J Periodontol • October 2007

Periodontal Disease at the

Biofilm–Gingival Interface
S. Offenbacher,* S.P. Barros,* R.E. Singer,* K. Moss,† R.C. Williams,* and J.D. Beck†

Background: A molecular epidemiologic study provided

the opportunity to characterize the biology of the biofilm–gin-
gival interface (BGI) in 6,768 community-dwelling subjects.
Methods: Disease classifications and multivariable models
were developed using clinical, microbial, inflammatory, and
host-response data. The purpose was to identify new clinical
categories that represented distinct biologic phenotypes

D
espite advances in our under-
based upon DNA checkerboard analyses of eight plaque bac- standing of the microbiology of
teria, serum immunoglobulin G (IgG) titers to 17 bacteria, and the biofilm, as well as the host
the gingival crevicular fluid (GCF) levels of 16 inflammatory cellular and molecular mechanisms of
mediators. Five BGI clinical conditions were defined using pathogenesis, diagnostic categories for
probing depths (PDs) and bleeding on probing (BOP) scores. periodontal classification remain based
Subjects with all PDs £3 mm were grouped as BGI-healthy almost exclusively upon medical and
(14.3% of sample) or BGI-gingivitis (BGI-G, 15.1%). Subjects dental history and clinical signs and
with one or more PDs ‡4 mm [deep lesion (DL)] were divided symptoms of disease.1-5 Unfortunately,
into low BOP (18.0%), moderate BOP (BGI-DL/MB, 39.7%), current disease classifications that are
and severe BOP (BGI-DL/SB, 12.9%). based upon clinical signs do not provide
Results: Subjects with BGI-G had increased levels of much insight into the underlying sub-
Campylobacter rectus–specific serum IgG levels (P = 0.01), clinical biology of the process that in-
and those with BGI-DL/SB had increased IgG levels to volves a complex interaction of the
Porphyromonas gingivalis (P < 0.0003) and C. rectus (P < biofilm with the host inflammatory and
0.01). BGI-DL/SB subjects had an excessive GCF interleukin immune responses.
(IL)-1b and prostaglandin E2 response and an enhanced Emerging medical models of nosology
chronic inflammatory response with significant increases in (disease classification), such as that pro-
GCF IL-6 and monocyte chemotactic peptide-1. Within BGI- posed by Casanova and Abel,6 offer a
DL/SB subjects, more severe pocketing and BOP were asso- conceptual framework for exploring dis-
ciated with higher levels of GCF IL-1b, not higher microbial ease definitions that recognize the in-
counts or plaque scores. terplay of host and microbial biologic
Conclusions: New BGI classifications create categories systems. The application of these con-
with distinct biologic phenotypes. The increased titers of cepts to define periodontal disease would
C. rectus IgG among 68.5% of the BGI-G subjects and elevated incorporate characterizing biologic sys-
P. gingivalis titers among BGI-DL/MB and BGI-DL/SB sub- tems that include unique individual expo-
jects (63.8% and 75.7%, respectively) are strongly supportive sures, e.g., bacterial infections, smoking,
of the microbial specificity of pathogenesis for BGI categories. and hyperglycemia of diabetes, that in-
J Periodontol 2007;78:1911-1925. teract with the genetic repertoire of the
individual host to create a biologic pheno-
KEY WORDS
type (a cascading cellular and molecular
Gingival crevicular fluid; microbiology; periodontal disease; process that includes the mechanism of
proteomics. pathogenesis), which, over time, results
in a clinical phenotype (the clinical pre-
sentation of disease). It was suggested
* Center for Oral and Systemic Diseases and Department of Periodontology, School of
Dentistry, University of North Carolina at Chapel Hill, Chapel Hill, NC. by Casanova and Abel6 that subtle ge-
† Department of Dental Ecology, School of Dentistry, University of North Carolina at Chapel netic immunodeficiencies underlie most
Hill.
microbial-based diseases in natural hu-
man conditions. These investigators

