted the association of periodontal diseases and cytokine gene
polymorphisms
157
. There is evidence for an association between certain cytokine gene
polymorphisms and human diseases that involve an inflammatory pathogenesis. Cytokine
polymorphisms may influence the level of cytokine secretion and may explain the
individual differences in the cytokine responses to bacterial stimuli. Moreover, allelic variation
in genes for cytokines and for factors regulating their expression may create phenotypic
differences in cytokine responses between individuals
158
.
There are three genes that regulate the production of IL-1, located in a 415 kb
region on the long arm of the chromosome 2ql3
159
. The IL-1 gene cluster includes IL-1a ,
IL-1b, and IL-1RN genes that code for IL-1 a, IL-1b, and IL-1 receptor antag onist (IL-lra),
respectively. Polymorphisms of the IL-1 gene cluster have been implicated with the
susceptibility and severity of various chronic inflamma tory diseases including marginal
periodontitis
160
.
Several studies
160, 161
have indicated a role for inter-leukin-1 gene cluster
polymorphism in the risk assess ment for adult periodontitis, specifically for individuals
carrying the allele 2 of the biallelic restriction frag ment length polymorphism of the
IL-Ib +3954 and the 1L-1a -889 loci in either the heterozygous or homologous state at
both loci. These subjects were found to have a significantly greater risk for developing
severe periodontitis when compared to a mild periodontitis group or to periodontally
healthy subjects.
32

The possible role of the IL-1a and IL-1B genetic polymorphisms were
evaluated in aggressive periodontitis in 28 African American and seven Caucasian
families using the transmission disequilibrium test
162
. Allele 1 of the IL-1A -889
polymorphism and allele 1 of allele IB +3954 polymorphism were transmitted
significantly more often than allele 2 in generalized aggressive periodontitis (GAgP). A
similar trend was reported for localized aggressive periodontitis (LAgP), although the
difference was not statistically significant. Evidence for linkage disequilibrium for
allele 1 of the IL-1b +3954 locus was even stronger for the GAgP. Allele 1 at IL-1b
+3954 was transmitted more often than allele 2 for LAgP cases, but the difference was
not statistically significant. It has been suggested that the IL-1b +3954 polymorphism
may be more important than the IL-1 A -889 polymorphism for the association with the
AgP phenotype. Walker et al.
163
found that 8% of African American patients with LAgP
and 14.5% of control subjects were genotype positive, and that the prevalence of the
IL-1b +3954 allele 1 polymorphism was higher than 99% in control individuals and
100% in LAgP patients. Hodge et al.
164
did not find an association between IL-1A and
IL-1B genetic polymorphisms with GAgP in untreated European Caucasian patients.
However, another study
165
found that the IL-1b genotype in combination with smoking
is risk factors for AgP. Genetic polymorphisms vary in different ethnic populations;
conclusions about disease association cannot be extended to other populations.
In study by Quappe et al. (2004)
166
prevalence of the positive composite IL-1
geneotype was higher in aggressive periodontitis patient (25%) than healthy subjects
(12%), but the difference was not significant. The IL-b 3954 homozygous for allele 1
33

frequency was higher in controls than in patients, suggesting a protective factor for
Aggressive Periodontitis (AgP). The heterozygous for allele 2 of the IL-b showed a
significant association with AgP.
In a study by Renxy and coworkers (2008)
167
interleukin-1 family polymorphisms
in aggressive periodontitis and their relative in China have been evaluated. When all
Aggressive periodontitis patients were stratified by the clinical sub type, the frequencies
of A1A1 gentotype and allele 1 at IL-b 511 were significantly increased in the localized
AgP patients compared to unaffected, while no significant difference was observed
between the generalized subtype and the unaffected. They concluded that polymorphisms
of IL-1B-511 may have an affect on the susceptibility of Localized Aggressive Patients in
Chinese population.
A plethora of genetic factors, ranging from single gene defects to more subtle
combinations of single nucleotide polymorphisms (SNPs), have been suggested as
predisposing to aggressive periodontitis (AgP) (Kinane et al. 2005)
168
. SNPs with a
supposed functional effect have been identified in the gene coding for the
proinflammatory cytokine interleukin-6 (IL-6) (Fishman et al 1998)
169
, and an association
with periodontitis and its treatment outcomes has been reported for the −174 and −572
SNPs in Caucasians (Treviatto et al. 2003)
170
. Interleukin-6 (IL-6) polymorphisms have
been shown to affect IL-6 promotor activity. A study by Nibalil and coworker (2008)
171

supports the hypothesis of a link between IL-6 genetic factors and AgP, and highlights
the importance of two IL-6 polymorphisms (-1363 and -1480) in modulating disease
phenotype and susceptibility. Also, Nibali L. et al (2008)
172
analyzed the disease
34

phenotypes in relatives of patients with aggressive periodontitis and explored the
distributions of genetic polymorphisms of interleukin-6 (IL-6) in AgP patients and their
healthy relatives. Their study confirmed a relatively high risk for relatives of AGP
patients to have AgP (10%). Genetic polymorphisms in the IL-6 (at the positions 174GG-
1480CC) may have an impact in aetiopathogenesis of this disease.
Bacteriology
The ability of microorganisms to form plaque appears to be prerequisite for the
initiation of periodontal destruction. For many years the etiology of periodontal disease
was based on the belief that microorganisms in dental plaque played a non-specific role
in the pathogenic procedure.
173
In other words, the total microbial load rather than the
qualitative characteristics of the dental plaque was the primary concern. Indeed,
controlling the deposition of bacteria on teeth and adjacent soft tissue has been shown to
be remarkably effective in the treatment and maintenance of most patients with plaque
associated periodontal diseases. However, a number of patients demonstrate certain forms
of periodontal disease where non-specific plaque control is not effective, either as a
treatment measure or for the maintenance of the treated patients. The increased
knowledge about the periodontal microbiota has been generated based on the belief that
specific periodontal pathogen may act as causative agents of condition such as
prepubertal periodontitis, rapidly progressive periodontitis, juvenile periodontitis, and
refractory periodontitis.
174, 175
Differences in microbial composition may also exist
between active and inactive lesions. The existence of the microorganisms in the
periodontal environment that may have greater pathogenic significance than others has
35

focused the attention of the investigator on relatively small number of species, fewer than
20. Among those are Aggregatibacter actinomycetemcomitans, (formerly named
Actinobacillus actinomycetemcomitans, A.a), Porphinomonas gingivalis, Tannerella
forsythia ( formerly named Bacteriod forsythus), Fusobacterium nucleatum,
Capnocytophaga spirocket, Eikenella corrodens, and Campilobater rectus.
Socransky and Newman
176
(1973) investigated the microbiota in one individual
with juvenile periodontitis. It was observed that the microbiota were quite different in a
10 mm deep pocket than in normal site with a pocket depth of 2mm. In the normal site in
the same individual it was the same as in healthy individuals, and consisted primarily of
the streptococcus sanguis, staphylococci, veillonella and gram-negative rods, whereas in
pathological sites the microbiota was dominated by gram-negative anaerobic rods. The
morphology of plaque in chronic periodontal disease and juvenile periodontitis was
studied with the electron microscope by Listgarten
177
(1976) and he found gram-negative
flora in most samples. Socrancky
178
(1977) in his extensive review article on the
microbiology of periodontal disease characterized the gram-negative bacteria. In addition,
as Irving and his coworkers (1978)
179
demonstrated, mono-infection of germ free rats
with these gram-negative strains could cause periodontal disease between the first and
second maxillary molars with migration of the epithelial attachment and destruction of
alveolar bone by osteoclasts.
The occurrence of subgingival Actinobacillus actinomycetemcomitans (A.a.) and
capnocytophaga in 12 localized juvenile periodontitis and 10 gingivitis patients from
Panama was determined using selective culture techniques. A.a. was present in all LJP
36

patients and was on average hundred-fold higher compare to gingivitis patients. But
capnocytophega was only recovered in approximately three times as many LJP patients
compared to gingivitis patients (Slots et al., 1983)
180
.

A study by Sirkka
181
(1985) showed that the occurrence of A.a is related to the
age of the LJP patients. In their study, twenty LJP patients were divided into three age
groups: 14-16, 17-19, and 20-25 years of age, and subgingival bacterial samples were
taken for cultivation of A.a and for dark field microscopic assessment of spirochetes. A.a.
was isolated in the youngest group more frequently than in the two older ones. But
spirochetes were not found to be related to the age of the group. They concluded that A.a
is possibly associated with the activity of the disease. Socranskey et al.
182
(1988) found
neutral average odds ratio between A. actinomycetemcomitans and Bacteriod intermedius
in periodontitis. Kornman and Robertson
183
(1985) inferred that B. intermedius hindered
elimination of A. actinomycetecomitans from localized juvenile periodontitis lesions.
Slots and coworkers
184
(1990) examined age relationship and mutual interrelationships
between cultivable A.a and bacteroides intermedius in 1624 periodontitis patients, 15 to
89 years of age. A.a occurred with higher prevalence (74%) in patients less than 25 years
old than in adult and geriatric patients (prevalence about 31%). A.a was detected in 85%
of localized juvenile periodontitis patients. B. intermedius was recovered from 45% of
study subjects, and showed no predilection for any age group. As determined from this
study these two microorganisms could occur both alone and in combination, and no
synergistic or antagonistic relationships between them could be delineated.
37

In a quantitative analysis of the subgingival distribution of Actinobacilus
actinomycetemcomitans in a patient with localized juvenile periodontitis by
Vanvinkenholf 1994
185
, of the 97 sites investigated, 28 (29%) showed attachment loss. 70
(73%) were positive for A. actinomycetemcomitans. Of the total number of A.
actinomycetemcomitans cells isolated from this patient, more than 99% were found at
sites with attachment loss, < 1% being present at sites without attachment loss. The mean
percentage of A. actinomycetemcomitans was 21.2% at sites with attachment loss and
0.45% at sites without attachment loss. The distribution of Porphyromonas gingivalis
showed a symmetrical pattern, being present at the 1st molar and 2nd premolar sites in all
quadrants and at the lower incisor sites.
In a study by Tinoco et al. (1997)
186
, a correlation was found between the number
of Actinobacillus actinomycetemcomitans cells and the clinical attachment level and
probing pocket depth in the Brazilian population. They found the prevalence of LJP to be
0.1-1.1% (average 0.3%) in different cities. Subgingival bacterial sample showed A.a was
present in 80% of patients, 35.3% of their parents and 43.9% of their siblings.
Micro flora of severe, moderate and minimal periodontal lesions in patients with
rapidly progressive periodontitis has been examined by Kamnia JJ et al (1995).
187
The
examination of the subgingival micro flora indicated that certain species, including
Porphyromonas gingivalis, Bacteroides forsythus, Fusobacterium nucleatum,
Actinobacillus actinomycetemcomitans, and Campylobacter species were found to be
predominant in sever periodontal lesions. B. forsythus, P. gingivalis, Prevotella
intermedia, F-nucleatum, and Capnocytophaga ochracea were predominant in medium
38

lesions while streptococcus species, Actinomyces species, C. ochracea, Hoemophilus
sanguis and veillonella parvula, were found in higher levels in minimal periodontal
lesions.
Actinobacillus actinomycetemcomitans strains with enhanced levels of production
of leukotoxin are characterized by a 530-bp deletion from the promoter region of the
leukotoxin gene operon. Haubek et al. (1997)
188
suggested that juvenile periodontitis in
some adolescents with African origins is associated with a particular clone of A.
actinomycetemcomitans. They analyzed the geographic dissemination of this clone by
examining 326 A. actinomycetemcomitans isolates from periodontally diseased but
otherwise health individuals as well as from patients with different types of extraoral
infections originating from countries worldwide. A total of 38 isolates, all belonging to
the same clone, showed the 530-bp deletion. Comparison of a 440-bp sequence from the
promoter region of the leukotoxin gene operon from ten of these strains revealed
complete identity, which indicates that the deletion originates from a single mutational
event. This particular clone was exclusively associated with localized juvenile
periodontitis (LJP). In at least 12 of the 28 families from which the clone was isolated,
more than one family member had LJP. Notably, all the subjects carrying this clone had a
genetic affiliation with the African population.
In another study by Lee et al.
189
(2003), prevalence of severe putative periodontal
pathogens in Korean patients with aggressive periodontitis has been evaluated. The
prevalence was 75% for A.a, 94% for Tannerella forsythensis (formerly named
bacteriods forsythus), 99.4% for Fusobacterium sp, 85.9% for Micromonas micros
39

