Saliva/pathogen biomarker signatures and periodontal disease progression.

The purpose of this study was to determine the role of saliva-derived biomarkers and periodontal pathogens during periodontal disease progression (PDP). One hundred human participants were recruited into a 12-month investigation. They were seen bi-monthly for saliva and clinical measures and bi-annually for subtraction radiography, serum and plaque biofilm assessments. Saliva and serum were analyzed with protein arrays for 14 pro-inflammatory and bone turnover markers, while qPCR was used for detection of biofilm. A hierarchical clustering algorithm was used to group study participants based on clinical, microbiological, salivary/serum biomarkers, and PDP. Eighty-three individuals completed the six-month monitoring phase, with 39 [corrected] exhibiting PDP, while 44 [corrected] demonstrated stability. Participants assembled into three clusters based on periodontal pathogens, serum and salivary biomarkers. Cluster 1 members displayed high salivary biomarkers and biofilm; 71% [corrected] of these individuals were undergoing PDP. Cluster 2 members displayed low biofilm and biomarker levels; 76% [corrected] of these individuals were stable. Cluster 3 members were not discriminated by PDP status; however, cluster stratification followed groups 1 and 2 based on thresholds of salivary biomarkers and biofilm pathogens. The association of cluster membership to PDP was highly significant (p < 0.0007). [corrected] The use of salivary and biofilm biomarkers offers potential for the identification of PDP or stability (ClinicalTrials.gov number, CT00277745).

MAPK signaling is required for LPS-induced VEGF in pulp stem cells.

Caries-induced pulpitis is typically accompanied by an increase in dental pulp microvascular density. However, the mechanisms by which dental pulp cells recognize lipopolysaccharides (LPSs) remain unclear. We hypothesized that Porphyromonas endodontalis and Escherichia coli LPSs induce vascular endothelial growth factor (VEGF) expression in dental pulp stem cells (DPSC) and human dental pulp fibroblasts (HDPF) through mitogen-activated protein kinase (MAPK) signaling. ELISA, semi-quantitative RT-PCR, immunofluorescence, and Western blots were used. Here, we observed that LPSs induced VEGF expression in DPSC and HDPF cells, and both cell types express Toll-like receptor 4 (TLR- 4). Notably, LPS-induced VEGF is associated with phosphorylation of protein kinase C (PKC zeta) and extracellular signal-regulator kinase (ERK1/2) and is dependent upon MAPK activation. Analysis of these data, collectively, unveils a signaling pathway responsible for synthesis of VEGF by pulp cells and suggests a novel therapeutic target for the management of vascular responses in teeth with pulpitis.

Differential display analysis of Porphyromonas gingivalis gene activation response to heat and oxidative stress.

BACKGROUND/AIMS: The etiologic relationship between periodontitis and Porphyromonas gingivalis is attributed to the ability of the organism to express a variety of virulence factors, many of which are cell surface components including lipopolysaccharide and arginine-specific cysteine proteases (Arg-gingipains, RgpA, and RgpB). P. gingivalis responds to the stress of rapid elevation in temperature by activating a set of genes to produce heat shock proteins that mediate the effects of sudden changes in environmental temperatures by repairing or eliminating cellular proteins denatured by that stress. METHODS: We used restriction fragment differential display (RFDD) to identify and measure the genes expressed by surrogates of environmental stresses, heat and oxidative stress. The results were then confirmed using quantitative reverse-transcription polymerase chain reaction. RESULTS: We selected 16 genes differentially induced from over 800 total expression fragments on the RFDD gels for further characterization. With primers designed from those fragments we found that a + 5 degrees C heat shock caused a statistically significant increase in expression compared 12 of 18 untreated genes tested. The exposure of P. gingivalis to atmospheric oxygen resulted in statistically significant increases in five of the target genes. These genes are likely involved in transport and synthesis of components of the lipopolysaccharide biosynthetic pathway important in anchoring the Arg-gingipains required for virulence-related activities. CONCLUSION: These results emphasize the need for studies to measure the coordinated responses of bacteria like P. gingivalis which use a multitude of interrelated metabolic activities to survive the environmental hazards of the infection process.