doi: 10.1902/jop.2007.060465

1911
Disease at the Biofilm–Gingival Interface
suggested that opportunistic infections pose a tre-
mendous diversity of potential bacterial exposures
that, in combination with subtle genotype variations
in the host, can result in a specific biologic phenotype
that induces disease. Many types of underlying bio-
logic phenotypes are probable because the genetic di-
versity in humans and the microbiota of the biofilm
permits perhaps hundreds of different combinations
of microbes/host response. Despite this possible de-
gree of complexity, there also may be shared path-
ways with similar cellular and molecular responses
that lead to common elements within the biologic phe-
notypes and similar clinical presentations. As pointed
out by Baelum and Lopez,7 the underlying diversity of
biologic phenotypes that are currently ‘‘subclinical’’
may not manifest as a variety of clinical phenotypes
using existing disease definitions. Thus, the challenge
facing us scientifically, and the goal of this study, was
to refine existing periodontal disease classifications in
a manner that will enable us to uncover and delineate
the various types of biologic phenotypes.
The strategy we used in this study was to supple-
ment clinical signs with diagnostic biomarker tests,
including microbial, inflammatory, and host response
assessments, to characterize the biologic phenotype
of each individual. We systematically examined how
clinical signs could be grouped to segregate different
clinical disease classifications that differ from each
other biologically but share a similar biologic pheno-
type within each group parsing a logical transition
from health to severe disease. Thus, in this study we
sought to create new nosological classifications that
allowed the clinical definitions to reflect the biologic
phenotype. The long-term rationale for attempting
to create new clinical definitions that share a common
underlying biologic phenotype is to improve the con-
sistency of diagnosis and prediction of therapeutic
response. Furthermore, by creating homogeneous
groups that share a common biologic phenotype,
we should be able to better identify the host and micro-
bial genetic contributions to the clinical conditions.
In previous publications,8-12 we provided clinical,
microbial, and host-response characteristics of a
large community-dwelling adult population, catego-
rizing subjects using traditional disease definitions
based upon clinical signs. In this report, we sought
to identify those clinical signs that would enable us
to segregate discrete clusters of individuals that could
be considered new, clinically relevant diagnostic cat-
egories or syndromes that also reflected what was
happening biologically at the biofilm–gingival inter-
face (BGI). We developed multivariable models to as-
certain which exposures or biologic characteristics
seemed to best describe the extent or severity of the
condition within each diagnostic category. In this re-
port, we provide a new analysis that has explored
1912
Volume 78 • Number 10
the clinical, microbial, and host-response characteris-
tics of this population. In addition, we present a de-
tailed proteomic analysis of 16 cytokines within the
gingival crevicular fluid (GCF) that was performed
on a representative subset of 180 subjects. We com-
bine all of these findings to demonstrate that new
disease categories can be defined that display signif-
icantly different patterns of microbial burden and host
response that define the biology at the BGI in a manner
that would have been indistinguishable using tradi-
tional disease classifications.
MATERIALS AND METHODS
Analytical Approach
The periodontal tissue–biofilm interface is bordered
on the gingival tissue side by the epithelium (sulcular,
pocket, and junctional) and the subgingival plaque
within the pocket. Linear probing depth (PD) provides
a reasonable approximation of the pocket surface
area when considered at six sites per tooth on all teeth
and expressed as summary or extent scores. The
deeper the probeable pocket, the better the environ-
ment for anaerobic subgingival biofilm growth, and
the relationship between higher periodontal pathogen
counts and increasing PD is well established.5 Thus,
PD can define one clinical attribute of the BGI that
should influence the subgingival microbial composi-
tion and growth density. When the pocket epithelium
is ulcerated or friable because of inflammatory
changes, bleeding on probing (BOP) is the associated
clinical sign. Thus, these two clinical signs (PD and
BOP) were used to define current clinical disease sta-
tus at the BGI. Although recession is an important
component of attachment loss, it does not contribute
to the anatomical boundaries of the BGI. Furthermore,
attachment level, a measurement that is made rela-
tive to a dental anatomical landmark, such as the ce-
mento-enamel junction (CEJ), is also independent of
the boundaries of the BGI and reflects historic attach-
ment loss rather than current status.
To determine the composition and magnitude of
the biofilm load, we determined the level of pathogens
present within the periodontal pocket using DNA
checkerboard methods in addition to traditional clini-
cal measurements of plaque. The local host inflam-
matory response is represented by measuring the
concentration of biochemical mediators of inflamma-
tion within the GCF. The patterns of these inflamma-
tory mediators reveal the specific qualitative and
quantitative aspects of the innate and chronic inflam-
matory response. As one might expect, the concen-
tration of inflammatory biomarkers within the GCF
generally increased with greater microbial burden,
even among subjects without any clinical signs of dis-
ease. However, by adjusting for the level of microbial
J Periodontol • October 2007
burden using plaque scores and microbial counts, we
can determine whether the level of inflammation rela-
tive to the level of biofilm exposure for a given BGI dis-
ease group is in excess compared to the BGI-healthy
(BGI-H) group response and, therefore, represents an
excessive or hyperinflammatory response. Finally,
the acquired immune response to the existing biofilm
was determined by measuring the serum antibody
level to each pathogen. The immunoglobulin G
(IgG) level to specific bacteria of the biofilm is an im-
portant parameter because it implicates specific bac-
teria as being potentially etiologic as related to each
biologic and clinical phenotype and reflects a sys-
temic level of exposure of the organism, albeit imper-
fectly. The purpose of this investigation was to create
clinical definitions of disease at the BGI that repre-
sented different patterns of microbial, inflammatory,
and immunologic parameters.
Periodontal Measurements
Periodontal examinations were performed on 6,768
individuals living in four United States communities.
This cross-sectional assessment of individuals also
included detailed medical history data and was de-
scribed in detail in several publications.13-15 Full-
mouth periodontal examinations were performed by
calibrated examiners on all teeth, including third mo-
lars. Clinical assessments included plaque index,16
gingival index (GI),17 PDs (rounding down to the near-
est millimeter), CEJ measurements, and attachment
levels (computed from PD and CEJ) and were deter-
mined in millimeters at six sites per tooth, as was BOP
(yes/no) at each site. The extent of BOP was ex-
pressed as a percentage of all sites.
Measurements of Bacterial Plaque and Serum
IgG Antibody
Levels of bacteria within plaque samples and serum
antibody titers to specific organisms were determined
on a random sample of 1,673 subjects using methods
described recently by Beck et al.18 One plaque sam-
ple was used from each subject, sampling the subgin-
gival mesio-buccal site of the maxillary right first
molar, and assayed for 18 organisms by DNA whole
chromosomal checkerboard arrays. For these ana-
lyses, the levels of eight periodontal pathogens and
the titers were used for disease model development, in-
cluding Porphyromonas gingivalis, Treponema denti-
cola, Tannerella forsythia (previously T. forsythensis),
Campylobacter rectus, Prevotella intermedia, Actino-
bacillus actinomycetemcomitans, and Fusobacterium
nucleatum. Levels of organism were expressed as
counts using known microbial standards. Serum
IgG levels were determined using immunochecker-
board arrays using these same seven organisms plus
Parvimonas micra (previously Peptostreptococcus
micros or Micromonas micros), Eikenella corrodens,
Offenbacher, Barros, Singer, Moss, Williams, Beck
Capnocytophaga ochracea, Veillonella parvula, Strep-
tococcus oralis, Streptococcus sanguinis, Streptococcus
intermedius, Selenomonas noxia, and Actinomyces
viscosus as whole-cell antigens and were expressed
as nanograms per milliliter of IgG-specific titers. Hu-
man IgG at three concentrations using membrane-
bound protein A was used on each membrane as an
internal standard. For the purposes of this analysis, the
level of the organisms and the titer were considered
continuous variables.
Statistical Analyses
Clinical signs were expressed as extent scores19 and
used as continuous variables to examine associations
with biologic variables to create composite biologic
phenotypes. Using extent PD, CAL (clinical attach-
ment level), or GI scores as continuous variables to
rank individuals based upon disease severity did not
create any consistent patterns in the levels of specific
bacteria, the concentration of GCF inflammatory me-
diators, or antibody titers. Only plaque levels and GCF
interleukin (IL)-1b levels were weakly positively asso-
ciated with extent of disease using PD, GI, or CAL as
continuous variables. Traditional definitions of peri-
odontal status based upon attachment loss definitions
resulted in creating subgroups that did not differ in
biomarker level and were non-informative in con-
structing a homogeneous biologic phenotype. Data
mining with self-assembling hierarchical clustering
methods resulted in biomarker subgroups that con-
tained a diverse range of periodontal disease levels
using traditional disease definitions or extent scores.
However, the extent BOP and PD scores were associ-
ated positively with various biomarkers and were used
initially to define clinical states. Most importantly, we
iteratively selected the clinical definitions and then
sought to determine whether there were underlying bi-
ologic phenotypes with these defined categories. In
other words, we intentionally reverse engineered def-
initions of clinical conditions that were based upon bi-
ologic data that defined the BGI. The final definitions
reflect cut-points in BOP and PD that resulted in sep-
arate biologic phenotypes and created gradient levels
of disease. Further subdivision of disease categories
did not result in different biologic phenotypes and,
therefore, were used as aggregate categories without
further subdivision. For example, segregating sub-
jects with or without attachment loss within each of
the BGI categories did not result in two groups with dif-
fering levels of GCF mediator or microorganism
counts. In all multivariate models that included bio-
logic phenotype markers, the use of indices, such
as GI, were supplanted by BOP in that BOP explained
a greater part of the variance of the final disease model
than GI, indicating that GI did not significantly im-
prove the overall model, i.e., not statistically significant
1913
Disease at the Biofilm–Gingival Interface
and affecting <5% of the total model variance. In a sim-
ilar manner, all CAL and/or CEJ measurements were
replaced by PD measurements. Thus, only PD and
BOP were used to define the final BGI clinical cate-
gories.
Differences between disease categories in patient-
based characteristics, periodontal clinical measures,
and GCF mediator levels were determined using
general linear models for continuous variables and
x2 tests for categorical variables. P values <0.05 were
considered statistically significant. To determine
whether there were overall differences among groups
and to correct for multiple comparisons, the Hotelling
T2 statistic was computed for levels of organisms and
titers. Once an overall difference was established by
Hotelling T2, group differences in levels and of each
periodontal organism were based upon non-paramet-
ric analysis of rank scores using general linear models
for overall significance and for group comparisons to
the healthiest group (BGI-H). Group differences in IgG
levels to specific organisms were computed using
general linear models. To define which independent
variables contribute to disease definition categories
compared to BGI-H, we used logistic regression to
create generalized logits models. Multivariate linear
regression models were developed to define the extent
BOP or extent PD ‡4 mm as the dependent variable
within each disease category.
GCF Analyses
GCF was collected from the mesio-buccal aspect of
each first molar or, if missing, an alternate site as re-
viewed in detail previously.20 Four GCF strips were
eluted and analyzed separately for each subject.
GCF analyte concentration data were pooled to pro-
vide a patient mean value in ng/ml for each analyte.
The mean GCF levels of prostaglandin E2 (PGE2)
and IL-1b were determined for 5,604 subjects. Site-
specific analyses of these data as related to clinical
signs are described elsewhere.21 In this report, the pa-
tient pooled mean level of each mediator was used as
a patient-level descriptor of whole-mouth periodontal
inflammatory response. For a more comprehensive
proteomic analysis of GCF mediators, we used a mul-
tiplex analytical platform as described previously.12
Multiple cytokine reagents permitted the analyses of
16 mediators simultaneously on each GCF sample.
For these proteomic GCF analyses, a random sample
of subjects was selected in each of the five BGI cate-
gories from the entire sample, matching frequency
distribution on age (within 5 years), race, and gender.
Smokers were omitted. A total of 180 subjects were
selected for these analyses. Each GCF sample (four
per patient at the disto-buccal aspect of each second
premolar) was analyzed using cytokine profiling by
multiplex immunoassay that permitted the simulta-
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Volume 78 • Number 10
neous measurement of 16 analytes in each GCF sam-
ple with the following mean minimum detection level
in nanograms per milliliter: interferon gamma (IFN-g),
0.31; IL-17, 0.39; IL-1b, 0.27; IL-1 receptor antago-
nist (IL-1ra), 2.06; regulated upon activation in nor-
mal T cells expressed and secreted (RANTES), 1.08;
monocyte chemotactic peptide 1 (MCP-1), 0.95; mac-
rophage inflammatory protein 1 beta (MIP-1b), 2.12;
epithelial-neutrophil activating peptide 78 (ENA-78),
2.71; IL-4, 1.75; IL-5, 0.33; IL-6, 0.36; IL-8, 0.39; tu-
mor necrosis factor-alpha (TNF-a), 0.47; granulocyte-
colony stimulating factor (G-CSF), 0.57; IL-10, 0.13;
and MIP-1a, 3.10. Means and SE were computed for
the five BGI groups. Samples with mediator levels that
were below the detection level or out of range exceed-
ing the maximum standard were included in these
analyses truncating the values using the lowest or
highest standard value, respectively. The mean val-
ues for each mediator were adjusted for age, gender,
and diabetes, and statistical differences between groups
tested using general linear models with a P value <0.05
considered significant.
RESULTS
Exploratory Analyses to Create Definitions of
Disease That Include Biologic Phenotype Data
Because all subjects in this adult population (mean
age, 62.4 [SD 5.6] years; range, 52 to 74 years)
had some attachment loss and could be considered
having periodontitis, we use the term BGI as a prefix
to clarify that the clinical state is defined without con-
sideration of attachment level or the position of the
free gingival margin relative to the CEJ. Five levels
of disease were defined based upon shallow (PD £3
mm) or deep (PD ‡4 mm) PDs in combination with
low or high bleeding scores.9 Two shallow PD groups
were defined: BGI-H was defined as all PDs £3 mm and
<10% BOP (971 subjects); BGI-gingivitis (BGI-G;
1,023 subjects) was defined as individuals with all
PDs £3 mm and ‡10% BOP. Three BGI deep lesion
(DL) groups were created based upon whole-mouth
PD and BOP scores. BGI-deep lesion/low bleeding
(BGI-DL/LB; 1,217 subjects) was defined as one or
more sites with PD ‡4 mm and BOP extent scores
<10%; BGI-DL/moderate bleeding (BGI-DL/MB;
2,685 subjects) was defined as one or more sites with
PD ‡4 mm and BOP extent scores between 10% and
50%; and BGI-DL/severe bleeding (BGI-DL/SB; 879
subjects) included subjects with one or more sites with
PD ‡4 mm and BOP extent scores ‡50%. These five
BGI categories created a gradient of disease severity
while creating categories that differed by biologic phe-
notype at site-based and patient-based levels.
The site-based analysis of the association between
PD and BOP for each of the five BGI categories
appears in Figure 1. The mean extent BOP score is
J Periodontol • October 2007
shown pooling all sites in all patients within each BGI
disease level at each PD to create a PD–bleeding-
response curve for each category. These curves are
similar to a dose-response function, whereby the dose
is the level of the PD and the response is the mean
bleeding score at that depth. Simply stated, the two
categories BGI-H and BGI-G are subjects with shallow
pockets with low BOP (<10%) or high BOP (‡10%),
respectively. The BGI-DL/LB, BGI-DL/MB, and BGI-
DL/SB subjects have deeper pockets with low,
moderate, and severe BOP, respectively. Although
patient-based PD and BOP summary scores were
used to create the five groups, the site-level bleeding
status differed significantly among groups across var-
ious PDs, except for the low BOP groups BGI-H and
BGI-DL/LB, which are indistinguishable for PDs £3
mm. For example, if one considers only 3-mm PD
sites, 12.4% exhibited BOP in BGI-H, 58.6% had BOP
among BGI-G, 10.7% had BOP among BGI-DL/LB,
45.5% had BOP in BGI-DL/MB, and 82.0% had BOP
in BGI-DL/SB. This finding suggests that the underly-
ing biology inducing the BOP response associated
with 3-mm pockets is fundamentally different among
subjects in the BGI-G, BGI-DL/MB, and BGI-DL/SB
categories compared to the two lowest BOP groups
(BGI-H and BGI-DL/LB). Alternatively, the underlying
biology may be identical, but simply more prevalent
at shallow sites among subjects with greater disease.
Similarly, at 5-mm PDs, 31.8% of the sites exhibited
BOP among subjects with BGI-DL/LB, which is signif-
icantly lower than the 67.2% BOP among BGI-DL/MB
subjects and the 91.9% BOP among BGI-DL/SB sub-
jects. Thus, combining PD and BOP measurements at
Figure 1.
The percentage of sites with BOP are shown as a function of PD for
each of the five BGI case definitions based upon PD and BOP scores
for each subject. Data represent pooled site-based variables for each
level for each BGI group: 971 BGI-H; 1,023 BGI-G; 1,217 BGI-DL/LB;
2,685 BGI-DL/MB; and 872 BGI-DL/SB.
Offenbacher, Barros, Singer, Moss, Williams, Beck
a site level to define a PD–BOP response curve creates
logical clinical disease categories based upon clinical
signs and suggests that the differences in bleeding
responses seen between clinical phenotypes based
on PD may reflect differing subject-level biologic phe-
notypes.
Clinical Characteristics of BGI Classifications
The subject characteristics for the five BGI category
distributions with 14.3% as BGI-H, 15.1% as BGI-G,
18.0% as BGI-DL/LB, 39.7% as BGI-DL/MB, and
12.9% as BGI-DL/SB are shown in Table 1. There were
significant BGI group differences with regard to race,
gender, diabetes, education, smoking (history and
pack-year), and body mass index (BMI).
A variety of periodontal measurements, the GCF
IL-1b and PGE2 levels, and the oral health behaviors
are shown for the five BGI categories in Table 2. As ex-
pected, there are significant differences among all
clinical measurements, and these values are provided
for descriptive and comparative purposes. The mean
extent of teeth with interproximal clinical attachment
loss (ICAL) ‡3 mm was 23.9%, even among the
healthiest BGI-H individuals, i.e., no clinically signifi-
cant BOP or pocketing; it increased across the more
diseased groups to 73.0% among the BGI-DL/SB
group. Within the BGI-H group of 971 subjects, only
205 (3% of the total) had no sites with ICAL ‡3 mm.
Within the 1,023 BGI-G subjects, only 161 (2.4% of
the total) had no sites with ICAL ‡3 mm. Within each
of the five BGI groups, those subjects without ICAL ‡3
mm were indistinguishable from those with one or
more sites in terms of biologic phenotype and, there-
fore, were considered in aggregate. Similarly, the
prevalence of disease using the Centers for Disease
Control and Prevention (CDC) and Oral Conditions
and Pregnancy (OCAP) definitions22,23 of disease
are shown for comparison. The levels of GCF IL-1b
and PGE2 also are lowest among those with BGI-H, in-
creasing significantly among subjects with BGI-G,
BGI-DL/LB, BGI-DL/MB, and BGI-DL/SB. Higher
levels of these GCF mediators are seen in high BOP
conditions (BGI-G and BGI-DL/SB). These five cate-
gories are clearly different from traditional definitions
of health, gingivitis, mild periodontitis, moderate peri-
odontitis, and severe periodontitis; however, they dis-
play a similar gradient with increasing severity and
extent of clinical signs from BGI-H through BGI-DL/SB
categories. However, there is an important exception
to this trend with the BGI-DL/LB category, which has
lower plaque (P = 0.03) and GI scores (P <0.001)
but deeper pocketing compared to the healthiest
group. The BGI-DL/LB category has an average of
5.69% sites with PD ‡4 mm, which is equivalent to an
average of eight sites per subject with PD ‡4 mm,
as computed using the mean number of teeth.
1915
Disease at the Biofilm–Gingival Interface Volume 78 • Number 10