(formerly named Peptostreptococcus micros), 96.8% for Porphyromonas gingivalis,
78.8% for Prevotella intermedia, and 96.8% for Treponema sp. The prevalence of these
bacteria was significantly higher in diseased sites than in healthy ones. They also found
that P. intermedia were more significantly associated with generalized aggressive
periodontitis in Korean populations.
However, a controversial study by Yasuo Takeushi et al. (2004)
190
did not show
a high prevalence of A.a in Japanese patients with Aggressive Periodontitis. In this study
the prevalence of A.a. was relatively low in localized (20%) and generalized (17.5%)
aggressive periodontitis patients. T. forsythensis, C.rectus, P. gingivalis, and T. denticola
were the predominant periodontopathic bacteria of aggressive periodontitis patients in
Japan. The prevalence and proportion of P. gingivalis correlated with the severity of
clinical attachment loss in both localized and generalized aggressive periodontitis.
In another study by Marta Gajard et al (2005)
191
in a Chilean population,
prevalence of periodontal pathogenic bacteria in Aggressive Periodontitis patients was
different. Result of this study showed Aggressive Periodontitis (AgP) patients had a
higher prevalence of C. rectus than Chronic Periodontitis (CP) patients. Also patients
with AgP showed a higher but not statistically significant prevalence of P. gingivalis, E
Corrodens, P. micros and Capnocytophaga sp. A similar prevalence in both groups of
patients was observed for F. nucleatum and P. intermedia. A. actinomycetemcomitans
was less prevalent in AgP than Chronic Periodontitis patients. In localized AgP, P.
intermedia/ nigrescens, E. corrodens, F. nucleatum and P. micros were the more prevalent
pathogens, in contrast to generalized AgP patients who harbored A.
40

actinomycetecomitans, P.gingivalis, and capnocytophaga sp. as the most prevalent
bacteria.
Analysis of periodontal pathogens in Japanese populations has been done by
Ohnishi (2006)
192
.The prevalence and proportions of Actinobacillus
actinomycetemcomitans, Tannerella forsythensis, Porphyromonas gingivalis and
Treponema denticola were determined in 15 patients with localized aggressive
periodontitis (LAgP), 25 patients with generalized aggressive periodontitis (GAgP) and
28 patients with chronic periodontitis (CP). The prevalence of A. actinomycetemcomitans
in saliva was significantly higher in LAgP patients (46.7%) and GAgP patients (40.0%)
than in CP patients (14.3%). The mean proportion of A. actinomycetemcomitans in LAgP
patients (4.42%) was significantly higher than that in GAgP patients (0.59%) and CP
patients (0.37%) in saliva. In subgingival plaque, LAgP patients showed a significantly
higher mean proportion of T. forsythensis (19.8%) than CP patients (7.45%). A.
actinomycetemcomitans was the more predominant periodontopathic bacteria in LAgP
than in GAgP and CP.
Also demographic microbial aspect of chronic and aggressive periodontitis in
Colombia from five regions of the country has been studied by G. Lafaurie
193
(2007). The
result showed that geographic regions do not influence the microbiota but that the
microbiota may vary by geographic region. P. gingivalis, T. forsythensis and C. rectus
were the most prevalent periodontophatic microorganisms in Colombia. A.
actinomycetecomitans was more common in AgP, and a large percentage of the
population studied had enteric rods in the subgingival plaque.
41

These different findings might be associated with different life style factors, such
as diet, frequency of dental visits, access to health services, consumption and self-
medication of antimicrobials, and water sanitation. They might also be related to race that
needs more studies to be done to establish their status on the differences observed.
Prevalence of A. actinomycetecomitans serotypes in populations from
geographically distant regions showed three predominant serotypes (a, b, c), and a lesser
frequency of two other serotypes (d and e). A study by Slot et al., (1983)
194
and one by
Yang and his coworkers (2004)
195
showed that the proportion of serotype b of
A.actinomycetemcomitans were significantly greater in culture positive patients with
aggressive periodontitits than in those with chronic periodontitis.
Most researchers agree that Actinobacillus actinomycetemcomitans plays an
important role in development of LJP. However, the virulence capacity of A.a and
protective antibodies in LJP patients would not provide sufficient explanation for the
onset of the disease at puberty, rapid periodontal destruction, and the disease predilection
to permanent first molar and incisors. Since the mid 1990s, herpes viruses have emerged
as putative pathogens in various types of periodontal disease. In particular, Human
Cytomegalovirus (HCMV) and Epstein-Barr virus seem to play important roles in the
ethiophatogenesis of severe types of periodontitis. Genomes of the two herpes viruses
occur at high frequency in progressive periodontitis in adults, localized and generalized
aggressive periodontitis, HIV-associated periodontitis, acute necrotizing ulcerative
gingivitis, periodontal abscesses, and some rare types of advanced periodontitis
associated with medical disorders
196
.
42

Studies during the past 10 years have associated herpes viruses with human
periodontitis. Kamma
197
investigated the occurrence of the DNA of HCMV, EBV-1 and
selected pathogenic bacteria in 16 patients with aggressive periodontitis from Greece.
The study revealed that herpes viruses can be detected in some but not others of the
periodontitis lesions of the same individual. HCMV, EBV-1 and HCMV-EBV1 co-
infection were statically associated with disease active periodontitis. Kubar et al.
198

found increased periodontal pocket depth and attachment loss in aggressive periodontitis
sites with HCMV presence, compared to periodontitis sites with a similar degree of
clinical inflammation but with no detectable HCMV. Yapar
199
described a close
relationship between herpes viruses and aggressive periodontitis, detecting HCMV in
65%, EBV-1 in 71% and HCMV-EBV co-infection in 47% of the deep lesions studied.
In aggressive periodontitis lesions, subgingival specimens averaged 4000-10000 HCMV
copies/ml and gingival tissue specimens yielded up to 750,000 copies. They detected a
lower qualitative and quantitative occurrence of herpesviruses in chronic periodontitis
lesions. Michalowicz
200
studied the presence of subgingival HCMV, EBV-1,
P.gingivalis and A.actinomycetemcomitans in 15 adolescents with localized aggressive
periodontitis lesions, 20 adolescents with incidental periodontal attachment loss and 65
randomly selected healthy controls. The study subjects were Africo-Caribbeans living in
Jamaica. The most efficient multivariate model for localized aggressive periodontitis
included HCMV and P.gingivalis. Apparently, HCMV and P.gingivalis are
independently and strongly associated with localized aggressive periodontitis in
Jamaican adolescents, and the two infectious agents seem to act synergistically to
43

influence the risk for both the occurrence and the severity of the disease. Ting et al.
201

studied the relationship between HCMV activation and aggressive Juvenile Periodontitis
in subjects between the ages of 10 and 23 years living in Los Angeles. The study found
HCMV activation and disease-active vs. disease-stable periodontitis in 11 patients with
aggressive Juvenile Periodontitis. The study found HCMV reactivation in some and
HCMV latency in other periodontal sites of the same patients, pointing to site specificity
in the oral HCMV transcription state. Periodontal sites demonstrating HCMV
reactivation also tended to exhibit elevated levels of A.actinomycetemcomitans,
apparently major pathogens of the disease; HCMV activation together with A.a
constitutes a significant pathogenic feature of the localized aggressive periodontitis
lesions in U.S. patients. To explain the discrete nature of tissue breakdown in localized
aggressive periodontitis, it is hypothesized that an active HCMV infection in tissue
surrounding the tooth damages the root surface structure during the time of root
formation of permanent incisor and first molars at 3-5 years of age. HCMV infections of
infants are known to have the potential to cause changes in tooth morphology, and teeth
affected by localized aggressive periodontitis frequently show cemental hypoplasia. It is
further hypothesized that localized aggressive periodontitis patients experience
reactivation of periodontal herpes viruses due to puberty-related hormonal changes, the
effect of which may be the overgrowth of resident periodotopathic bacteria and
subsequent tissue breakdown occur more frequently and progress more rapidly in
herpesvirus infected than in herpesvirus-free periodontal site. Herpesvirus may cause
periodontal pathosis as a direct result of virus infection and replication, or as a
44

consequence of virally induced impairment of the periodontal immune defense, resulting
in heightened virulence of resident bacterial pathogenesis (Slots, 2000)
196
. It is assumed
that the ability of herpesviruses to express cythopatogenetic effects, immune evasion,
immunopathogenicity, latency, reactivation from latency, and tissue tropism could be
relevant for the development of periodontal disease.
Immunology
The role of the immune mechanisms in the pathogenesis of periodontal disease
has been extensively investigated. Lymphocyte and plasma cells predominate in the
periodontium in established or advanced disease, and the central role for these cells in the
pathogenesis has been postulated (Page & Schroder, 1982)
202
.

However, neutrophils are
present through all four stages of periodontal lesions. These phagocytic cells with their
production of lysosomal enzymes and metabolic prouducts usually play a protective role
in periodontal tissue. Defects in neutrophil function may explain severe periodotitis in
young adults.
PMNs are the frontline in the acute host response against bacterial infection. Their
functions can be categorized as adherence, chemotaxis, phagocytosis, and microbicidal
activity. Prior to adherence, cells migrate to the site of attack through the bloodstream.
This migration is facilitated by several factors such as complement (C5a), interleukin-8
(IL-8), leukotrienes, and bacterial antigens. Extravasation of PMNs through the
endothelium of the capillary wall takes place in three steps: (a) initial rolling of PMNs along
the endothelium; (b) PMN activation and adhesion; and (c) transendothelial migration
(Adams et al., 1994)
203
. PMN rolling is mediated by selectins. Of these sialylated
45

carbohydrate determinants, L-selectin is expressed on the surface of neutrophils and on
other leukocytes, E-selectin on endothelial cells, and P-selectin on platelets and
endothelium (Hardwell)
204
. Through the interaction of selectins, PMNs not only roll in the
blood vessel, but also binds to the endothelial surface
205
. PMN adherences to the
endothelium takes place at the endothelial interface through specific molecules located on
both cell types. While beta-2 integrins that include a common beta-2 chain (CD18) and one
of three separate alpha chains (GDI la, CDllb, and CDllc) regulate the PMN adherence
(Arnaout MA, 1990)
206
, the principal endothelial cell ligand counterpart is an intercellular
adhesion molecule (1CAM-1). Following adherence, PMNs migrate to the site of injury. This
event is defined as chemotaxis and is mediated by specific chemotactic molecules.
Complement proteins play a major role in chemotaxis
207
. When PMNs arrive at the site of
injury, they recognize specific molecules on the surface of the invader. This recognition is
mediated by molecules called opsonins and takes place prior to the phagocytosis of the
foreign body via invagination of the plasma membrane. Phagocytosis is facilitated if
microbes are coated with opsonic proteins such as immunoglobulins, complement
fragment C3b, and binding lectin, for which the neutrophil expresses multiple specific
receptors. Phagocytosis is mediated by a separate and distinct receptor on the cell surface.
These chemotaxis receptors; N-Formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP)
and C5a and phagocytosis receptors (C3b) mediate other cell functions, such as specific
granule release and superoxide anion production. Phagocytosed microorganisms are killed
by the PMNs via 2 types of microbicidal mechanisms: non-oxidative and oxidative killing.
In non-oxidative killing, hydrolases from the granules in the PMNs are secreted. These
46

molecules are essential elements of non-oxidative activity. The respiratory burst is an
important pathway for microbial killing and involves the generation of superoxide,
hydrogen peroxide, hydroxyl radical and, subsequently, hypochlorous acid and
chloramines. Degranulation involves release of the contents of the cell granules and
contributes to "oxygen-independent bactericidal activity." The enzymes associated with
the oxidative burst leading to hydrogen peroxide production, in conjunction with
myeloperoxidase and halide ions, are responsible for oxidative killing
208
.
Lactoferin (LF) is a protein found in nutrophils that is located exclusively in the
specific granules. Lactoferin has been shown to be bactericidal and enhance hydroxyl
radical production in neurophil particulate fraction and in an enzymatic H2O2-generating
system. Patients lacking specific granules and LF are subjects to recurrent infections
209
,
including abnormalities in FMLP-stimulated chemotaxis, adherence, aggregation, ability
to decrease cell-surface charge, and impaired hydroxyl radical production in response to
phagocytois. Superoxidase anion is a potentially bactericidal agent as are other unstable
oxygen intermediates produced by neutrophils upon stimulation of chemotactic (FMLP)
and phagocytic (C3b) receptors.
The best-characterized periodontal disease with impaired neutrophil function is
LAgP. Defect in neutrophil functions may explain aggressive periodontitis in young
adults. The observations of a study by Suzuki et al.
210
(1984) indicated that patients with
Juvenile Periodontitis have abnormalities in the neutrophil functions of chemotaxis,
phagocytosis and spore germination.
47

Patients with Localized Juvenile Periodontitis (LJP) exhibit defective neutrophil
functions for a variety of environmental and host stimuli. In a study of 23 patients with
LJP by Van Dyke et al. (1986)
211
, the results indicated that all 23 patients exhibited
chemotaxis depression to N-formyl-methionyl-L-leucyl-L-Phenylalanine (FMLP) and
Endotoxin- Activated Serum (EAS). Also fourteen of the 14 LJP patients tested exhibited
defective phagocytosis. But both granule release and superoxidas production was normal.
Occupancy of chemoattractant receptor on PMNs by ligand results in the
utilization of a guanine nucleotide regulatory (G) protein and phospholipase C to cleave
phosphotidylinositol 4, 5-biphosphate into intracellular messengers, 1, 2-diacylglycerol
and inositol 1, 4, 5- biphosphate, which mobilize intracellular Ca2+. This in turn activates
protein kinase C and leads to eventual cell activation
212, 213
.