Quantitative real-time PCR detection of oral Enterococcus faecalis in humans.

OBJECTIVE: Enterococcus faecalis is consistently associated with recurrent root canal infections. Only low concentrations of E. faecalis in the human mouth have been demonstrated by culture techniques. Quantitative detection strategies more sensitive than culturing, such as quantitative PCR (qPCR), could provide more illuminating data. DESIGN: Thirty outpatients attending the University of Michigan Graduate Endodontic Clinic for endodontic treatment provided oral rinse samples that were analysed for E. faecalis using qPCR and microbiological culturing. A SYBR Green I qPCR protocol was developed for the quantifiable detection of E. faecalis and total bacteria in oral rinse samples using primers designed to target the 16S rRNA gene. Annealing temperature and primer, magnesium ion, and dimethyl sulfoxide concentrations were investigated for optimisation of the protocol; a minimum sensitivity limit of 23 rRNA copies (an estimated six E. faecalis cells) was established for E. faecalis in pure culture, and 104 rRNA copies (an estimated 26 E. faecalis cells) in mixed culture. RESULTS: In qPCR assays, based on extrapolation from estimated rRNA gene copy numbers, E. faecalis comprised 0.0006-0.0047% of a total bacteria load that ranged from 5.92 x 10(5) to 5.69 x 10(7) cells/ml of oral rinse. E. faecalis was detected in five (17%) samples in concentrations from 114 to 490 cells/ml. In parallel culture assays E. faecalis were detected in only two samples (7%) of the five identified by qPCR and in concentrations 30 and 240 CFU/ml. CONCLUSIONS: qPCR reported a higher incidence of E. faecalis in oral rinse samples than culture techniques and afforded greater sensitivity.

Monocyte differentiation in localized juvenile periodontitis is skewed toward the dendritic cell phenotype.

Several lines of evidence indicate that the monocytes of subjects with localized juvenile periodontitis (LJP) are functionally distinct from cells of age- and race-matched nonperiodontitis (NP) subjects. Among the abnormalities are the propensity to secrete large amounts of prostaglandin E(2) and the induction of immunoglobulin G2 (IgG2) antibodies. The experiments described here were performed to further characterize the LJP monocytes and to determine if these cells mature differently than NP monocytes. When adherent monocytes from LJP subjects were cultured in the presence of human serum, both macrophages and cells with the morphology of immature monocyte-derived dendritic cells (MDDC) were observed. Within 4 days the prevalence of the immature MDDC was approximately twofold higher in LJP cultures than in NP cultures. In addition to their dendritic morphology, these cells were CD11c(+) and CD14(-) or CD14(low) and stimulated potent autologous mixed leukocyte reactions, consistent with differentiation to the MDDC phenotype. Like LJP monocytes, cultures of MDDC generated with interleukin-4 and granulocyte-macrophage colony-stimulating factor selectively induced IgG2 in cultures of pokeweed mitogen-stimulated NP leukocytes. Together, these data suggest that the monocytes of LJP subjects have a propensity to differentiate into MDDC and that this differentiation may be related to the high levels of IgG2 that are observed in the sera of LJP subjects. As high levels of circulating IgG2 are correlated with less severe disease, the propensity of LJP monocytes to differentiate into MDDC may have important implications for both the host response against oral pathogens and the progression of LJP.

Catecholamines in murine bone marrow derived mast cells.

Cultured murine bone marrow derived mast cells (BMMC) were found to store high levels of dopamine (3753+/-844 pg/10(7) cells) and occasionally produce norepinephrine and epinephrine. The catecholamine synthesis inhibitor, alpha-methyl-para-tyrosine, decreased intracellular catecholamine concentrations, and activation with ionomycin stimulated dopamine release. Neither dopaminergic receptor antagonists nor exogenous dopamine < or =10 microM affected IL-3-induced cell proliferation. High exogenous dopamine (20-100 microM) decreased proliferation and increased apoptosis, and the anti-oxidant ascorbic acid prevented these effects. Increased expression of the anti-apoptotic factor Bcl-2 or loss of pro-apoptotic Bax expression attenuated dopamine-induced apoptosis, suggesting the apoptosis proceeds through a mitochondrial pathway.