Table 1.
Subject Characteristics by BGI Level

Subject Characteristic BGI-H BGI-G BGI-DL/LB BGI-DL/MB BGI-DL/SB P Value

Subjects (N [% total]) 971 (14.3%) 1,023 (15.1%) 1,217 (18.0%) 2,685 (39.7%) 872 (12.9%)
Age (years; mean [SE]) 62.2 (0.2) 62.4 (0.2) 62.4 (0.2) 62.4 (0.1) 62.8 (0.2) 0.20
Race: African American 314 (32.3%) 257 (25.1%) 140 (11.5%) 314 (11.7%) 275 (31.5%)

Race: white 657 (67.7%) 766 (74.9%) 1,077 (88.5%) 2,371 (88.3%) 597 (68.5%) <0.0001
Gender: male 261 (26.9%) 378 (37.0%) 586 (48.2%) 1,352 (50.4%) 516 (59.2%)
Gender: female 710 (73.1%) 645 (63.1%) 631 (51.9%) 1,333 (49.7%) 356 (40.8%) <0.0001
Diabetes: yes 105 (10.9%) 164 (16.1%) 128 (10.6%) 357 (13.4%) 180 (20.9%)

Diabetes: no 855 (89.1%) 853 (83.9%) 1,081 (89.4%) 2,316 (86.6%) 680 (79.1%) <0.0001
Education: basic 141 (14.5%) 170 (16.7%) 86 (7.1%) 305 (11.4%) 215 (24.7%)
Education: intermediate 401 (41.3%) 440 (43.1%) 489 (40.2%) 1,239 (46.2%) 344 (39.5%)

Education: advanced 428 (44.1%) 410 (40.2%) 641 (52.7%) 1,139 (42.5%) 311 (35.8%) <0.0001
Smoker: current 102 (10.6%) 88 (8.6%) 179 (14.7%) 324 (12.1%) 147 (17.0%)
Smoker: former 355 (36.8%) 342 (33.5%) 560 (46.1%) 1,142 (42.6%) 345 (39.9%)

Smoker: never 509 (52.7%) 590 (57.8%) 476 (39.2%) 1,215 (45.3%) 372 (43.1%) <0.0001
Pack-years (mean [SE]) 11.3 (0.7) 9.2 (0.6) 16.2 (0.6) 13.7 (0.4) 17.2 (0.7) <0.0001
BMI (kg/m2; mean [SE]) 28.5 (0.2) 28.8 (0.2) 27.7 (0.2) 28.6 (0.1) 29.3 (0.2) <0.0001
The subject characteristics for the five BGI levels defining periodontal status using only PD and BOP extent scores. These disease classifications do not
incorporate attachment level measurements. Basic education includes up through high school; intermediate education is some college; and advanced is
some graduate education.