Therefore, relative changes in
any of the second messengers, such as Ca
2+
, inositol triphosphate, or diacylglycerol can
be used as a measure of postreceptorial signal transduction. A study by Agrawal (1989)
214

showed that lower chemotactic response in PMNs from JP patients could be associated
with a defect in intracellular signal transduction, as measured by stimulus-induced
changes in free calcium (Ca
2+
) mobilization. FMLP stimulation induced higher levels of
free cytosolic Ca
2+
mobilization in PMN cells from healthy control subjects as compared
to LJP patients. Also, the putative effect or mechanisms for chemotactic receptor of
neutrophils include the possible activation of phospholipase, protein kinase c,
methyltransferase, or adenylate cyclase.
The intracellular transduction mechanisms that follow receptor-ligand coupling on
the neutrophil surface and which lead to chemotaxis are not clearly established.
48

Chemotaxis and phagocytosis are modulated by a variety of receptors and involve
several activation pathways; the role of intracellular calcium as a presumptive second
messenger and as a mediator of these events is well established
215
. The putative effector
mechanisms for the chemotactic receptor of neutrophils also include the possible
activation of a phospholipase, protein kinase C, methyltransferase, or adenylate cyclase.
In normal neutrophils, a phosphoinositide pathway initiated by phospholipase C, which
results in the activation of protein kinase C via diacylglycerol and the generation of IP3,
has been implicated.
216
A study by Daniel et al. (1993)
217
showed that the early phase of
the calcium response affiliated with the release of intracellularly sequestered calcium
appears intact in LJP neutrophils. However, the second phase of calcium response,
associated with membrane channel activation and an influx of extra cellular calcium, is
comprised in the neutrophils of the LJP population.
A study by Cogen et al. (1993)
218
showed that neutrophil defects in LJP patients
are under genetic control. They compared thirteen LJP patients and GJP and their
matched healthy controls with respect to selected leukocyte functions and HLA (genes at
MHC) phenotypic frequencies. The result showed that all JP patients displayed intrinsic
cell defects in chemotaxis compared with control; also there appeared to be a significant
association between JP patients with HLA-DR2 and HLA-A33 phenotypes. Fifty percent
of the JP patients were HLA-DR2 positive, whereas only six percent of the matched
controls were positive. Thirty-six percent of JP patients were HLA-A33 positive, whereas
none of the controls were positive.
49

Protein kinase C (PKC) is a key molecule in neutrophil signal transduction after
receptor stimulation by soluble bioactive molecule. PKC activity in neutrophils has been
reported to be functionally related to chemotaxis, lysosomal enzyme release, respiratory
burst, and hexose transport. Investigation of the signal transduction pathways in
neutrophils from patients with LJP has revealed lowered activity of diglyceride kinase
and high intracellular levels of diacylglycerol (DAG), the natural stimulator for PKC
220
.
The high DAG level might affect PKC activity in neutrophils from patients with LJP.
Neutrophils treated with phorbol 12-myristate 13-acetate (PMA), a potent PKC
stimulator, demonstrated low chemotaxis in vitro in most
221, 222
, but not all
223
studies. The
investigation of PKC activity in neutrophils from patients with LJP and depressed
chemotaxis is relevant not only to the pathogenesis of LJP but also to the role of PKC in
neutrophil chemotaxis. Kurihana et al. (1998)
224
evaluated the Calcium-dependent
protein kinase c activity of peripheral blood neutrophils from 12 patients with LJP. The
calcium-dependent protein kinase c activity was found to be lower from patients with LJP
than from healthy subjects.
Locomotion of neutrophils to extraellular space is facilitated by a family of
adhesion molecules
225
. Three distinct, but structurally and functionally related, adhesion
molecules belonging to CD11/CD18 family of proteins have been characterized on
neutrophils. Mac-1 molecules, identical to complement receptor 3 (CR3) are present on
monocytes and neutrophils and mediate adhesion, migration and phagocytsis
226
.
Leukocyte function-associated antigens (LFA-1) are present primarily on monocytes,
neutrophils and lymphocytes and natural killer cells mediated killing and in adherence of
50

immune cells to endothelium.
227
The third type of adhesion molecules, the p150, 95 is a
complement receptor. All three adhesion molecule (Mac-1, LFA-1 and p150, 95) have
been shown to be present in the intracellular vesicular compartment as well as on the cell
surface
228
. Inflammatory mediators such as chemoattractants and tumor necrosis factor-a
(TNFa) interleukin 1B(IL-1B) stimulate a 5 to 10 fold increase in Mac-1, LFA-1 and
p150, 95 expression on the surface of neutrophils
229
. The upregulation of adhesion
molecules from the intracellular vesicular compartment to the cell surface on neutrophils
is rapid and does not require protein synthesis.
230
Agrawal and his coworkers (1994)
231

showed that the neutrophils from localized Juvenile periodontitis patients with decreased
chemotaxis and increased adherence exhibit increased expression of the CD11/CD18
family of adherence molecules, Mac-1, leukocyte function-associated antigen (LFA-1)
and P150, 95, as compared with neutrophils obtained from systemically and periodontally
healthy controls. Pretreatment of localized juvenile periodontitis serum with anti-tumor
necrosis factor and anti-interleukin-1 antibodies was able to at least partially inhibit
induction of adherence molecules in these patients.
Adhesion of circulating neutrophil to capillary endothelium is an early key event
in a sequence of reactions which allows neutrophils to leave the circulation. The initial
adhesion or rolling of leukocytes over the endothelium is mediated by the neutrophil L-
selection
232
. The firm adhesion and the subsequent extravasion is facilitated by the
adhesion receptors of the B
2
integrin family, of which the CD11b/ CD 18 (CR3 or Mac-1)
is perhaps the most important in human neutrophils
233
.
51

Enhanced adhesion of neutrophils to endothelial cells contributes to vascular and
tissue injury and also inhibits migration of neurophils to the infection site. A study by
Huritall et al. (1998)
234
suggested that a defect in diacylglycerol (DAG) kinase activity
could lead to neutrophil anomalies, which then, in turn, would promote the development
of the periodontal disease in a part of the LJP patient’s population. DAG-kinase is an
upstream regulator of protein kinase c (PKC). Due to inappropriate PKC activation by
uncontrolled accumulation of endogenous DAG, neutrophil would become hyperadherent
and incapable of properly migrating to the site of infection and may promote the
development of LJP by causing tissue damage in the periodontium.
Nitric Oxide (NO) and its enzyme, Nitric oxide synthase (NOS), have been
suggested to be involved in chemotaxis. NO is a gas with diverse biological activities
produced from L-arginine by nitric oxide synthase (NOS). NO induces cyclic guanosine
3
/
, 5
/
-monophosphate (cGMP) production via guanylate cyclase activation. Various
important roles for NO in immune regulation and inflammation have been suggested
235
.
NOP activity and NOS mRNA, and NOS protein has been detected in human
neutrophils
236
. Intracellular accumulation of cGMP has been suggested to regulate
neutrophil chemotaxis
237
. NO, NOS, N-nitro-L-arginine methyl ester (L-NAME), a NOS
inhibitor, have been suggested to be involved in neutrophil chemotaxis. It is known that
NO is involved in acute and chronic inflammation
238
. In a study on 10 Localized
Aggressive Periodontitis patients (LAgP), Kazuyki and his coworkers suggested that
NOS is present in human neutrophil and may be involved in FMLP-induced chemotaxis
52

in normal neutrophils because NOS was increased in LAgP and was negatively correlated
to chemotaxis response.
Until recently, defects associated with neutrophil functions were believed to
predispose to infection. However there is a growing body of data implying that this
presumption may not to be valid. Under new paradigm, neutrophil abnormalities in LAP
are the result of a chronic hyperactivated neutrophil. Based on recent studies, neutrophils
are not hypofunctional or deficient, they are hyperfunctional, and their amplified activity
is responsible for the tissue destruction in periodontal disease (Alpdogan Kantarci et al.,
2003)
239
. From the extensive literature available it seems that any impairment neutrophil
function will lead to some degree of increased susceptibility to infection. Periodontium is
perhaps the tissue most sensitive to pathological changes in oral cavities. In the cases of
severe neutrophil dysfunction, there is also a severe periodontal breakdown. However, in
cases of mild neutrophil dysfunction, where there is no other infection, such as in patients
with LAP, there is a severe periodontal breakdown. Hyperresponsiveness of the
neutrophil, due to cell priming/predisposition, results in enhanced tissue damage. Studies
demonstrating the familial nature of both the neutrophil chemotactic disorder and clinical
entity represented by LAP, point to a strong role of genetic determinants in this disease.
The analysis of lymphocyte subpopulations in blood and other body fluids or
tissues is a well-established method for characterizing immunological profiles in various
infectious diseases. In addition, in patients with periodontal diseases, analyses have been
carried out to investigate the proportions of lymphocyte subpopulations in the peripheral
blood
240
as well as in biopsies of periodontal tissue
241
. Little is known, however, about
53

the proportions of crevicular lymphocytes in different forms of periodontitis. In contrast,
there are extensive data on polymorphonuclear leukocytes.
It is generally accepted that the crevice is an important region of the local host
defense
242
. The occurrence of different lymphocyte patterns in this area has been little
investigated. Although different studies on crevicular fluid cells report the presence of
lymphocytes, to date the crevicular lymphocyte subpopulations have not been
examined
243
.
It is known that the healthy gingiva contains a CD4
+
/CD8
+
ratio comparable with
that of the peripheral blood
244
. However, a reduced CD4
+
/CD8
+
ratio has been described
in periodontal lesions, compared with that of the peripheral blood
245
. Several researchers
assume that T lymphocytes are predominant in gingivitis, whereas B-cell dominance
develops during the formation of a periodontal lesion
246
. Kinane and his coworkers
(1989)
247
, in a study of 12 patients with aggressive periodontitis found a decreased ratio
of helper (CD4
+
) to suppressor (CD8
+
) T cell compared to the periodontally healthy
control. Several researchers assume that T lymphoctes are predominant in gingivitis,
whereas B-cell dominance develops during the formation of periodontal lesion
246, 248
.
Pietruska et al. observed T lymphocytes more frequently in aggressive periodontitis
lesions and B cells more frequently in generalized chronic periodontitis lesions
249
. Suarez
et al.
250
, who compared periodontally healthy patients to patients with aggressive
periodontitis, employing immuno-histochemistry and reverse transcription–polymerase
chain reaction (RT–PCR) for cytokines, reported that the tissue of patients with
aggressive periodontitis contains fewer CD3
+
and CD4
+
cells, but only a minimal
54

difference in the number of CD8
+
cells was observed between the groups
250
. They
proposed that the role of CD8
+
cells in aggressive periodontitis lesions should be
investigated, and Petit et al. also demand further research on the role of CD8
+
cells
251
. In
the study by Sigusch and his coworkers (2006)
252
, patients with LAP and patients with
GAP were found to have increased numbers of crevicular T-suppressor/cytotoxic and B
cells. This supports the hypothesis of a changed immune pathology in patients with
aggressive periodontitis.
Remarkable aspects of the biology of aggressive periodontitis include the
production of very high concentrations of serum antibodies reactive with a limited
number of periodontal disease pathogens and the increased concentrations of IgG found
in the sera of such patients. Both the high levels of antibodies specific for Actinobacillus
actinomycetemcomitans and Porphyromonas gingivalis and elevated IgG, especially
IgG2, serum concentrations have been observed by several investigators. Initial studies
by Ranney et al.
253
of the relationships between the antibody response to
A. actinomycetemcomitans and clinical status led to the hypothesis that the presence of
such an antibody in patients with aggressive periodontitis protects them against
periodontal attachment loss. Assessment of the presence or absence of a precipitating
antibody reactive with A. actinomycetemcomitans strains Y4 and N27 showed that the
patients with aggressive periodontitis who had precipitating antibody present had lower
numbers of teeth with severe (≥ 5 mm) or moderate (≥ 2 mm) attachment loss. In addition,
a higher proportion of patients with localized disease than with generalized disease
demonstrated such antibodies. In a subsequent study, concentrations of serum antibodies
55