Compared to the BGI-DL/MB and BGI-DL/SB groups,
the BGI-DL/LB group showed a significant trend for a
greater prevalence of reported brushing two or more
times per day, flossing daily, and visiting the dentist
within the last year, a finding that is consistent with
the lower plaque and GI scores.
Biologic Phenotype of BGI Classifications
The levels of periodontal biofilm organisms for each
BGI group appear in Table 3 and the IgG titers appear
in Table 4. The group differences in bacterial level and
IgG titers are significant overall by the Hotelling T2 test
(P <0.0001), which considers multiple comparisons.
Relative to the BGI-H category, BGI-G had signifi-
cantly higher total counts and orange complex
counts, as well as specific elevations in P. intermedia,
Prevotella nigrescens, and C. rectus (Table 3). Within
this group, the dominant microbial shift is the signifi-
cant twofold increase in C. rectus. The total bacterial
counts and the total red and orange complex counts
are significantly lower among BGI-DL/LB subjects
compared to the BGI-H group, consistent with the
clinical presentation of lower plaque scores and lower
mean GI scores but the trend for higher BOP and PDs
(Tables 2 and 3). All three of the red complex bacteria
are significantly lower in the BGI-DL/LB group com-
pared to the BGI-H group, as are the counts of C. rec-
tus, F. nucleatum, and A. actinomycetemcomitans.
The BGI-DL/MB subjects had significantly higher to-
tal, red complex, and orange complex microbial
counts compared to BGI-H. Among the BGI-DL/MB
subjects, the dominant red complex organisms were
T. forsythia and T. denticola, which were elevated two-
fold and 1.8-fold, respectively. Among the BGI-DL/
SB subjects, the total bacterial count was elevated
2.5-fold compared to BGI-H subjects. This increase
extended across the entire biofilm, including orga-
nisms of the red and orange complexes and A. actino-
mycetemcomitans.
A similar, but not identical, pattern for the BGI
group differences in serum IgG titers to the plaque
bacteria are shown in Table 4, with a significant in-
crease in total IgG levels in the two most severe dis-
ease categories. Compared to the BGI-H group, the
BGI-G group had increased titers to C. rectus and a
trend for higher V. parvula titers. The BGI-DL/LB
group had significantly elevated titers to S. sanguinis
and A. viscosus compared to BGI-H. Stronger IgG

1916
J Periodontol • October 2007 Offenbacher, Barros, Singer, Moss, Williams, Beck

Table 2.
Periodontal Clinical Measurements (mean [SE]), GCF IL-1b and PGE2 Levels
(mean ng/ml [SE]), and Oral Health Behaviors by BGI Category

All PD £3 mm 1+ Sites With PD ‡4 mm

BOP £10% BOP >10% BOP £10% >10% BOP <50% BOP ‡50%
Periodontal Measure BGI-H BGI-G BGI-DL/LB BGI-DL/MB BGI-DL/SB P Value

Teeth (N) 19.7 (0.2) 18.5 (0.2) 23.9 (0.2) 23.5 (0.1) 19.4 (0.2) <0.0001
Plaque score 0.34 (0.02) 0.54 (0.02) 0.29 (0.01) 0.48 (0.01) 0.99 (0.02) <0.0001
GI score 0.33 (0.02) 0.56 (0.02) 0.16 (0.01) 0.35 (0.01) 1.15 (0.02) <0.0001
Extent BOP 3.3 (0.38) 30.1 (0.36) 4.6 (0.34) 26.1 (0.23) 71.3 (0.40) <0.0001
PD 1.47 (0.01) 1.52 (0.01) 1.84 (0.01) 2.00 (0.01) 2.69 (0.02) <0.0001
Extent PD ‡4 mm – – 5.69 (0.27) 8.65 (0.18) 22.61 (0.32) <0.0001
Extent PD ‡5 mm – – 1.61 (0.20) 3.38 (0.13) 12.97 (0.23) <0.0001
AL 1.38 (0.03) 1.41 (0.03) 1.67 (0.03) 1.76 (0.02) 2.93 (0.03) <0.0001
Extent AL ‡3 mm 12.5 (0.66) 13.6 (0.64) 21.41 (0.59) 23.4 (0.40) 48.7 (0.70) <0.0001
Extent AL ‡4 mm 4.2 (0.53) 4.9 (0.52) 9.6 (0.47) 11.0 (0.32) 30.9 (0.56) <0.0001
Extent of teeth with ICAL ‡3 mm 23.9 (0.84) 27.1 (0.81) 39.7 (0.75) 44.9 (0.50) 73.0 (0.88) <0.0001
Extent of teeth with ICAL ‡3 mm – – 6.5 (0.40) 11.7 (0.30) 31.7 (0.50) <0.0001
and PD ‡5 mm
CDC definition* (n [%])*
Healthy 796 (82.0%) 832 (81.3%) 412 (33.9%) 701 (26.1%) 62 (7.1%)
Moderate 175 (18.0%) 191 (18.7%) 654 (53.7%) 1,403 (52.3%) 361 (41.4%)
Severe 0 (0.0%) 0 (0.0%) 151 (12.4%) 581 (21.6%) 449 (51.5%) <0.0001
OCAP definition† (n [%])
Health 613 (63.1%) 219 (21.4%) 0 (0.0%) 0 (0.0%) 0 (0.0%)
Mild 358 (36.9%) 804 (78.6%) 1,043 (85.7%) 1,905 (71.0%) 390 (44.7%)
Moderate/severe 0 (0.0%) 0 (0.0%) 174 (14.3%) 780 (29.1%) 482 (55.3%) <0.0001

GCF mediators (ng/ml)

IL-1b 104.1 (4.8) 148.7 (4.7) 122.5 (4.3) 141.4 (2.9) 194.7 (5.3) <0.0001
PGE2 198.9 (6.3) 249.0 (6.3) 218.1 (5.3) 234.7 (3.6) 254.4 (6.8) <0.0001

Oral health behaviors

Brush two or more 755 (78.3) 725 (71.4) 890 (73.4) 1,822 (68.1) 516 (59.4) <0.0001
times/day (n [%])
Floss daily (n [%]) 689 (71.5) 619 (61.5) 929 (76.6) 1,693 (63.3) 387 (44.6) <0.0001
Dental visit £1 year (n [%]) 736 (76.5) 745 (73.3) 1,049 (86.6) 2,173 (81.3) 531 (61.4) <0.0001
AAP = American Academy of Periodontology; AL = attachment loss; – = not applicable.
* CDC/AAP definition of periodontal disease was derived independently during a 2003 meeting by a Working Group on Surveillance Systems for Periodontal
Infections meeting. This three-level definition is as follows: severe periodontitis: at least two interproximal sites (not on same tooth) with CAL ‡6 mm and
at least one interproximal site with PD ‡5 mm; moderate periodontitis: at least two interproximal sites with CAL 4 or 5 mm (not on same tooth) and
no interproximal sites with CAL ‡6 mm; and healthy/gingivitis: individuals not meeting the above definitions.
† In OCAP, periodontal disease was defined as a three-level variable: health, mild disease, or moderate-severe disease. Mothers with periodontal health were
defined as having all PDs £4 mm, with no 3- or 4-mm sites showing BOP. Moderate-severe periodontitis subjects had 15 or more sites with PDs >4 mm; the
remaining subjects were placed in the mild category.

responses are seen among the BGI-DL/MB group that
displayed elevated titers for the total biofilm, red
complex organisms (T. forsythia and T denticola),
C. rectus, P. micra, A. actinomycetemcomitans, and
S. sanguinis compared to the BGI-H group. In the
BGI-DL/SB group, the total microbial counts in-
creased 2.5-fold compared to the BGI-H group (Table
3). This was accompanied by a 27% increase in IgG
level titer against the total biofilm. However, two bac-
terial IgG responses are rather dramatic, with P. gingi-
valis titers increasing 3.1-fold and C. rectus IgG levels
increasing 1.8-fold (followed by A. actinomycetemco-
mitans titers, 1.4-fold), compared to BGI-H. BGI-DL/
SB also demonstrated moderately increased titers to
T. denticola, P. intermedia, P. micra, and C. ochracea.
To determine whether there were different underly-
ing biologic phenotypes for each of the four BGI dis-
ease categories, we combined the patient background

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Disease at the Biofilm–Gingival Interface Volume 78 • Number 10

Table 3.
Level of Selected Periodontal Organisms by BGI Category Median Counts ·103
(interquartile range)

BGI-H BGI-G BGI-DL/LB BGI-DL/MB BGI-DL/SB

IgG Antibody (n = 186) (n = 204) (n = 261) (n = 562) (n = 218) P Value*
Total biofilm counts 27.3 (11.8 to 77.9) 37.8 (17.9 to 108.1)† 22.0 (9.7 to 44.9)†‡ 40.3 (16.4 to 186.7)†‡ 67.1 (26.4 to 335.3)†‡ <0.0001
† †‡
P. gingivalis 1.3 (0.0 to 4.0) 1.1 (0.0 to 3.2) 0.7 (0.0 to 2.3) 1.5 (0.0 to 4.4) 2.3 (0.2 to 6.9) <0.0001
T. forsythia 1.0 (0.0 to 3.9) 1.5 (0.0 to 4.8) 0.5 (0.0 to 2.4)† 2.0 (0.0 to 7.1)†‡ 3.3 (0.8 to 14.7)†‡ <0.0001
T. denticola 2.1 (0.0 to 8.0) 3.0 (0.0 to 9.8) 1.2 (0.0 to 5.7)† 3.8 (0.0 to 15.3)† 7.0 (1.4 to 24.5)†‡ <0.0001
Red biofilm counts 5.2 (1.9 to 15.8) 7.1 (2.5 to 16.5) 3.6 (1.3 to 10.1)† 8.1 (2.7 to 25.5)†‡ 13.6 (4.6 to 50.6)†‡ <0.0001

P. intermedia 3.7 (0.0 to 16.9) 6.3 (0.0 to 18.9) 2.7 (0.0 to 10.4) 6.1 (0.0 to 23.8) 10.1 (0.0 to 43.5)†‡ <0.0001
P. nigrescens 2.7 (0.0 to 9.9) 4.6 (0.0 to 17.3)† 1.1 (0.0 to 7.2) 5.8 (0.0 to 24.7)†‡ 8.1 (0.5 to 34.4)†‡ <0.0001
C. rectus 2.0 (0.0 to 9.3) 4.0 (0.8 to 13.6)† 1.2 (0.0 to 5.7)† 3.9 (0.0 to 18.3)†‡ 6.8 (1.8 to 29.3)†‡ <0.0001
F. nucleatum 5.3 (0.0 to 22.7) 9.0 (0.0 to 34.0) 2.5 (0.0 to 11.4)† 9.9 (0.0 to 65.2)†‡ 14.8 (0.6 to 93.8)†‡ <0.0001
Orange biofilm counts 21.4 (6.3 to 61.5) 29.1 (11.2 to 90.1)† 15.1 (5.3 to 31.5)†‡ 29.4 (9.1 to 140.6)†‡ 48.8 (16.2 to 247.1)†‡ <0.0001

A. actinomycetemcomitans 1.6 (0.0 to 5.8) 2.4 (0.2 to 7.8) 1.1 (0.0 to 4.0)† 2.7 (0.3 to 8.5)† 3.5 (1.0 to 10.8)†‡ <0.0001
* P values are for group differences as determined using non-parametric one-way analysis of rank scores.
† Statistically significant compared to BGI-H at P <0.05.
‡ Statistically different compared to BGI-H at P <0.01.

characteristics, plaque levels, GCF inflammatory re- cantly increased plaque, and the subjects tended to
sponse, microbial plaque composition, and serum be white, male, and diabetic. Plaque, race, gender,
IgG response (Table 5) and developed generalized lo- and diabetes were significant in all four BGI disease
gits models. Compared to BGI-H, BGI-G had signifi- models, with plaque, gender, and diabetes having

Table 4.
Biofilm-Specific Serum IgG Levels (ng/ml [SD]) by BGI Level

IgG Antibody BGI-H BGI-G BGI-DL/LB BGI-DL/MB BGI-DL/SB P Value*

‡ †‡
Total biofilm IgG 1,067 (53) 1,140 (51) 1,143 (50) 1,204 (32) 1,353 (55.5) 0.003
P. gingivalis 53.8 (9.1) 50.7 (8.8) 60.9 (8.4) 69.4 (5.5) 168.4 (9.5)†‡ <0.0001
T. forsythia 37.8 (2.8) 37.8 (2.7) 40.1 (2.6) 46.6 (1.7)†‡ 45.5 (2.9) 0.009
T. denticola 28.9 (1.6) 31.0 (1.5) 28.1 (1.5) 33.4 (1.0)†‡ 36.5 (1.6)†‡ 0.003
Red biofilm IgG 122.1 (11.3) 120.2 (10.9) 130.6 (10.5) 150.9 (6.9)‡ 251.8 (11.7)†‡ <0.0001
P. intermedia 82.2 (5.8) 74.5 (5.7) 77.2 (5.4) 83.1 (3.5) 108.0 (6.1)‡ 0.0005
P. nigrescens 138.2 (8.6) 153.6 (8.4) 128.1 (8.0) 143.7 (5.3) 144.0 (9.0) 0.27
C. rectus 25.7 (2.4) 33.1 (2.3)‡ 25.5 (2.2) 33.1 (1.4)†‡ 47.4 (2.5)†‡ <0.0001
F. nucleatum 16.5 (1.3) 19.7 (1.3) 14.2 (1.2) 17.0 (0.8) 16.3 (1.4) 0.04
P. micra 88.3 (6.4) 96.6 (6.2) 104.6 (5.9) 108.8 (3.9)†‡ 112.1 (6.7)†‡ 0.03
Orange biofilm IgG 351.2 (18.6) 377.7 (18.0) 350.2 (17.3) 386.3 (11.3) 428.2 (19.4)†‡ 0.02
A. actinomycetemcomitans 122.8 (7.8) 136.0 (7.6) 120.5 (7.3) 146.7 (4.8)†‡ 177.2 (8.2)†‡ <0.0001
E. corrodens 18.8 (1.6) 21.6 (1.5) 15.3 (1.5) 18.8 (1.0) 21.8 (1.6) 0.02
C. ochracea 31.6 (2.0) 34.7 (2.0) 30.3 (1.9) 34.3 (1.2) 42.0 (2.1)†‡ 0.0006
V. parvula 11.0 (1.1) 14.1 (1.1)‡ 12.6 (1.0) 12.0 (0.7) 13.6 (1.1) 0.23
S. intermedius 92.6 (9.7) 91.4 (9.4) 108.4 (9.0) 112.0 (5.9) 94.8 (10.1) 0.20
S. oralis 61.6 (7.1) 76.0 (6.9) 68.1 (6.7) 77.1 (4.3) 81.7 (7.5) 0.24
S. sanguinis 65.0 (6.9) 69.0 (6.7) 88.4 (6.4)‡ 87.0 (4.2)†‡ 78.1 (7.2) 0.02
S. noxia 92.5 (11.9) 108.7 (11.6) 95.0 (11.1) 69.9 (7.2) 70.7 (12.5) 0.03
A. viscosus 76.0 (7.8) 79.3 (7.5) 104.7 (7.2)†‡ 92.2 (4.7) 80.3 (8.1) 0.03
Other biofilm IgG 574.4 (33.2) 631.8 (32.2) 643.8 (30.8) 651.0 (20.2)‡ 662.4 (34.7) 0.33
Bold text highlights statistically significant findings.
* P values are for group differences as determined by least square procedures.
† Statistically significant compared to BGI-H at P <0.01.
‡ Statistically different compared to BGI-H at P <0.05.

1918
J Periodontol • October 2007
Table 5.
Generalized Logits Models for BGI-G, BGI-DL/LB, BGI-DL/MB, and BGI-DL/SB Using
the BGI-H Level as a Reference
Variable BGI-G
† †
Extent PI >1 (10%) 1.15 (1.10 to 1.19)
Race (white) 3.09 (2.11 to 4.52)†
Gender (male) 1.74 (1.30 to 2.33)†
Diabetes 1.76 (1.17 to 2.66)†
GCF IL-1b (0.1 ng/ml) 1.43 (1.24 to 1.66)†
GCF PGE2 (0.1 ng/ml) 1.18 (1.10 to 1.27)†
C. rectus IgG (0.1 mg/ml) 1.44 (1.14 to 1.83)†
P. gingivalis IgG (0.1 mg/ml) 1.00 (0.90 to 1.11)
PI = plaque index.
* Also adjusted for age, education, number of teeth, smoking (current, former, never, and pack-years), and BMI.
† Statistically significant at P <0.05.
larger effects in the BGI-DL/SB group. Smoking his-
tory or pack-years were not significantly different
among these four BGI groups compared to BGI-H.
Compared to BGI-H subjects, all four disease groups
had significantly increased GCF levels of IL-1b. GCF
PGE2 was significantly elevated in the BGI-G, BGI-DL/
MB, and BGI-DL/SB, groups compared to the BGI-H
group. In all four BGI models, the serum IgG level sup-
planted the level of organism present. The BGI-G
group had significantly increased serum levels of C.
rectus IgG. The C. rectus–specific antibody was se-
lected among all IgG titers as the single best predictor
of BGI-G using logistic regression to select the best
single model. For the BGI-DL/LB, BGI-DL/MB, and
BGI-DL/SB groups, P. gingivalis was the best single
predictor. P gingivalis followed by C. rectus were the
two best IgG predictors of BGI-DL/MB or BGI-DL/
SB using logistic regression to identify the best two
predictors. For this reason, the IgG titers for both or-
ganisms are included in these models; however, the
P. gingivalis titer does not contribute significantly to
BGI-G, and the C. rectus titer does not contribute sig-
nificantly to the BGI-DL/LB model. Collectively, these
data strongly support the concept that BGI-G is asso-
ciated with an increased C. rectus titer without signif-
icant P. gingivalis exposure and that BGI-DL/MB and
BGI-DL/SB are characterized by the exposure of C.
rectus and the additional microbial antigenic expo-
sure of P. gingivalis. This statement is supported fur-
ther by the fact that two independent methods (IgG
titer and DNA-based microbial counts) both selected
for these two microbes. Furthermore, in these com-
posite phenotypes that define the BGI disease states,
Offenbacher, Barros, Singer, Moss, Williams, Beck
Odds Ratio (95% confidence interval)*
BGI-DL/LB BGI-DL/MB BGI-DL/SB
†
1.05 (>1.00 to 1.09) 1.22 (1.18 to 1.27) 1.38 (1.32 to 1.45)†
2.32 (1.57 to 3.42)† 3.97 (2.80 to 5.61)† 2.79 (1.83 to 4.25)†
2.46 (1.87 to 3.23)† 2.56 (1.99 to 3.29)† 4.25 (3.09 to 5.84)†
1.54 (1.01 to 2.34)† 1.65 (1.14 to 2.41)† 2.22 (1.44 to 3.43)†
1.22 (1.05 to 1.41)† 1.47 (1.29 to 1.69)† 1.80 (1.55 to 2.08)†
1.05 (0.97 to 1.14) 1.11 (1.03 to 1.18)† 1.16 (1.07 to 1.26)†
1.02 (0.79 to 1.32) 1.31 (1.05 to 1.64)† 1.45 (1.13 to 1.87)†
1.14 (1.04 to 1.25)† 1.17 (1.07 to 1.27)† 1.25 (1.14 to 1.37)†
it is clear that within each diagnostic category there
seem to be different biologic aspects related to the
inflammatory and antibody response that we collec-
tively refer to as a biologic phenotype.
The relatively large sample size of this molecular
epidemiologic study enabled us to describe those
biologic phenotypes within each of the disease cate-
gories (Table 5) and to define those that contribute
to more severe disease within each BGI group (Table
6). Using multivariable linear regression models to ex-
plain the extent of BOP and extent PD ‡4 mm as de-
pendent variables, the relative contribution of each
biologic parameter was examined adjusting for other
factors. With each 10% increase in plaque extent
scores above 1, there was a significant 1.2% increase
in extent BOP scores in the BGI-G subjects. The extent
of plaque had significant, but smaller, effects on
bleeding scores among BGI-DL/LB, BGI-DL/MB,
and BGI-DL/SB subjects. Increasing plaque levels
were associated with greater extent of PDs (‡4 mm)
only among the BGI-DL/LB and BGI-DL/MB groups,
and the effects of plaque on BOP were much stronger
than the effects on PDs. Plaque scores had no effect on
the extent PD ‡4 mm among BGI-DL/SB subjects.
Greater BOP and extent PD ‡4 mm scores were found
among African American BGI-DL/SB subjects (nega-
tive b coefficient), whereas white BGI-DL/LB subjects
were more likely to have higher BOP levels. Diabetes
did not significantly increase the extent of BOP or
extent PD ‡4 mm within disease groups. Former
smoking history significantly inhibited BOP among
BGI-DL/MB subjects, and current smoking had a
major impact on extent PD ‡4 mm among BGI-DL/
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Disease at the Biofilm–Gingival Interface Volume 78 • Number 10