to 25 bacterial strains commonly cultured from subgingival plaque, measured by
radioimmunoassay, were assessed in 99 patients with aggressive periodontitis, and
associations with loss of attachment were examined
254
. Eleven of these strains were
found to be positively or negatively associated with attachment levels. After correction
for plaque score and age, strong inverse associations were found between attachment loss
and antibody levels for both A. actinomycetemcomitans and P. gingivalis in both patients
with localized aggressive periodontitis, and in those with generalized aggressive
periodontitis. Furthermore, the combination of antibody reactive to both
A. actinomycetemcomitans and P. gingivalis was the most favorable with respect to
attachment loss in aggressive periodontitis. In a further analysis of the value of antibody
concentrations in predicting or differentiating periodontal diagnoses, Gunsolley et al.
255
observed that antibody responses to five bacterial strains (A. actinomycetemcomitans
strains Y4 and N27, P. gingivalis, Fusobacterium nucleatum and Eubacterium brachy)
could discriminate between patients with localized aggressive periodontitis, generalized
aggressive periodontitis, chronic periodontitis, and periodontal health. However, the
generalized aggressive periodontitis group was both clinically and immunologically
heterogeneous; a group of patients with the most attachment loss had no antibody
reactive with the five organisms. These results strongly suggested that an antibody
reactive with periodontal disease pathogens, particularly with A. actinomycetemcomitans
and P. gingivalis, may protect patients with existing aggressive periodontitis from
progressive attachment loss.
56

In a review of the apparent protective effect of high antibody concentrations,
studies carried out by Califano et al.
256, 257
attempted to define the antigens of
A. actinomycetemcomitans that induced protection against progression of periodontitis in
patients with existing aggressive disease. Initial studies defined this antigen in
A. actinomycetemcomitans strain Y4 as the serotype-specific carbohydrate. Later studies
revealed that a carbohydrate antigen was the immunodominant antigen for
A. actinomycetemcomitans serotypes b and c. These carbohydrate antigens were later
found to be contained in the O-antigen of A. actinomycetemcomitans lipopolysaccharide.
The nature of the antibody response to these antigens was explored by Lu et al.
258
,
who determined the isotype and subclass responses of aggressive periodontitis to the
immunodominant carbohydrate antigen of A. actinomycetemcomitans Y4 (serotype b).
Using a limiting dilution Western blot analysis they determined that the dominant
response to A. actinomycetemcomitans serotype b was an IgG response against the
immunodominant carbohydrate identified by Califano and his coworkers
256, 257
.
Furthermore, it was shown that the IgG response was predominantly an IgG2 response,
with IgG2-specific antibody concentrations of 65.7 μg/ml far exceeding the mean IgG1
antibody concentration of 8.8 μg/ml. Later studies by Califano et al.
258
examined the
antibody response to the serotype-specific carbohydrate antigens of P. gingivalis. They
showed that generalized aggressive periodontitis patients, as well as chronic periodontitis
patients, produced high serum IgG2 antibody responses to one or more of the serotype-
specific antigen K1 through K6.
57

Relationships between the antibody response to specific antigenic virulence
factors, specifically the IgG2 response, and clinical measures were also studied. Since
antibody to A. actinomycetemcomitans was previously found to be associated with less
attachment loss in aggressive periodontitis, and the dominant antibody response to
A. actinomycetemcomitans was to the serotype-specific carbohydrate of the O-antigen,
Califano et al.
259
explored the impact of antibody reactive with
A. actinomycetemcomitans lipopolysaccharide on attachment loss in patients with
generalized aggressive periodontitis. They first demonstrated that patients with the
highest concentration of serum antibody reactive with a whole-cell antigen preparation of
A. actinomycetemcomitans had less attachment loss than those with intermediate or low
antibody concentrations. They further observed that the subset of patients with
generalized aggressive periodontitis who had the highest serum concentrations of anti-
serotype b lipopolysaccharide also had less attachment loss than those with intermediate
or low levels, and that these subject groupings were identical to those in the patient
groupings defined by their response to the whole-cell antigen preparations. In addition,
the antilipopolysaccharide avidity was higher in the patients with the highest antibody
concentrations. Since the vast majority of these antibodies are of the IgG2 subclass, these
data argue for a role for IgG2 as protective in generalized aggressive periodontitis.
Further studies were carried out to examine the role of antibody responses to two
additional A. actinomycetemcomitans virulence factors, leukotoxin and hemagglutinin
256,
257
. The leukotoxin response was dominated by IgG1 antibody while the hemagglutinin
response was mainly IgG1 and IgG3. In both cases, these responses were only weakly
58

associated with attachment levels or not associated at all. In their study, Takeuchi and his
coworkers 2006)
260
demonstrated that P. gingivalis infection elicited an IgG subclass
antibody response in all groups of periodontal disease (localized aggressive, generalized
aggressive and chronic periodontitis); however a significant increase of IgG2 level was
not observed in localized aggressive periodontitis. Anti-fimbriae IgG sub class levels of
IgG1, IgG2 and IgG4 did not differ among bacterium-positive subjects in all groups,
while the anti-fimbriae IgG3 level in generalized chronic periodontitis was significantly
higher than that in localized and generalized aggressive periodontitis.
These studies suggested an important role for antibodies reactive with
carbohydrate antigens in the response to the aggressive periodontitis microbial pathogens
A. actinomycetemcomitans and P. gingivalis. Furthermore, they also suggest that the
IgG2 response that is typical against carbohydrate antigens may be important in the
protective immune response in aggressive periodontitis.
It is likely that AgP has a complex etiology, with interactions of multiple
susceptibility genes and environmental factors. It is difficult to explain the pathogenesis
of early onset periodontitis based on a single risk factor.
In spite of a relatively rare occurrence, Localized Aggressive Periodontitis has
been the focus of the many investigations aimed at understanding its etiology and
pathogenesis. There are many studies about the genetic basis, immunology and bacterial
characteristics of this disease. But these studies do not seem to provide sufficient
explanation for the onset of the disease at puberty, the remarkably rapid periodontal
59

destruction, and the disease predilection to permanent first molars and incisors. Finding
answers for these questions warrants more research.
60

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81

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82

Chapter 2: Methods and Materials
Objective
The objective of this study is to explore underlying mechanisms involved in
selective sites of infection and the subsequent inflammatory process in Localized Aggressive
Periodontitis (LAP). The presence of major periodontal pathogens, Cytomegalovirus,
cytokines such as TNFa, adhesion molecules such as ICAM1, and NFKb have been
evaluated in sites with deep pockets and bone loss, and in healthy sites in same
individuals with this disease.
Research Questions
1) Are there any differences in inflammatory processes (such as the presence of
NFKb, ICAMI, and TNFa) in sites affected by LAP when compared to non-affected sites
in the same individual?
2) Could LAP be a site specific disease?
Hypothesis
Periodontitis is characterized by cellular infiltration into the deep pockets of gum
tissue, leading to inflammatory lesions, which are mediated by the activated immune
response to microbial agents. In LAP preferentially this pathogenesis has a tendency to
present itself at a selective site. Upregulation of various components such as adhesion
molecules; ICAM1 plays a critical role in recruitment and extravasation of circulating
immune cells at the site of infection to initiate the inflammatory process. Subsequently,
inflammatory cells undergo activation to produce cytokine (i.e. TNFa) and to activate
nuclear proteins, such as the NF-KB/rel complex, for further expression of adhesion
83

molecules and cytokine and other biologically active molecules which damage the
surrounding tissue. Over expression of P65, not P50, activates the ICAM1 transcription.
Whereas P65 mediated transactivated ICAM-1 is suppressed when is co expression of
P50.
Based on this information we hypothesize that there is upregulation of gene PP65
with active expression of adhesion molecules at inflammatory sites. In contrast, in normal
sites with healthy gums we expect to find minimal or no adhesion molecule expression.
Alternatively, homodimmer P50 or P65 or heterodimmer P65/50 are differentially
expressed at different sites of the gums. In such a manner the lesion site is associated with
P65 and adhesion molecules whereas normal sites are associated with P50 or P65/50
heterodimmer, with minimal or no adhesion molecule expression.
This finding may be of value in early diagnosis, prevention of inflammatory
process, as well as treatment of early lesion using specific antibody to block NFKb and
associated adhesion molecule to suppress leukocyte infiltration and periodontitis
development.
Patients
Ten LAP patients participated in this study (3 male and 7 female), with ages
ranging from 13-32 years. Patients were systemically healthy and had not received
antibiotic therapy in the six months prior to the study. Medical history and demographic data
were determined by chart review and patient interview.
Diagnosis of Localized Aggressive Periodontitis was based on full mouth
periodontal probing and analysis of the alveolar bone level on the periapical and
84

interproximal radiographs. Each patient revealed at least two permanent first molars or
incisors with periodontal probing depth of 5 mm or more.
Sample Collection
Samples were taken from one tooth with the deepest pocket for a test site and one
tooth with healthy periodontium for a control site from each individual. Before sampling,
supragingival plaque was removed with sterile cotton pellets. Six paper points were inserted
to the depth of the periodontal test site and retained for 40s. For controls, pooled paper point
samples were obtained from healthy sites of the same individual in the same manner.
Sampling was been done twice. The first samples were transferred to 2 ml vial containing
VMGA III transport medium for bacterial culture. The second samples were placed into
separate empty sterile silicon plastic vials and have been frozen in minus 70
o
C until will
be ready for Real Time Polymerase Chain reaction (RT- PCR) processing .
Laboratory Procedure
Bacterial culture
Anaerobic microbiological isolation and identification of putative periodontal
pathogens were carried out with no knowledge of the source of the specimens, following
previously described procedures. Microorganisms were mechanically dispersed from the
paper points with a Vortex mixer at the maximal setting for 45 seconds, and were then
10-fold serially diluted in VMG I anaerobic dispersion solution. Using a sterile bent glass
rod, 0.1 ml aliquots from 10
3
to 10
5
dilutions were plated onto non-selective 4.3%
brucella agar supplemented with 0.3% bactoagar, 5% defibrinated sheep blood, 0.2%
hemolyzed sheep red blood cells, 0.0005% hemin, and 0.00005% menadione. Total
85

viable counts and proportions of specific bacteria in relationship to the total viable counts
were determined. Aliquots diluted in VMGA III medium were plated onto TSBV
medium for culture of A. actinomycetemcomitans, enteric Gram-negative rods, and
yeasts. The non-selective blood agar was incubated at 35°C in an anaerobic chamber
containing 85% N2-10% H2-5% CO2 for 10 days. TSBV medium was incubated in 10%
CO2 in air at 35°C for 4 days. Presumptive identification of representative colonies of
each group of organisms that morphologically resembled the study species was
performed according to the methods described by Slots

and by the use of a micromethod
system. Organisms examined included A. actinomycetemcomitans, P. intermedia, P.
gingivalis, Tannerella forsythia, Campylobacter species, Fusobacterium species, M.
micros, enteric Gram-negative rods, and Candida species. Bacteria designated as major
periodontal pathogens included A.actinomycetemcomitans, P.gingivalis, D.pneumosintes
and T.forsythia. The percentage recovery of periodontal pathogens was determined by the
colony count of each microbial taxon in relation to total viable count.
Real Time Polymerase Chain reaction (RT- PCR)
Samples were frozen at -70
o
C and carried on dry ice to the laboratory for
processing. Cell lysing has been done by using the QIAGEN RNeasy Plus Microkit,
containing Mercapto Ethanol to inhibit RNAase activity (Valencia, CA). Subsequently,
nucleic acid cell lysate was treated with equal volumes of cold 70% Ethanol, and then
placed in a QIGEN RNeasy spin column and centrifuged at 8000 x g to immobilize Ribo
Neucleic Acid (RNA). Demmoblized neucleic acid was washed 3 times with QIAGEN
RNeasy buffers, to remove DNA and Proteins from the columns and centrifuged at 1000
86

x g for 1 minute. This was followed by cDNA preparation from the total RNA Template.
With this regard RNA Template was mixed with a QIAGEN RT primer mix containing
revese transcriptase, oligodt primer, nucleotides (Timidin, Cytosine, Guanine and
Adenine) and Magnesium Chloride. Then the mixed samples were incubated 15 minutes
at 42
o
C and 3 minutes at 95
o
C. Each prepared cDNA (3 micro liters) was mixed with A.a,
ICAM1, CMV, NFKb, TNFa and beta actin primers (2 micro liters), 10 microliters of
water, and 15 micro liters of master mix (containing DNA polymerase, nucleotide, Mg
Chloride and cyber green).
To amplify the template, samples were placed in Real Time PCR (Bio Rad I-
Cycler) with the following program;
Initial step: 50
o
C for 2 minutes.
Second step: 95
o
C for 15 minutes to eliminate any carry over Dump
contamination and activate polymerase respectively.
Third step: 94
o
C for 15 seconds and 54
o
C for 30 seconds and 72
o
C for 30 seconds
for denaturation, annealing and elongation respectively.
Also Beta actin has been used as housekeeping positive control and corresponding
negative control without primer.
Statistical Methods
Descriptive statistics were calculated for all study variables. The results of each
PCR test was classified as either not present, not significant, or significant. A 3x2
contingency table analysis by group (test or control tooth) was used to test the association
between PCR result and group, using the chi square statistic. A Bonferroni correction was
87

used to control for inflation of experimentwise error, so alpha = .01. A Mann-Whitney U
nonparametric test of group differences was conducted on the 14 cultures taken by group.
A Bonferroni correction was used to control for inflation of experimentwise error, so
alpha=.0036.
88