Table 6.
Multivariable Regression Models* for Extent of BOP and PD ‡4 mm Within Each BGI
Disease Level

Extent BOP Extent PD ‡4 mm

Variable BGI-G BGI-DL/LB BGI-DL/MB BGI-DL/SB BGI-DL/LB BGI-DL/MB BGI-DL/SB

Extent PI >1 1.21 (<0.0001) 0.18 (<0.0001) 0.50 (<0.0001) 0.74 (0.001) 0.16 (0.03) 0.31 (<0.0001) 0.27 (0.30)
(per 10%)
Race (white) -0.24 (0.92) 0.95 (0.04) 0.70 (0.50) -8.86 (<0.0001) 0.01 (0.99) 0.29 (0.68) -8.01 (0.0004)

Diabetes 3.46 (0.11) 0.51 (0.18) -0.45 (0.59) 1.53 (0.38) -0.38 (0.54) -0.25 (0.65) 3.63 (0.08)
Smoking: Ref Ref Ref Ref Ref Ref Ref
never
Smoking: -3.30 (0.11) 0.15 (0.61) -1.50 (0.04) -1.24 (0.49) -0.39 (0.43) 0.26 (0.58) 1.13 (0.60)
former
Smoking: -2.00 (0.59) 0.44 (0.33) -2.26 (0.055) 0.62 (0.82) 0.99 (0.18) 4.14 (<0.0001) 8.84 (0.005)
current
Smoking: -0.05 (0.95) -0.12 (0.13) 0.02 (0.91) 0.04 (0.92) 0.61 (<0.0001) 0.56 (<0.0001) 0.50 (0.28)
pack-years
BMI -0.08 (0.54) 0.03 (0.22) 0.01 (0.80) 0.19 (0.15) 0.02 (0.70) 0.15 (<0.0001) 0.23 (0.16)

GCF IL-1b 1.51 (0.01) 0.21 (0.11) 0.23 (0.40) 0.64 (0.004) 0.80 (0.0002) 0.47 (0.01) 1.05 (0.0001)
(0.1 ng/ml)
P. gingivalis IgG 0.31 (0.57) 0.19 (0.002) 0.33 (0.04) 0.29 (0.02) 0.14 (0.14) 0.35 (0.0008) 0.70 (<0.0001)
(0.1 mg/ml)
Bold values highlight statistically significant findings.
PI = plaque index; Ref = reference group to which other categories are compared.
Table shows only significant independent variables, parameter estimates (b coefficient), and (P values).
* Also adjusted for age, gender, smoking, education, number of teeth, GCF PGE2, and C. rectus IgG titer.

MB and BGI-DL/SB subjects. BMI had a significant ef-
fect on extent PD ‡4 mm within the BGI-DL/MB group.
Diabetes and BMI had a non-significant trend toward
increasing the extent PD ‡4 mm in BGI-DL/SB. In-
creased GCF levels of IL-1b significantly increased
BOP and extent PD ‡4 mm across all disease cate-
gories. For example, for each 1 ng/ml increase in
GCF IL-1b level, there was a 10.5% increase in extent
PD ‡4 mm among BGI-DL/SB, which, on average,
was 12 more sites with PD ‡4 mm. The BGI-DL/LB
subjects had greater extent PD ‡4 mm with increasing
GCF IL-1b. Although GCF PGE2 levels were higher
among all three disease groups (Table 5), the levels
were not statistically significantly associated with in-
creased disease expression within these diseased
groups.
Serum titers to C. rectus, although predictive of the
presence of BGI-G (Table 5), did not significantly pre-
dict the severity of the condition, nor did they contrib-
ute to the extent of BOP or PD ‡4 mm among any BGI
level. In contrast, the IgG levels to P. gingivalis were
increased with higher BOP scores among the BGI-
DL/MB and BGI-DL/SB groups, as well as increased
extent PD ‡4 mm. The increased P. gingivalis IgG ti-
ters had the largest effect on increasing PDs in the
BGI-DL/SB subjects.
The GCF levels of 16 inflammatory mediators
among the subset of 180 individuals within the five
BGI groups (randomly selected, but balanced on
age, race, and gender) appear in Table 7. These mean
values are adjusted for the extent of PD ‡4 mm, race,
gender, age, diabetes, and BMI. Thus, these may re-
flect subject-level differences in the qualitative and
quantitative nature of the inflammatory response, in-
dependent of the level of disease or microbial burden
present. Several mediators were not significantly dif-
ferent among groups, including IL-10, -4, -5, -8, -17,
and -1ra, MIP-1a and -1b, IFN-g, and ENA-78. The
adjusted means confirm that GCF IL-1b showed a
trend for increases across diseased groups, showing
statistically significant increases among the BGI-
DL/MB and BGI-DL/SB groups using a multiplex
analytical platform. GCF PGE2 levels showed similar
increases, with a significant 1.75-fold increase among
the BGI-DL/SB group (P = 0.006; data not shown).
These data also demonstrated that there were addi-
tional inflammatory responses within the BGI-DL/
SB group, with a 4.5-fold increase in MCP-1 and a
6.2-fold increase in IL-6. The 2.1-fold increase in
TNF-a should be interpreted with caution because there
were no overall statistical differences for the group
(P = 0.29), nor were there overall group differences

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J Periodontol • October 2007 Offenbacher, Barros, Singer, Moss, Williams, Beck

Table 7.
Detailed Proteomic GCF Inflammatory Mediator Analysis of Subset of Subjects
From BGI Groups

GCF Mediators BGI-H BGI-G BGI-DL/LB BGI-DL/MB BGI-DL/SB

(ng/ml; mean [SE]) (n = 60) (n = 60) (n = 10) (n = 20) (n = 30) P Value*
IL-10 1.40 (0.12) 1.52 (0.12) 1.48 (0.28) 1.35 (0.20) 1.54 (0.22) 0.90
† †‡
IL-1b 253.6 (40.4) 342.3 (39.6) 291.5 (91.4) 437.9 (66.7) 518.6 (70.9) 0.03

IL-4 1.07 (0.32) 1.50 (0.32) 0.73 (0.73) 1.80 (0.53) 2.06 (0.57) 0.46
IL-5 0.06 (0.03) 0.07 (0.03) 0.08 (0.06) 0.13 (0.04) 0.05 (0.05) 0.68
IL-6 0.90 (0.64) 1.93 (0.63) 2.22 (1.44) 1.74 (1.05) 5.62 (1.12)†‡ 0.02
IL-8 1,205 (140) 1,211 (137) 1,155 (316) 1,430 (231) 1,698 (245) 0.53

TNF-a 1.78 (0.39) 2.12 (0.38) 2.35 (0.88) 2.75 (0.64) 3.70 (0.68)† 0.29
†
G-CSF 82.30 (15.0) 108.1 (14.7) 65.7 (33.9) 126.9 (24.7) 148.7 (26.3) 0.17
MIP-1a 25.6 (3.94) 32.42 (3.87) 27.8 (8.93) 28.6 (6.51) 40.6 (6.92) 0.37

MIP-1b 29.3 (5.17) 38.6 (5.07) 29.81 (11.70) 27.15 (8.53) 41.2 (9.07) 0.47
RANTES -0.13 (0.36) 0.33 (0.35) 0.28 (0.81) 0.12 (0.59) 2.45 (0.63)†‡ 0.02
MCP-1 3.18 (1.59) 7.12 (1.56) 6.97 (3.59) 5.61 (2.62) 14.44 (2.78)†‡ 0.02

IFN-g 2.03 (0.73) 2.12 (0.72) 2.18 (1.66) 3.56 (1.21) 4.60 (1.29) 0.56
IL-17 0.28 (0.11) 0.55 (0.11) 0.30 (0.25) 0.40 (0.18) 0.66 (0.20) 0.29
ENA-78 84.9 (9.5) 92.1 (9.4) 57.8 (21.6) 58.9 (15.7) 86.7 (16.7) 0.30

IL-1ra (/100) 21.5 (0.97) 23.6 (0.95) 21.6 (2.20) 24.2 (1.60) 25.6 (1.70) 0.25
Significant values are highlighted in bold.
Data have been adjusted for extent PD ‡4 mm, race, gender, age, diabetes, and BMI.
* P values are for group differences as determined by mean least squares.
† Statistically elevated compared to BGI-H at P <0.05.
‡ Statistically elevated compared to BGI-H at P <0.01.

in G-CSF. Although there was a statistically signifi-
cant increase in RANTES in BGI-DL/SB, this GCF
mediator was detectible in only 1.7% of all 720 GCF
samples analyzed.
DISCUSSION
These data strongly support the concept that defining
periodontal status using clinical measurements of PD
and BOP levels that reflect the integrity of the BGI can
create diagnostic subgroups of individuals who have
differing biologic phenotypes, even after considering
traditional clinical risk factors for periodontal disease.
The BGI categories make sense clinically because
they logically create a natural gradient in disease se-
verity that can be identified easily using simple clinical
measurements. Furthermore, because the categories
display different biologic phenotypes with regard to
the underlying qualitative and quantitative aspects
of the microbial and inflammatory response, these
groupings eventually may be shown to provide useful
categories for studying response to treatment. For ex-
ample, in this United States population, the levels of
plaque, gender, diabetic status, and race were signif-
icant contributors to the presence of BGI disease ex-
pression, but the impact of these were not uniform
across different BGI categories. Subjects with deep
lesions, but low BOP, have less plaque than BGI-H
subjects but more biochemical inflammation, raising
the question as to whether anti-inflammatory strategies
may be more efficient therapeutic approaches than
antimicrobial treatments in these subjects.
Levels of specific bacteria are undoubtedly signifi-
cant contributors to the presence or absence of dis-
ease in these BGI models, but the nature of the
microbial challenge and the host innate and acquired
immune response to the oral biofilm also seem to be
critical to disease presentation. Increased expression
of IL-1b and PGE2, which are important biochemical
mediators of periodontal tissue destruction,20,21 is