Tables: Demographics
Table 1: Patients ID
Frequency Percent Valid Percent
Cumulative
Percent
Valid Patient 1
1 10.0 10.0 10.0
patient 10
1 10.0 10.0 20.0
patient 2
1 10.0 10.0 30.0
patient 3
1 10.0 10.0 40.0
patient 4
1 10.0 10.0 50.0
patient 5
1 10.0 10.0 60.0
patient 6
1 10.0 10.0 70.0
patient 7
1 10.0 10.0 80.0
patient 8
1 10.0 10.0 90.0
patient 9
1 10.0 10.0 100.0
Total
10 100.0 100.0

Table 2: Patients Sex
Frequency Percent Valid Percent
Cumulative
Percent
Valid Female
7 70.0 70.0 70.0
Male
3 30.0 30.0 100.0
Total
10 100.0 100.0

Table 3: Patients Age
Frequency Percent Valid Percent
Cumulative
Percent
Valid 15
1 10.0 10.0 10.0
16
1 10.0 10.0 20.0
17
3 30.0 30.0 50.0
22
1 10.0 10.0 60.0
28
1 10.0 10.0 70.0
30
1 10.0 10.0 80.0
32
2 20.0 20.0 100.0
Total
10 100.0 100.0

89

Table 4: Patients Race
Panel A
Frequency Percent Valid Percent
Cumulative
Percent
Valid Asian
1 10.0 10.0 10.0
Black
5 50.0 50.0 60.0
Hispanic
4 40.0 40.0 100.0
Total
10 100.0 100.0

Panel B

PCR Analysis == Collapsed Coding for Clinically Significant Values

Group

Frequency Percent Valid Percent
Cumulative
Percent
Valid Test
10 50.0 50.0 50.0
Control
10 50.0 50.0 100.0
Total
20 100.0 100.0

90

Tables: PCR Analyses
Table 5: PCR Analysis for A.a
Panel A
Frequency Percent Valid Percent
Cumulative
Percent
Valid .0 Not present
19 95.0 95.0 95.0
1.0 Not clinically
significant
1 5.0 5.0 100.0
Total
20 100.0 100.0

Panel B

PCR Analysis == Collapsed Coding for Clinically Significant Values

rA.a

Frequency Percent Valid Percent
Cumulative
Percent
Valid .00 Not present
19 95.0 95.0 95.0

1.00 significant
1 5.0 5.0 100.0
Total
20 100.0 100.0

Table 6: PCR Analysis for CMV
Panel A

Frequency Percent Valid Percent
Cumulative
Percent
Valid .0 Not present
20 100.0 100.0 100.0

Panel B

PCR Analysis == Collapsed Coding for Clinically Significant Values
rCMV

Frequency Percent Valid Percent
Cumulative
Percent
Valid .00 Not present
20 100.0 100.0 100.0

91

Table 7: PCR Analysis for NFKb
Panel A

Frequency Percent Valid Percent
Cumulative
Percent
Valid .0 Not present
4 20.0 20.0 20.0
1.0 Not clinically
significant
5 25.0 25.0 45.0
21.0
1 5.0 5.0 50.0
23.5
1 5.0 5.0 55.0
27.0
1 5.0 5.0 60.0
28.0
1 5.0 5.0 65.0
28.2
1 5.0 5.0 70.0
28.8
1 5.0 5.0 75.0
30.9
2 10.0 10.0 85.0
31.0
1 5.0 5.0 90.0
31.1
1 5.0 5.0 95.0
32.6
1 5.0 5.0 100.0
Total
20 100.0 100.0

Panel B

PCR Analysis == Collapsed Coding for Clinically Significant Values
rNFKb

Frequency Percent Valid Percent
Cumulative
Percent
Valid .00 Not present
4 20.0 20.0 20.0
1.00 Not significant
5 25.0 25.0 45.0
2.00 significant
11 55.0 55.0 100.0
Total
20 100.0 100.0

92

Table 8: PCR Analysis for TNFa
Panel A

Frequency Percent Valid Percent
Cumulative
Percent
Valid .0Not present
20 100.0 100.0 100.0

Panel B

PCR Analysis == Collapsed Coding for Clinically Significant Values
rTNFa

Frequency Percent Valid Percent
Cumulative
Percent
Valid .00Not present
20 100.0 100.0 100.0

93

Table 9: PCR Analysis for ICAM1
Panel A

Frequency Percent Valid Percent
Cumulative
Percent
Valid .0Not present
6 30.0 30.0 30.0

1.0Not significant
3 15.0 15.0 45.0
28.5
1 5.0 5.0 50.0
28.7
1 5.0 5.0 55.0
31.1
1 5.0 5.0 60.0
31.6
1 5.0 5.0 65.0
31.7
1 5.0 5.0 70.0
32.4
1 5.0 5.0 75.0
32.8
2 10.0 10.0 85.0
33.0
1 5.0 5.0 90.0
35.8
1 5.0 5.0 95.0
37.6
1 5.0 5.0 100.0
Total
20 100.0 100.0

Panel B

PCR Analysis == Collapsed Coding for Clinically Significant Values

rICAM_I

Frequency Percent Valid Percent
Cumulative
Percent
Valid .00Not present
6 30.0 30.0 30.0
1.00Not significant
3 15.0 15.0 45.0
2.00Clinically significant
11 55.0 55.0 100.0
Total
20 100.0 100.0

94

Tables: Crosstabs
rA.a * Group: No significant association between A.a category and “tooth.”
Table 10: Crosstab for A.a.
Panel A

Group
Total 1.0Test 2.0Control
rA.a .00Not present Count
9 10 19
% within rA.a
47.4% 52.6% 100.0%
% within Group
90.0% 100.0% 95.0%
1.00Not
significant
Count
1 0 1
% within rA.a
100.0% .0% 100.0%
% within Group
10.0% .0% 5.0%
Total Count
10 10 20
% within rA.a
50.0% 50.0% 100.0%
% within Group
100.0% 100.0% 100.0%

Panel B

Chi-Square Tests

Value Df
Asymp. Sig.
(2-sided)
Exact Sig.
(2-sided)
Exact Sig.
(1-sided)
Pearson Chi-Square
1.053(b) 1 .305
Continuity Correction(a)
.000 1 1.000
Likelihood Ratio
1.439 1 .230
Fisher's Exact Test
1.000 .500
Linear-by-Linear
Association
1.000 1 .317
N of Valid Cases
20

a Computed only for a 2x2 table

b2 cells (50.0%) have expected count less than 5. The minimum expected count is .50.

rCMV * Group:No variance in CMV category (none present), so statistical test cannot be
performed.

95

Table 11: Crosstab for CMV
Panel A

Group
Total 1.0Test 2.0Control
rCMV .00Not present Count
10 10 20
% within
rCMV
50.0% 50.0% 100.0%
% within
Group
100.0% 100.0% 100.0%
Total Count
10 10 20
% within
rCMV
50.0% 50.0% 100.0%
% within
Group
100.0% 100.0% 100.0%

Panel B

Chi-Square Tests

Value
Pearson Chi-Square
.(a)
N of Valid Cases 20

aNo statistics are computed because rCMV is a constant.

rNFKb * Group:Test teeth are significantly more likely to have significant NFKb than
control teeth.

96

Table 12: Crosstab for NFKb
Panel A

Group Total
1.0Test 2.0Control
rNFKb .00Not present Count
0 4 4
% within rNFKb
.0% 100.0% 100.0%
% within Group
.0% 40.0% 20.0%
1.00Not significant Count
2 3 5
% within rNFKb
40.0% 60.0% 100.0%
% within Group
20.0% 30.0% 25.0%
2.00significant Count
8 3 11
% within rNFKb
72.7% 27.3% 100.0%
% within Group
80.0% 30.0% 55.0%
Total Count
10 10 20
% within rNFKb
50.0% 50.0% 100.0%
% within Group
100.0% 100.0% 100.0%

Panel B

Chi-Square Tests

Value Df
Asymp. Sig.
(2-sided)
Pearson Chi-Square
6.473(a) 2 .039
Likelihood Ratio 8.105 2 .017
Linear-by-Linear
Association
6.131 1 .013
N of Valid Cases
20

a4 cells (66.7%) have expected count less than 5. The minimum expected count is 2.00.

rTNFa * Group: No variance in TNFa, so no statistical test can be done regarding test and
control teeth.

97

Table 13: Crosstab for TNFa
Panel A

Group
Total 1.0Test 2.0Control
rTNFa .00Not present Count
10 10 20
% within
rTNFa
50.0% 50.0% 100.0%
% within
Group
100.0% 100.0% 100.0%
Total Count
10 10 20
% within
rTNFa
50.0% 50.0% 100.0%
% within
Group
100.0% 100.0% 100.0%

Panel B

Chi-Square Tests

Value
Pearson Chi-Square
.(a)
N of Valid Cases 20

aNo statistics are computed because rTNFa is a constant.

rICAM_I * Group:Test teeth are more likely to have more ICAM I than control teeth, but
not statistically significant. (p=.072).

98

Table 14: Crosstab for ICAM1
Panel A

Group Total
1.0Test 2.0Control
rICAM_I .00Not present Count
1 5 6
% within rICAM_I
16.7% 83.3% 100.0%
% within Group
10.0% 50.0% 30.0%
1.00Not significant Count
1 2 3
% within rICAM_I
33.3% 66.7% 100.0%
% within Group
10.0% 20.0% 15.0%
2.00significant Count
8 3 11
% within rICAM_I
72.7% 27.3% 100.0%
% within Group
80.0% 30.0% 55.0%
Total Count
10 10 20
% within rICAM_I
50.0% 50.0% 100.0%
% within Group
100.0% 100.0% 100.0%

Panel B

Chi-Square Tests

Value Df
Asymp. Sig.
(2-sided)
Pearson Chi-Square
5.273(a) 2 .072
Likelihood Ratio 5.609 2 .061
Linear-by-Linear
Association
4.886 1 .027
N of Valid Cases
20

a4 cells (66.7%) have expected count less than 5. The minimum expected count is 1.50.

Further Collapsed PCR

Crosstabs
r2A.a * Group:No variance in A.a so no statistical test can be done regarding test and
control teeth.

99

Table 15: Crosstab for A. a.
Panel A

Group
Total 1.0Test 2.0Control
R2A.a 1.00Not present
or significant
Count
10 10 20
% within r2A.a
50.0% 50.0% 100.0%
% within Group
100.0% 100.0% 100.0%
Total Count
10 10 20
% within r2A.a
50.0% 50.0% 100.0%
% within Group
100.0% 100.0% 100.0%

Panel B

Chi-Square Tests

Value
Pearson Chi-Square
.(a)
N of Valid Cases 20

aNo statistics are computed because r2A.a is a constant.

r2CMV * Group: No variance in CMV, so no statistical test can be done regarding test
and control teeth.

100

Table 16: Crosstab for CMV
Panel A

Group
Total 1.0Test 2.0Control
R2CMV 1.00Not present
or significant
Count
10 10 20
% within
r2CMV
50.0% 50.0% 100.0%
% within Group
100.0% 100.0% 100.0%
Total Count
10 10 20
% within
r2CMV
50.0% 50.0% 100.0%
% within Group
100.0% 100.0% 100.0%

Panel B

Chi-Square Tests

Value
Pearson Chi-Square
.(a)
N of Valid Cases 20

a No statistics are computed because r2CMV is a constant.

r2NFKb * Group: Test teeth are more likely to have more NKFb, but not statistically
significant.

101

Table 17: Crosstab for NFKb
Panel A

Group Total
1.0Test 2.0Control
R2NFKb 1.00Not present
or significant
Count
2 7 9
% within r2NFKb
22.2% 77.8% 100.0%
% within Group
20.0% 70.0% 45.0%
2.00significant Count
8 3 11
% within r2NFKb
72.7% 27.3% 100.0%
% within Group
80.0% 30.0% 55.0%
Total Count
10 10 20
% within r2NFKb
50.0% 50.0% 100.0%
% within Group
100.0% 100.0% 100.0%

Panel B

Chi-Square Tests

Value Df
Asymp. Sig.
(2-sided)
Exact Sig.
(2-sided)
Exact Sig.
(1-sided)
Pearson Chi-Square
5.051(b) 1 .025
Continuity Correction(a)
3.232 1 .072
Likelihood Ratio
5.300 1 .021
Fisher's Exact Test
.070 .035
Linear-by-Linear
Association
4.798 1 .028
N of Valid Cases
20

a Computed only for a 2x2 table

b2 cells (50.0%) have expected count less than 5. The minimum expected count is 4.50.

r2TNFa * Group:No variance in TNFa, so no statistical test can be done regarding test
and control teeth.

102

Table 18: Crosstab for TNFa
Panel A

Group Total
1.0Test 2.0Control
R2TNFa 1.00Not
present or
significant
Count
10 10 20
% within r2TNFa
50.0% 50.0% 100.0%
% within Group
100.0% 100.0% 100.0%
Total Count
10 10 20
% within r2TNFa
50.0% 50.0% 100.0%
% within Group
100.0% 100.0% 100.0%

Panel B

Chi-Square Tests

Value
Pearson Chi-Square
.(a)
N of Valid Cases 20

No statistics are computed because r2TNFa is a constant.

r2ICAM_I * Group:Test teeth are more likely to have more ICAM I, but not statistically
significant.

103

Table 19: Crosstab for ICAM1
Panel A

Group Total
1.0Test
2.0Contr
ol
R2ICAM
_I
1.00Not present or
significant
Count
2 7 9
% within r2ICAM_I
22.2% 77.8% 100.0%
% within Group
20.0% 70.0% 45.0%
2.00significant Count
8 3 11
% within r2ICAM_I
72.7% 27.3% 100.0%
% within Group
80.0% 30.0% 55.0%
Total Count
10 10 20
% within r2ICAM_I
50.0% 50.0% 100.0%
% within Group
100.0% 100.0% 100.0%

Panel B

Chi-Square Tests

Value Df
Asymp. Sig. (2-
sided)
Exact Sig. (2-
sided)
Exact Sig.
(1-sided)
Pearson Chi-Square
5.051(b) 1 .025
Continuity
Correction(a)
3.232 1 .072
Likelihood Ratio
5.300 1 .021
Fisher's Exact Test
.070 .035
Linear-by-Linear
Association
4.798 1 .028
N of Valid Cases
20

a Computed only for a 2x2 table

b2 cells (50.0%) have expected count less than 5. The minimum expected count is 4.50.

104

Tables: Microbiology (Bacterial Cultures)
Table 20: Bacterial Culture for A. a.
Frequency Percent Valid Percent
Cumulative
Percent
Valid 0
20 100.0 100.0 100.0

Table 21: Bacterial Culture for P. gingivalis
Frequency Percent Valid Percent
Cumulative
Percent
Valid .0
14 70.0 70.0 70.0
3.6
1 5.0 5.0 75.0
3.8
3 15.0 15.0 90.0
4.6
1 5.0 5.0 95.0
7.7
1 5.0 5.0 100.0
Total
20 100.0 100.0

Table 22: Bacterial Culture for P. intermedia
Frequency Percent Valid Percent
Cumulative
Percent
Valid .0
14 70.0 70.0 70.0
3.8
3 15.0 15.0 85.0
6.2
1 5.0 5.0 90.0
6.9
1 5.0 5.0 95.0
10.8
1 5.0 5.0 100.0
Total
20 100.0 100.0

105

Table 23: Bacterial Culture for T. forsythia
Frequency Percent Valid Percent
Cumulative
Percent
Valid 0
12 60.0 60.0 60.0
3.1
1 5.0 5.0 65.0
3.8
2 10.0 10.0 75.0
4.6
1 5.0 5.0 80.0
5.4
2 10.0 10.0 90.0
5.5
1 5.0 5.0 95.0
7.7
1 5.0 5.0 100.0
Total
20 100.0 100.0

Table 24: Bacterial Culture for Campylobacter species
Frequency Percent Valid Percent
Cumulative
Percent
Valid .0
11 55.0 55.0 55.0
2.3
1 5.0 5.0 60.0
2.7
1 5.0 5.0 65.0
3.1
1 5.0 5.0 70.0
4.6
2 10.0 10.0 80.0
5.4
2 10.0 10.0 90.0
5.5
1 5.0 5.0 95.0
15.4
1 5.0 5.0 100.0
Total
20 100.0 100.0

Table 25: Bacterial Culture for Eubacterium Species
Frequency Percent Valid Percent
Cumulative
Percent
Valid 0
20 100.0 100.0 100.0

106

Table 26: Bacterial Culture for Fusobacterium species
Frequency Percent Valid Percent
Cumulative
Percent
Valid .0
11 55.0 55.0 55.0
4.6
2 10.0 10.0 65.0
5.4
1 5.0 5.0 70.0
5.5
1 5.0 5.0 75.0
6.2
3 15.0 15.0 90.0
6.4
1 5.0 5.0 95.0
7.7
1 5.0 5.0 100.0
Total
20 100.0 100.0

Table 27: Bacterial Culture for P. micros
Frequency Percent Valid Percent
Cumulative
Percent
Valid 0
19 95.0 95.0 95.0
4
1 5.0 5.0 100.0
Total
20 100.0 100.0

Table 28: Bacterial Culture for Enteric gram negative rod
Frequency Percent Valid Percent
Cumulative
Percent
Valid 0
19 95.0 95.0 95.0
11
1 5.0 5.0 100.0
Total
20 100.0 100.0

Table 29: Bacterial Culture for Beta hemolytic streptococci
Frequency Percent Valid Percent
Cumulative
Percent
Valid 0
19 95.0 95.0 95.0
7
1 5.0 5.0 100.0
Total
20 100.0 100.0

107

Table 30: Bacterial Culture for Yeast
Frequency Percent Valid Percent
Cumulative
Percent
Valid 0
20 100.0 100.0 100.0

Table 31: Bacterial Culture for Eikenlla corrodens
Frequency Percent Valid Percent
Cumulative
Percent
Valid 0
20 100.0 100.0 100.0

Table 32: Bacterial Culture for Staphylococcus species
Frequency Percent Valid Percent
Cumulative
Percent
Valid 0
19 95.0 100.0 100.0
Missing System
1 5.0
Total
20 100.0

Table 33: Bacterial Culture for D. pneumosintes
Frequency Percent Valid Percent
Cumulative
Percent
Valid 0
18 90.0 94.7 94.7
4
1 5.0 5.3 100.0
Total
19 95.0 100.0
Missing System
1 5.0
Total
20 100.0

Non-parametric test of differences between test and control teeth. The test is based on
average ranking, as shown in table below. A high rank represents more colonies (?).

Mann-Whitney Test

108

Table 34: Ranks
Group N Mean Rank Sum of Ranks
A. a. 1test
10 10.50 105.00
2control
10 10.50 105.00
Total
20
P.gingivalis 1test
10 13.50 135.00
2control
10 7.50 75.00
Total
20
P.intermedia 1test
10 14.50 145.00
2control
10 6.50 65.00
Total
20
Campilobacter.sp 1test
10 15.00 150.00
2control
10 6.00 60.00
Total
20
Eubacterium sp 1test
10 10.50 105.00
2control
10 10.50 105.00
Total
20
Fusobacterim sp 1test
10 15.00 150.00
2control
10 6.00 60.00
Total
20
P. micros 1test
10 11.00 110.00
2control
10 10.00 100.00
Total
20
Enteric G- rod 1test
10 11.00 110.00
2control
10 10.00 100.00
Total
20
Beta hemolytic
streptococci
1test
10 11.00 110.00
2control
10 10.00 100.00
Total
20
Yeast 1test
10 10.50 105.00
2control
10 10.50 105.00
Total
20
Eikenella
corrodens
1test
10 10.50 105.00
2control
10 10.50 105.00
Total
20
Staphylococcus 1test
9 10.00 90.00
2control
10 10.00 100.00
Total
19
D.pneumosintes 1test
9 10.56 95.00
2control
10 9.50 95.00
Total
19

109

Table 34 (continued)

You can interpret the U, W, or Z statistic for significance. Of the 14 microbes, there is a
significant test-control difference for P. intermedia; T. forsythia; Campilobacter sp.;
P.gingivalis; and Fusobacteria sp.

Table 35: Statistics Tests for Periodontal Pathogens

Mann-
Whitney U
Wilcoxon
W Z
Asymp. Sig. (2-
tailed)
Exact Sig. [2*(1-tailed
Sig.)]
A. a.
50.000 105.000 .000 1.000 1.000(a)
P. gingivalis
20.000 75.000 -2.802 .005 .023(a)
P. intermedia
20.000 75.000 -2.802 .005 .023(a)
T. forsythia
10.000 65.000 -3.416 .001 .002(a)
Campilobacter
sp
5.000 60.000 -3.727 .000 .000(a)
Eubacteriumsp
50.000 105.000 .000 1.000 1.000(a)
Fusobacterium
sp
5.000 60.000 -3.732 .000 .000(a)
P.micros
45.000 100.000 -1.000 .317 .739(a)
Enteric G- Rod
45.000 100.000 -1.000 .317 .739(a)
Beta hemolytic
strep
45.000 100.000 -1.000 .317 .739(a)
Yeast
50.000 105.000 .000 1.000 1.000(a)
Eikenella
corrodens
50.000 105.000 .000 1.000 1.000(a)
Staphylococcus
sp
45.000 100.000 .000 1.000 1.000(a)
D. pneumosintes
40.000 95.000 -1.054 .292 .720(a)

Means: For clarity, here are the means for each of the 14 microbes, by group

110

Table 36: Means for Periodontal Pathogens

1test 2control Total
Mean N
Std.
Deviation
Mea
n N
Std.
Deviati
on Mean N
Std.
Deviation
A. a.
.00 10 .000 .00 10 .000 .00 20 .000
P.gingivalis
2.730 10 2.6289 .000 10 .0000 1.365 20 2.2880
P. intermedia
3.530 10 3.6727 .000 10 .0000 1.765 20 3.1095
T. forsythitia
3.9300 10 2.42810
.000
0
10 .00000 1.9650 20 2.61861
Campilobacter sp
4.900 10 4.0844 .000 10 .0000 2.450 20 3.7710
Eubacterium sp
.00 10 .000 .00 10 .000 .00 20 .000
Fusobacterium sp
5.280 10 2.0687 .000 10 .0000 2.640 20 3.0600
P. micros
.38 10 1.202 .00 10 .000 .19 20 .850
Enteric G- Rod
1.14 10 3.605 .00 10 .000 .57 20 2.549
Beta hemolytic
strep
.69 10 2.182 .00 10 .000 .35 20 1.543
Yeast
.00 10 .000 .00 10 .000 .00 20 .000
Eikenella
corrodens
.00 10 .000 .00 10 .000 .00 20 .000
Staphylococcus sp
.00 9 .000 .00 10 .000 .00 19 .000
D. pneumosntes
.42 9 1.267 .00 10 .000 .20 19 .872