1921
Disease at the Biofilm–Gingival Interface
an essential characteristic of patients with disease at
the BGI. This is true even for the BGI-G subjects, sug-
gesting that this represents an inflammatory state that
may not be innocuous. The level of GCF mediators of
the innate immune response is high, even adjusting
for the level of plaque or the level of organism present,
compared to subjects with BGI-H; for that reason, we
use the term ‘‘excessive’’ or ‘‘hyperinflammatory’’ as
a descriptor. In essence, this represents a shift in the
patient’s dose-response relationship whereby low
levels of microbes (dosage) might not trigger IL-1b re-
lease (response) in a BGI-H subject, but the same
level of microbial challenge will trigger a robust IL-
1b response in BGI-DL subjects. For example, con-
sider that the periodontal sulcus of a typical BGI-H
subject has a total microbial count of ;27,300 bacte-
ria (median) and a mean GCF level of IL-1b of 104 ng/
ml. When one does the calculation (molecular weight
IL-1b: ;10,162 Da), there are ;1 trillion (1012) mol-
ecules of IL-1b within the GCF for every bacterium
present within the pocket. However, if one compares
the response in the mildly upregulated BGI-DL/LB
group, there are total counts of ;22,000 bacteria with
a mean GCF level of IL-1b of 122 ng/ml. This com-
putes to almost 1.4 times as many IL-1b molecules
per bacterium for this BGI-DL/LB group and >1.6 tril-
lion IL-1b molecules per single bacterium in the
pocket for BGI-DL/SP. Therefore, it is not just an in-
crease in concentration of IL-1b within the pocket, it
is an increase in concentration relative to the micro-
bial load.
We are unsure as to the mechanisms that underlie
this upregulated inflammatory response because
many explanations are plausible. Nonetheless, this
observation in a large community-dwelling popula-
tion provides a robust demonstration that subjects
with disease have a high innate inflammatory re-
sponse locally at the BGI, which is in excess of that
modeled by plaque scores or levels of organisms as
determined by DNA checkerboard methods. It also
is possible that the excessive inflammatory response
may be attributable to the presence of unidentified,
perhaps uncultivable, microorganisms that were not
represented by the plaque scores or the checkerboard
analyses in this investigation. However, this study also
indicated that there is a characteristic systemic anti-
body response to selected organisms within the bio-
film, in addition to the high microbial counts that
discriminate between BGI disease classifications,
thereby implicating specific organisms in pathogene-
sis among certain subjects within these BGI groups.
Microbial and Seroreactive Phenotypes of
Shallow and Deep BGI Lesions
This investigation demonstrated that BGI-G is associ-
ated with an acquired immune response to C. rectus.
1922
Volume 78 • Number 10
Among seropositive BGI-G subjects, 68.5% had ele-
vated titers to C. rectus (above the median level of
BGI-H subjects). This is a well-known orange complex
periodontal pathogen that was associated with gingi-
vitis and early periodontitis in previous studies by
Tanner et al.24 and is related closely phylogenetically,
i.e., 96% homology, to the other gastrointestinal
pathogen Helicobacter pylori.25 The association of
this pathogen with high bleeding scores in shallow
pockets raises the possibility that C. rectus may in-
duce ulceration of the pocket epithelium and there-
fore, BOP, in a manner similar to that elicited by the
ulcerogenic H. pylori on the gut epithelium. The level
of organism is correlated highly to the serum antibody
response, but the serum IgG response is associated
more strongly with disease, probably reflecting the
fact that this organism is evading host defenses
and, thereby, eliciting local inflammation and BOP;
this may result in a greater systemic exposure and an-
tibody response. Collectively, these microbial and IgG
responses provide two independent lines of evidence
to strongly implicate the role of C. rectus in gingival in-
flammatory changes that occur in the shallow gingival
pocket.
In a similar manner, the P. gingivalis IgG response
emerges as a dominant player in the more advanced
BGI deep lesion of BGI-DL/MB and BGI-DL/SB, with
63.8% and 75.7% of subjects having elevated titers,
respectively, compared to the BGI-H median. In-
creases in IgG titer to P. gingivalis were associated
with greater bleeding scores and extent of deep pock-
eting in subjects with BGI-DL/MB and BGI-DL/SB dis-
ease, with GCF IL-1b levels enhancing BOP among
BGI-DL/SB. The presence of elevated titers to C. rec-
tus as the second best IgG predictor (following P. gin-
givalis titers) for the BGI-DL/MB and BGI-DL/SB
categories suggested that the role of C. rectus in me-
diating the shallow lesion also may persist in these
deep lesion categories.
Smoking, a well-established traditional risk fac-
tor,26 also plays a major role in disease as an ‘‘inhib-
itor’’ of bleeding scores in BGI-DL/MB and a strong
‘‘enhancer’’ of greater pocketing (extent PD ‡4 mm).
Our findings also are consistent with an earlier report
by Saito et al.,27 regarding the role of obesity as a con-
tributing factor to periodontal disease expression.
One BGI classification seems to mirror a common
clinical presentation of a patient with deep pockets,
but minimal BOP, e.g., a ‘‘healthy or stable’’ periodon-
tal recall patient. BGI-DL/LB subjects have pocketing,
but minimal clinical inflammation. This clinical pre-
sentation is consistent with the observation that these
subjects have lower plaque scores and greater dental
healthcare use patterns. Although we have no data
regarding specific periodontal treatment history,
many in this group might have had periodontal care
J Periodontol • October 2007
included in their dental treatments. These subjects are
quite similar to the BGI-H group, except they have
deeper pockets and show higher IL-1b levels within
the GCF and higher P. gingivalis titers, despite having
lower counts of most organisms, including P. gingiva-
lis and C. rectus (Table 3). This BGI-DL/LB group also
is distinguished by the lack of increased C. rectus
titers, a trait that is seen among all other disease cat-
egories. The low C. rectus counts and serum IgG anti-
body in this group are consistent with low BOP scores,
especially compared to the BGI-G group. Further-
more, the BGI-DL/LB group had the highest ratio of
IgG/bacteria for P. gingivalis and C. rectus compared
to all other BGI groups; this suggested that the anti-
body response may be more effective in dampening
the microbial counts and clinical inflammatory re-
sponse, i.e., low BOP, seen in this group of patients
compared to BGI-DL/MP and BGI-DL/SP. It also is
possible that the increase in the (IgG/bacteria) ratio
may be due to these subjects having more periodontal
therapy. Although these data are cross-sectional in
nature, in a clinical setting, these patients with pock-
eting but with minimal inflammation might be sug-
gestive of a more stable clinical phenotype, i.e.,
less bleeding and, therefore, less likely to show future
disease progression.
Perhaps the most intriguing findings from these
molecular epidemiologic analyses are the character-
istics of the biologic phenotype that is associated with
the most severe form of disease, the BGI-DL/SB con-
dition that affects 12.9% of this population. Disease
extent of PD ‡4 mm within this BGI-DL/SB group is
not influenced by the level of supragingival plaque.
BGI-DL/SB subjects have an excess innate acute in-
flammatory response characterized by higher PGE2
and especially IL-1b, which is even greater than that
of BGI-G or BGI-DL/MB subjects. These BGI-DL/SB
subjects have much higher P. gingivalis IgG re-
sponses (and subgingival P. gingivalis load) with
more extensive disease compared to BGI-DL/MB
and have differing qualitative inflammatory responses
across a broader spectrum of inflammatory mediators
(Table 7). In addition to increased levels of GCF IL-1b
and PGE2, the BGI-DL/SB condition is associated with
an increased expression of several key mediators of
chronic inflammation that are not elevated among
other subjects with BGI-deep lesions, indicating that
the nature of the inflammatory response is qualita-
tively different, not just quantitatively different, among
these subjects. For example, increased levels of IL-6
are critical in that this molecule regulates the transi-
tion from acute to chronic inflammation and induces
the synthesis of MCP-1.28 These individuals have
much greater levels of RANTES and MCP-1 (C-C che-
mokines) that enhance monocytic recruitment and
activation and promote dendritic cell formation.
Offenbacher, Barros, Singer, Moss, Williams, Beck
RANTES was elevated significantly among BGI-SP
subjects; however, this increase was not a common
event because only 1.7% of GCF samples examined
had detectible levels of RANTES.
Although further characterization seems war-
ranted, these findings indicate that the underlying in-
flammatory characteristics of BGI-DL/SB are quite
different, displaying a coordinated enhanced expres-
sion of IL-6 and MCP-1. The combination of IL-1b and
-6 and PGE2 is capable of inducing bone and connec-
tive tissue destruction. This is consistent with the find-
ing that these subjects have approximately five times
the amount of interproximal bone loss compared to
BGI-DL/MB patients, as calculated from the differ-
ence in extent of teeth with ICAL £3 mm plus PD ‡5
mm. The levels of these mediators, as determined
by this survey method, should be interpreted with cau-
tion; the numbers of subjects (180) and GCF samples
(720) represented a relatively small nested case-con-
trol sample of the population. Analyses of additional
subjects should reveal additional patterns. Smoking
also seems to have a much greater impact on disease
levels among the BGI-DL/SB subjects, contributing to
a 2.6-fold greater extent PD ‡4 mm levels compared
to those with less periodontal disease (BGI-MP). It is
not clear whether smoking further upregulates the
local inflammatory response or whether the effects
of smoking are worsened in the presence of an upre-
gulated immunoregulatory state. However, limiting
analyses to never smokers suggested that the same
increased inflammatory response is present (data
not shown). In any event, both effects are important
because those who are exposed to smoking and ex-
cessive inflammation have greater disease expression
than subjects with either exposure alone, i.e., interac-
tion effects are significant.
It is not known whether the excessive local produc-
tion of inflammatory mediators among the BGI groups
are attributable to differences in underlying geno-
type,29 epigenotype,30 or the presence of an un-
known, perhaps non-cultivable organism that was
not identified by the methods used in this study. How-
ever, establishing distinct clinical groups that provide
unique biologic phenotypes is an important step
toward establishing linkages with the underlying sin-
gle nucleotide polymorphisms patterns of key in-
flammatory or transcriptional regulatory genes or the
possible epigenetic modifications of chromatin struc-
ture that may arise from microbial or environmental
factors.29,30 In other words, specific biofilm organisms
or exposures may have the capacity to alter the ‘‘in-
flammatory set point’’ of the local tissues in certain
individuals via epigenetic mechanisms.29,30 Alterna-
tively, the broad activation of these innate mediators
of chronic inflammation and bone resorption (IL-6
and MCP-1) may reflect the increased load, and
1923
Disease at the Biofilm–Gingival Interface
perhaps tissue invasion, of P. gingivalis. Further ex-
periments are needed to address these issues.
Despite the large size and richly comprehensive na-
ture of this molecular epidemiologic dataset, certain
limitations regarding the general applicability of these
findings should be emphasized. The population is a
sample of four United States communities of older
adults with a mean age of 62.4 years. With advancing
age, the clinical and biologic phenotype associated
with periodontal conditions might change and the
effects of possible treatments in many individuals
might be more apparent. Thus, the applicability of
these BGI definitions to other populations may not
yield similar biologic phenotype characteristics. Fur-
thermore, considering the age range of the popula-
tion, it may not include the most aggressive forms
of disease that might have resulted in tooth loss and
edentulism. We included numbers of teeth in all rele-
vant models to, in part, adjust for the effect of tooth
loss due to disease. Although this cross-sectional
study cannot establish temporality, the BGI-G condi-
tion is consistent with an inflammatory gingival lesion
in shallow pockets or perhaps an early gingival lesion,
even in the presence of extensive loss of attachment
with recession. However, it is not known whether this
shallow lesion develops into a deep lesion over time,
remains stable, or migrates toward the root apex re-
taining shallow PDs. The gingival and alveolar dimen-
sions that define the hard and soft tissue biomass, or
the ‘‘periodontal biotype,’’ may be important in deter-
mining whether inflammation results in pocketing or
recession. In fact, the time-dependent relationships
or transitions within or between any of these disease
categories are unknown during disease progression
or in response to treatment.
Finally, our goal of achieving homogeneity within
each disease group, although clearly better than tra-
ditional definitions, is not perfect. For example, cer-
tain subjects within the BGI-DL/SB group clearly
showed selective elevations in A. actinomycetemco-
mitans counts and titers, rather than P. gingivalis or
P. gingivalis plus C. rectus. Patients within the BGI-
DL/MB group also demonstrated higher counts of spi-
rochetes, and certain subjects also had significantly
elevated IgG titers to these organisms. Thus, pa-
tient-level diagnostics and further refinement in our
nosological identification schema are indicated.
Nonetheless, we suggest that this approach for as-
sessing disease groupings using clinical signs that
are common to dental practices seems to enable
the partial characterization of underlying biologic
phenotypes. To assign a patient to one of the five
BGI categories, one only needs to identify whether
the patient has one or more PDs ‡4 mm (rounding
down to the nearest millimeter) and compute the per-
centage of total sites that bleed upon probing, i.e.,
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Volume 78 • Number 10
(number of sites with BOP/total number of sites in
mouth) · 100, and then classify by referring to the
shallow versus deep lesion pocketing and percentage
BOP groupings described above. We do not know
whether patients move from one BGI category to an-
other category as disease progresses or in response to
treatment. Although treatment responses cannot be
predicted from these data, the fact that these BGI clas-
sifications and biologic phenotypes differ in response
to microbial load and inflammatory response sug-
gests the likelihood that treatment responses may
vary among differing phenotypes. This is an important
question for future studies. Furthermore, creating
clinical definitions that reflect the biology of patho-
genesis ultimately will enable us to better define the
microbial and host genotypes associated with disease
and personalize diagnostics and therapeutics.
CONCLUSIONS
We provided evidence for the creation of new disease
definitions that enable us to identify four distinct bio-
logic phenotypes of BGI disease. All four definitions of
disease are influenced by the presence of plaque,
race, gender, and diabetes. The BGI-G lesion is char-
acterized by an increased GCF level of IL-1b and PGE2
and exposure to C. rectus. The BGI-DL/LB condition
is associated with elevated GCF IL-1b and P. gingiva-
lis IgG titers, but lower counts of most organisms.
BGI-DL/MB and BGI-DL/SB are associated with an
elevated innate inflammatory phenotype (increased
IL-1b and PGE2) and exposure to C. rectus and P. gin-
givalis. In addition, BGI-DL/SB is associated with an
excessive innate inflammatory response for the level
of organism present that is characterized by even
higher levels of IL-1b and an increased expression
of mediators of chronic inflammation (IL-6 and
MCP-1, as well as the less frequent elevation in
RANTES). Thus, this study provides strong molecular
epidemiologic evidence that periodontal conditions
reflect an excessive inflammatory response relative
to the level of the microbial burden presented by the
oral biofilm, as measured by plaque scores and sub-
gingival DNA checkerboard analyses, and that there
is microbial specificity in disease presentation and
pathogenesis.
ACKNOWLEDGMENTS
The authors thank Dr. Ken Kornman, president and
chief scientific officer, Interleukin Genetics, Waltham,
MA, for his helpful comments. This work was sup-
ported by National Institutes of Health/National In-
stitute of Dental and Craniofacial Research grants
DE-13079 and RR-00046.
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Correspondence: Dr. Steven Offenbacher, Center for Oral
and Systemic Diseases, University of North Carolina at
Chapel Hill School of Dentistry, CB #7455, Dental Re-
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Chapel Hill, Chapel Hill, NC 27599-7455. Fax: 919/966-
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Submitted November 22, 2006; accepted for publication
April 25, 2007.
1925