111

Chapter 3: Results
Ten patients with localized aggressive periodontitis participated in this study.
Table 1, 2, 3, 4 lists the characteristics of these patients. Study patients ranged in age
from 15 to 32 years. The male to female ratio was 3: 7. Five patients were Black, four
were Hispanic, and one was Asian. Localized aggressive sites (test) were first molars that
exhibited attachment loss of 5 to 11 mm and probing depth of 6 to 11 mm. Control sites
were either premolars or canines that revealed no evidence of attachment loss and
showed pocket depth of 3 mm or less.
Bacterial culture was done to evaluate fourteen periodontal pathogens in test and
control teeth. No periodontal pathogens were present in the control teeth sites. All of the
test teeth revealed some periodontal pathogens. Aggrigatibacter actinomycetemcomitans
were not present in any test samples. The mean percentages of other pathogens in test
teeth were 5.28 for Fusobacterium species, 4.90 for Campilobacter species, 3.93 for T.
forsythia, 3.53 for P. intermedia, 2.73 for P. gingivais, 1.40 for Enteric gram negative rod,
0.69 for Beta hemolytic streptococci, 0.38 for D. pneumosintes, and 0.00 for Yeast,
Eubacterium species, Eikenella corrodens, and Staphilococcus species (Table 36). Of the
fourteen periodontal pathogens, there was a significant test-control difference for
Fusobacterium species, P. intermedia, Campilobacter species, T. forsythia, and
Fusobacterium species P. intermedia, and P.gingivalis (Table 35).
RT-PCR analysis has been done for evaluating the presence of Aggrigatibacter
actinomycetemcomitans (A.a) RNA, cytomegalovirus RNA, TNFa, NFKb and ICAM1 in
test and control sites. CMV RNA was not present in any of the test or control samples
112

(Table 6). A.a RNA was present in only one test sample (16 year old Hispanic female)
but was not significant (Table 5).
Measurement of NFkb by RT-PCR showed that there was a marked increase in
expression of NFKb mRNA in the affected sites when compared with control sites (Table
7). Ten out of 10 samples from affected sites showed a starting cycle of 20.96 to 33.8
whereas only 6 out of 10 control samples showed a starting cycle 28.6 to 36.1. Pearson
Chi-square test showed test teeth are more likely to have significant level of NFKb than
control teeth that is statistically significant at the level of 0.039 (Table 12). Also Fisher’s
Exact test showed test teeth are more likely to have more NFKb, but not statistically
significant at the level of 0.070 (Table 17). In a parallel study, ICAM 1 expression was
markedly increased in the affected site compare with control site (Table 9). Nine of 10
affected sites showed starting cycles of 28.5 to 34.6, whereas five of 10 control samples
showed a starting cycle of 31.1 to 37.6, respectively. Pearson Chi-square test (p=0.072)
and Fisher’s Exact test (p=0.070) showed test teeth are more likely to have ICAM1 than
control teeth, but not satistically significant (Tables 14, 19).
In spite of the high expression of NFKb in affected sites, TNFa was not present in
any of the test or control samples (Table 8).
113

Chapter 4: Discussion
The ability of microorganisms to form plaque appears to be prerequisite for the
initiation of periodontal destruction. Most researchers agree that Aggrigatibacter
actinomycetemcomitans (A.a.) plays an important role in development of Localized
Aggressive Periodontitis (Sirkka
181
1985; Slots et al., 1983
180
). A study by Asikainen
Sirkka
181
(1985) showed that the occurrence of A.a. was related to the age of the LJP
patients. In that study, twenty LJP patients were divided into three age groups: 14-16, 17-
19, and 20-25 years of age. A. a. was isolated in the youngest group more frequently than
in the two older groups. Slots and coworkers
184
(1990) also examined age relationship
and mutual interrelationships between cultivable A. a. and bacteroides intermedius in
1624 periodontitis patients, 15 to 89 years of age. A. a. occurred with higher prevalence
(74%) in patients less than 25 years old than in adult and geriatric patients (prevalence
about 31%). A. a. was detected in 85% of localized juvenile periodontitis patients. But in
contrast, some other studies showed other periodontal pathogen to be more significant in
pathogenesis of LAP. For example, a study by Yasuo Takeushi et al. (2004)
190
did not
show high prevalence of A. a. in Japanese patients with Aggressive Periodontitis. Also, in
another study, done by Marta Gajard et al. (2005)
191
in a Chilean population, the
prevalence of periodontal pathogenic bacteria in Aggressive Periodontitis patients was
different. Results of that study showed that Aggressive Periodontitis patients (AgP) had a
higher prevalence of C. rectus than chronic periodontitis patients. Also, patients with AgP
showed a higher, but not statistically significant prevalence of P. gingivalis, E Corrodens,
P.micros and Capnocytophaga sp.
114

In this study, A. a. was not present in the cultures of any sample sites. P.
intermedia, T. forsythia, Campilobacter species, Fusobacterium species and P gingivalis
had significant differences in test and control sites. In testing for A. a. RNA with PCR
technique the significant presence of A. a. was not confirmed in any samples. A. a. was
present but, not significant, in only one test sample (16 years old Hispanic patient) by
PCR.
Conflicting findings by different investigators might be associated with different
lifestyle factors, such as diet, frequency of dental visits, access to health services,
consumption and self-medication of antimicrobials, and water sanitation. They might also
be related to race. More studies need to be done to establish the status on the observed
differences.
Also, in this study, the age range of patients was greater than in some of the
studies that showed a high percentage of A. a. in LAP patients. Some studies have shown
that A. a. is present in the active phase of LAP (Slots & Ting, 2000)
201
. However, in this
study we did not look at presence or absence of lamina dura to evaluate the activeness of
the disease and progression of bone loss.
Some investigation supports the role of CMV in localized aggressive periodontitis
(Michalowicz
200
2000, Ting & Slots et al.
201
, 2000). However in this study we did not
observe any CMV RNA that was representative of active CMV. The previous studies
that cited support presences of CMV in localized aggressive periodontitis were looking
only at CMV DNA. Because primary HCMV infection, which is thought to commence
at about 4 years of age, affects 89.1% of 4-7 years old children
231
, and a high percentage
115

of population might have patent HCMV, we decided to look only for actively replicating
HCMV in our subjects by using Real Time-PCR (RT-PCR). As mentioned before, we
did not look for the presence or absence of lamina dura to evaluate the activeness of the
disease and progression of bone loss. There is possibility that patients of this study were
in a silent not active phase of disease, but we can not confirm this statement.
Measurement of NFkb by RT PCR showed a marked increased in expression of
NFKb mRNA in disease-affected sites when compared with the control site (Table 7).
Ten out of 10 samples from affected sites showed a starting cycle of 20.96 to 33.8,
whereas only six out of 10 control samples showed starting cycles of 28.6 to 36.1.
Nuclear factor kb (NFkb) is a protein transcription factor that functions to
enhance the transcription of a variety of genes, including cytokines and growth factors,
adhesion molecules, immunoreceptors, and acute-phase proteins. NFkb is required for
maximal transcription of many cytokines, including tumor necrosis factor a (TNFa),
interleukin-1 (IL-1), IL-6, and IL-8, which are thought to be important in the generation
of acute inflammatory responses
261
.
NFKb consists of two members of the Rel family of proteins. As with other
transcription factors, NFkb attaches to DNA in the promoter regions of target genes as a
dimmer composed of two Rel family proteins, p50 (NFkb1) and RelA (p65). In the NFkb
heterodimer, both subunits contact DNA, but only RelA contains a transactivation
domain in the C-terminal end of the protein that activates transcription by direct
interaction with the basal transcription apparatus
262
. The Rel family contains other
members, including c-Rel, RelB, and p52 (NFkb2), which, in combination with p50 and
116

RelA, exist in a wide variety of cell types and can form various hetero and homodimers.
Although NFkb is classically defined as a p50/ RelA heterodimer, other combinations of
Rel proteins can function identically to NFkb. For this reason, any combination of Rel
proteins that bind to NFkB binding sites, called as NFkb. In quiescent cells, NFkb is
sequestered in the cytoplasm through its interaction with the inhibitors IkBa, IkBb, or
kBe. In addition, p105, which is the precursor of p50, and p100, which is the precursor of
p52, can bind RelA and thus function as NKkb inhibitors. The C-terminal portions of
p105 and p100 have been designated IkBg and IkBd, respectively. These inhibitory units
contain ankyrin repeat domains that allow interaction with NFkB in configurations that
mask nuclear localization signal domains, thereby preventing nuclear transport
263
. IkBa
interacts only weakly with p50 homodimers and does not efficiently prevent their
translocation to the nucleus
264
. NFkb can be activated in cells by a variety of stimuli,
including bacterial endotoxin, TNFa, IL-1 b, mitogens, viral proteins, ionizing radiation,
UV light, and certain chemical agents
264
. Following activation, the inhibitory units are
phosphorylated and degraded, unmasking nuclear localization signals that allow NFkb to
be transported to the cell nucleus, where its dimers are free to bind DNA. The mechanism
of processing of the inhibitory units has been a subject of much recent investigation, and
is currently best understood for I kBa.
Phosphorylation of IkBa leads to recognition of the IkBa molecule by the
proteasome complex and subsequent degradation of the IkBa molecule, freeing NFkb to
translocate to the nucleus. Because NFkb is an integral and critical regulator of cytokine-
mediated inflammation, the activation of NFkb is a tightly controlled event. Feedback
117

control of NFkb activation occurs both intracellularly and extracellularly. Positive
feedback may occur through extracellular mechanisms that serve to amplify
inflammatory signals. NF-kb activation enhances the transcription of TNFa and IL-1b,
and both of these cytokines are in turn known to activate NFkb. An inflammatory signal,
such as a bacterial endotoxin, can cause cells to activate NFkb, which enhances TNFa
and IL-1b production and release, and presumably could amplify the original
inflammatory signal. Negative feedback control is essential in regulating NFkb activation.
Both intracellular and extracellular mechanisms are responsible for limiting NFkb
activation in response to a given stimulus. Intracellularly, NFkb activation leads to
transcriptional upregulation of the IkBa and p105 genes, since both of these genes have
NFkb-responsive elements in their promoters
265, 266
. Increased production of inhibitory
units presumably helps trap NFkB in the cytoplasmic compartment, and downregulates
activated nuclear NFkb, thus terminating new cytokine transcription and limiting the
inflammatory response. An interesting effect of increased production of p105 is that p50
homodimer formation is also increased, which may diminish NFkb-mediated responses to
subsequent stimuli. Since p50 homodimers do not bind efficiently to IkB, and lack
transcription- activation domains, they can translocate to the nucleus and function as
inhibitors of NFkb-mediated gene expression by competing with other Rel proteins for
access to NFkb binding sites.
Investigations into the role of NFkb in human disease have only recently been
undertaken. Schwartz and coworkers
267
have reported that NFkb in alveolar macrophages
from patients with acute respiratory distress syndrome (ARDS) is activated to a
118

significantly higher degree than in alveolar macrophages from critically ill patients with
other diseases. In addition, Asahara and colleagues
268
showed that NFkb is activated in
the synovium of patients with rheumatoid arthritis as compared with patients with
osteoarthritis. Currently, experimental evidence linking NFkb activation in specific cells
or tissues to other inflammatory diseases in humans is lacking. Several animal models
have been developed to evaluate the role of NFkb in the production of inflammatory
events. NFkb activation has been shown in rat microglial cells in a model of autoimmune
encephalomyelitis
269
. In a rat model of glomerulonephritis, NFkb activation has been
shown in glomeruli
270
. A study by Robert A. et al. (2001)
271
found that infection of
quiescent fibroblasts with human cytomegalovirus (HCMV) causes a rapid activation of
cellular phosphatidylinositol 3-kinase (PI3-K). Maximum PI3-K activation occurred from
15 to 30 min post-infection. This activation was transient, and by 2 h post-infection (hpi),
PI3-K activity had declined to pre-infection levels. However, at 4 hpi, a second tier of
PI3-K activation was detected, and PI3-K activity remained elevated relative to that of
mock-infected cells for the remainder of infection. The cellular kinases Akt and p70S6K
and the transcription factor NF-kB were activated in a PI3-K-dependent manner at similar
times following HCMV infection.
In this study it is intriguing to note that NFKb expression is increased in the
affected sites when compared with corresponding control sites. Research on the role of
transcription factors in periodontal diseases is in its very early stages. Further
investigation is warranted to determine whether the intensity of NFkb activation is useful
as a marker for the severity of inflammation in this disease, and whether NFkb activation
119

could be useful as a surrogate marker for assessing the efficacy of therapeutic
interventions.
Another important finding in this study is expression of ICAM 1 in affected sites.
In the test sites, nine of ten samles showed starting cycles of 28.5 to 34.6, whereas in the
control sites, five of ten samples showed starting cycles of 31.1 to 37.6 (Table 9).
ICAM1 (Inter Cellular Adhesion Molecule 1), also known as CD54 (Cluster of
Differentiation 54), is a human gene. The protein encoded by this gene is a type of
intracellular adhesion molecule continuously present in low concentrations in the
membranes of leukocyte and endothelial cells. Upon cytokine stimulation, the
concentrations greatly increase. ICAM-1 can be induced by IL-1 and TNFα, and is
expressed by the vascular endothelium, machrophges and lymphocyts. ICAM-1 is a
ligand for LFA-1 (integrin), a receptor found on leukocytes. When activated, leukocytes
bind to endothelial cells via ICAM-1/LFA-1 and then transmigrate into tissues
272
.
Leukocyte emigration from the bloodstream into tissues at sites of inflammation
is controlled by sequential intercellular adhesion events with endothelial cells that line the
vascular wall. The initial rolling steps are mediated by members of the selectin family,
including endothelial leukocyte adhesion molecule 1 (ELAM1 or E-selectin) and L-
selectin
273
. Vascular cell adhesion molecule 1 (VCAM1) and intercellular cell adhesion
molecule 1 (ICAM1), located on the surface of cytokine-activated endothelium, belong to
the immunoglobulin supergene family and are considered to be involved in the next step
of leukocyte-endothelium interaction, where a tighter adhesion takes place
274
. Some
studies have indicated that ELAM1, VCAM1, and ICAM1 can be detected on endothelial
120

cells adjacent to the junctional epithelium early in the course of experimentally induced
gingivitis
275
, suggesting that they are involved in crucial processes which direct leukocyte
migration into the tissues and toward the gingival sulcus. Further, ICAM1 on oral
epithelial cells has recently been shown to be susceptible to proteolysis by gingipains
276
.
The importance of cell adhesion molecules is highlighted by the rapid and severe
periodontitis that characterizes leukocyte adhesion deficiency, where polymorphonuclear
leukocytes (PMNs) are unable to migrate through the endothelium of gingival blood
vessels
277
.
Of further interest is to find that the magnitude of NFKB and ICAM1 was directly
correlated (Tables 7 and 9). Samples with lower starting cycles showed earlier ICAM 1
cycles and vice versa. In addition, we found no samples with TNFa expression (Table 8).
Based on these findings, we speculate that NFKb induced ICAM1 expression and, in turn,
ICAM1 promoted translocation of inflammatory cells into the affected site.
In spite of the high expression of NFKb, in affected sites, TNFa was not present
in any of test or control samples table. Tumor necrosis factor alpha (TNFa) is a cytokine
involved in systemic inflammation, and is a member of a group of cytokines that
stimulate the acute phase reaction
278
. The primary role of TNFa is in the regulation of
immune cells. TNFa is also able to induce apopotic cell death, to induce inflammation,
and to inhibit tumor ingeneis and viral replication. As mentioned before, TNFa has an
important role in the upregulation of activeNFKb. However, TNFa-induced cell death
plays only a minor role compared to its overwhelming functions in the inflammatory
process
279
. Its death-inducing capability is weak compared to other family members (such
121

as Fas), and often masked by the anti-apoptotic effects of NFKb. Furthermore, NFKb
may exert an inhibitory influence on TNFa in order to initiate the apoptotic process,
leading to continuous inflammatory and osteoclast activity and bone resorption.
Activation of nuclear factor kb (NFkb) is thought to be required for cytokine production
by lipopolysaccharide (LPS)-responsive cells. Mitsuhiro Fujihara et al. (2000)
280
,
investigated the contribution of NFkb in preventing LPS-induced transcription of the
tumor necrosis factor a (TNFa) gene in a murine macrophage cell line, P388D1, when
tolerance was induced in the cells with a short exposure to a higher dose of LPS.
Electrophoretic mobility shift assays with the kB elements of the murine TNFa promoter
and enhancer revealed that nuclear mobilization of heterodimers of p65/p50, c-rel/p50
and p65/c-rel, and homodimers of p65 was markedly reduced in LPS-tolerant cells,
whereas that of p50 homodimers was only slightly increased. Western blot analysis
showed that the phosphorylation of Ser32 on IkBa and its transient degradation did not
occur in LPS-tolerant cells. These results thus suggest that desensitization of the TNFa
gene expression in this LPS-tolerant state is closely associated with the down-regulation
of transactivating NFkb and may involve a defect in the LPS-induced IkBa kinase
pathway. However more research is needed to evaluate effect of upregulation of NFKb
on TNFa.
Minimal expression of NFKb and ICAM1 in control site may also occur by NFKb
inactive variant such as homodimer P50/p50 rather than by active dominant p65.
122

Chapter 4 Endnotes
180- Eisenmann AC, Eisenmann R, Sousa O, Slots J. Microbiological study of localized
juvenile periodontitis in Panama. J Periodontol, 1983 Dec; 54(12):712-3.

181- Asikainen S. Occurrence of Actinobacillus actinomycetemcomitans and spirochetes
in relation to age in localized juvenile periodontitis. J Periodontol. 1986 Sep;
57(9):537-41

184- Slots, J, Felik D and Rams TE. Actinobacillus actinomycetemcomitans and
bacteriod Intermedius in human periodontitis, age relationship and mutual
association. J Clin Periodontol, 190, 17; 659-662.

190- Takeuchi Y, Umeda M, Ishizuka M, Huang Y, Ishikawa I. Prevalence of
periodontopathic bacteria in aggressive periodontitis patients in a Japanese
population. J Immunol, 2004 Feb 1; 172(3):1856-61.

200- Michalowicz BS, Ronderos M, Camara-Silva R, Contreras A, Slots J. Human
herpesviruses and Porphyromonas gingivalis are associated with early-onset
periodontitis. J Periodontol 2000: 71: 981–988.

201- Ting M, Contreras A, Slots J. Herpesviruses in localized juvenile periodontitis. J
Periodontal Res 2000: 35: 17–25.

231- Agarwal S, Suzuki JB, Piesco NP, Aichelmann-Reidy MB. Neutrophil function in
Juvenile Periodontitis. Oral Microbiol Immunol, 1994: 9: 262-271

261- Timothy S. Blackwell and John W. Christman. The Role of Nuclear FactorkB in
Cytokine Gene Regulation, Am. J. Respir. Cell Mol. Biol. Vol. 17, pp. 3–9, 1997

262- Schmitz, M. L., and P. A. Baeuerle. 1991. The p65 subunit is responsible forthe
strong transcription activating potential of NF-kB. EMBO J. 10:3805.

263- Miyamoto, S., and I. M. Verma. 1995. REL/NF-kB/IkB story. Adv. Cancer Res.
66:255–287.

264- Siebenlist, U., G. Franzoso, and K. Brown. 1994. Structure, regulation and function
of NF-kB. Annu. Rev. Cell. Biol. 10:405–455.

265- Kazantsev, and Baldwin, Jr. 1994. Three NF-kappa B sites in the I kappa B-alpha
promoter are required for induction of gene expression by TNF alpha. Nucleic
Acids Res. 22:3787–3792.

123

266- Cogswell, P. C., R. I. Scheinman, and A. S. Baldwin, Jr. 1993. Promoter of the NF-
kappa B p50/p105 gene. Regulation by NF-kappa B subunits and by c-REL. J.
Immunol. 150:2794–27804.

267-Schwartz, M. D., E. E. Moore, F. A. Moore, R. Shenkar, P. Moine, J. B. Haenel, and
E. Abraham. 1996. Nuclear factor-kB is activated in alveolar macrophages from
patients with acute respiratory distress syndrome. Crit. Care Med. 24:1285–1292.

268- Asahara, H., M. Asanuma, N. Ogawa, S. Nishibayashi, and H. Inoue. 1995. High
DNA-binding activity of transcription factor NF-kB in synovial membranes of
patients with rheumatoid arthritis. Biochem. Mol. Biol. Int. 37:827–832.

269- Kaltschmidt, C., B. Kaltschmidt, J. Lannes-Vieira, G.W. Kreutzberg, H. Wekerle, P.
A. Baeuerle, and J. Gehrmann. 1994. Transcription factor NFkappa B is activated
in microglia during experimental

270- Sakurai, H., Y. Hisada, M. Ueno, M. Sugiura, K. Kawashima, and T. Sugita. 1996.
Activation of transcription factor NF-kappa B in experimental glomerulonephritis
in rats. Biochem. Biophys. Acta 1316:132–138.

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124

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125

Chapter 5: Conclusion
NFkb has been shown to be a tightly regulated agent for initiating the
transcription of a wide variety of genes involved in the production of acute inflammation.
Difficulty in obtaining LAP patients limited the present study to 10 subjects. The small
sample size precludes any definitive conclusions.
The preliminary findings of this study suggest that upregulation of active NFKb
and ICAM 1 plays an important role in LAP. Research on the role of transcription factors
in periodontal diseases is in its very early stages. Further investigation is warranted to
determine whether the intensity of NFkb activation is useful as a marker for the severity
of inflammation in this disease, and whether NFkb activation could be useful as a
surrogate marker for assessing the efficacy of therapeutic interventions. Specific
inhibitors of NFkb would be beneficial in further dissecting the role of NFkb in the
complex acute inflammatory response, and could be clinically useful in treating
inflammatory diseases such as periodontal disease.
126

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Abstract (if available)

Abstract Introduction: Localized Aggressive Periodontitis (LAP) has a bacterial etiology, with infection of the gingival tissues by several bacterial pathogens. However, in addition to bacterial etiology, host immune and inflammatory factors are implicated in the pathogenesis and progression of localized aggressive periodontitis. Objective: The objective of this study is to explore underlying mechanisms involved in selective sites of infection, and the subsequent inflammatory process in LAP. Methods and Materials: Ten LAP patients participated in this study (3 male and 7 female), with an age range of from 13- 32 years. Samples were taken from one tooth with the deepest pocket for a test site and one tooth with healthy periodontium for a control site from each individual.

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Technological advancements in microbial analyses of periodontitis patients: focus on Illumina® sequencing using the Miseq system on the 16s rRNA gene: a clinical and microbial study

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Prevalence and distribution of facial alveolar bone fenestrations in the anterior dentition: a cone beam computed tomography analysis

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Parvimonas micra adhesion on different implant surfaces: an in vitro pilot study

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Cytokine levels, microbiology, and virology in healthy implants and teeth

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Fractal analysis on dental radiographs to detect trabecular patterns in patients affected by periodontitis

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Longitudinal analysis of peri-implant keratinized mucosa using computer imaging

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Effect of 0.25% sodium hypochlorite oral rinse and subgingival irrigation on periodontal clinical parameters

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A prospective observational study of immediately loaded platform switched 3i implants

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Human histological analysis of autogenous block grafts

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Efficacy of fibrin-assisted soft-tissue promotion (FASTP) in treatment of multiple gingival recession defects: a retrospective 3-D volumetric analysis

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Local and systemic responses to craniofacial osteolytic defects in an animal model

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Aggregatibacter actinomycetemcomitans evolves in vivo in patients with periodontal disease

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Cytomegalovirus induced amelogenesis imperfecta

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Reproducibility of digital and analogue assessment methods used for quantitative measurements of periodontal soft tissues dimensions

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Periodontal regeneration utilizing antibody mediated osseous regeneration (AMOR): supra-alveolar critical sized defect model

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A randomized controlled clinical trial evaluating the efficacy of grafting the facial gap at immediately placed implants in the anterior maxilla: 3D analysis of bone and soft tissue changes

Asset Metadata

Creator Zandi, Mahnaz (author)

Core Title Inflammatory mechanisms in localized aggressive periodontitis

School School of Dentistry

Degree Master of Science

Degree Program Craniofacial Biology

Publication Date 06/19/2009

Defense Date 04/15/2009

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag cmv,ICAM1,Locaized aggressive periodontitis,NFKb,OAI-PMH Harvest,TNFa

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Nowzari, Hessam (committee chair), Navazesh, Mahvash (committee member), Rich, Sandra (committee member)

Creator Email mahzandi2002@yahoo.com,mzandi@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-m2308

Unique identifier UC1499443

Identifier etd-Zandi-2957 (filename),usctheses-m40 (legacy collection record id),usctheses-c127-244716 (legacy record id),usctheses-m2308 (legacy record id)

Legacy Identifier etd-Zandi-2957.pdf

Dmrecord 244716

Document Type Thesis

Rights Zandi, Mahnaz

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Repository Name Libraries, University of Southern California

Repository Location Los Angeles, California

Repository Email cisadmin@lib.usc.edu