The role of Th2 cytokines in mast cell homeostasis.

Homeostatic mechanisms regulating mast cell numbers and function in peripheral tissues have largely focused on cytokines, such as stem cell factor, interleukin (IL)-3, IL-4, and IL-10, which regulate mast cell maintenance and proliferation. Despite these advances, little attention has been paid to the mechanisms that mediate mature mast cell turnover, and control of mast cell hyperplasia generated during Th2-mediated responses. These are important issues, as mast cells are now known to be multi-functional effector cells, that have the capacity to mediate both innate and Th2-induced immune responses. Numerous secretagogues may elicit mast cells to release a large number of important mediators that can cause chronic inflammation. Therefore, how mast cell homeostasis is regulated may have significant effects on normal physiology, and contribute to the genesis of inflammatory disease. Our laboratory has characterized an in vitro model of mast cell homeostasis, by which long-term exposure of murine bone-marrow-derived mast cells to the Th2-derived cytokines IL-3, IL-4, and IL-10, will induce downregulation of critical mast cell effector proteins such as Kit and Fcepsilon-RI, followed by mast cell apoptosis. These data offer a novel role for Th2 cytokines, acting to both initiate and resolve mast cell activation and proliferation. Loss of these signals may contribute to a multitude of diseases, such as mastocytosis and allergy

Combined stimulation with the T helper cell type 2 cytokines interleukin (IL)-4 and IL-10 induces mouse mast cell apoptosis.

Mast cells are found in connective and mucosal tissues throughout the body. Their activation via immunoglobulin E (IgE)-antigen interactions is promoted by T helper cell type 2 (Th2) cytokines and leads to the sequelae of allergic disease. We now report a mechanism by which Th2 cytokines can regulate mast cell survival. Specifically, we find that interleukin (IL)-4 and IL-10 induce apoptosis in IL-3-dependent bone marrow-derived mast cells and peritoneal mast cells. This process required 6 d of costimulation with IL-3, IL-4, and IL-10, and expression of signal transducer and activator of transcription 6 (Stat6). Apoptosis was coupled with decreased expression of bcl-x(L) and bcl-2. While this process occurred independent of the Fas pathway, culture in IL-3+IL-4+IL-10 greatly sensitized mast cells to Fas-mediated death. Additionally, we found that IgE cross-linkage or stimulation with stem cell factor enhanced the apoptotic abilities of IL-4 and IL-10. Finally, IL-3-independent mastocytomas and mast cell lines were resistant to apoptosis induced by IL-3+IL-4+IL-10. These data offer evidence of Th2 cytokine-mediated homeostasis whereby these cytokines both elicit and limit allergic responses. Dysregulation of this pathway may play a role in allergic disease and mast cell tumor survival.

Prevalence of periodontal pathogens in localized and generalized forms of early-onset periodontitis.

The primary objectives of this study were to investigate the prevalence of 8 putative periodontal pathogens in subjects with early-onset periodontitis (EOP) and to evaluate the microbial differences between localized and generalized forms of this periodontal disease condition. Thirty-one females and 11 males with a mean age of 30.3 (s.d. 4.0) years were examined. Seventeen subjects had generalized (GEOP) and 25 had localized early-onset periodontitis (LEOP). Subgingival plaque samples were assayed using PCR which provided subject prevalence data for the pathogens; Bacteroides forsythus 78.6%, Treponema denticola 88.1%, Actinobacillus actinomycetemcomitans 19.0%, Porphyromonas gingivalis 16.7%, Prevotella intermedia 40.4%, Prevotella nigrescens 61.9%, Eikenella corrodens 42.3% and Campylobacter rectus 92.8%. Only 3 healthy sites harbored one or more of these periodontal pathogens. Seven of the 8 subjects positive for A. actinomycetemcomitans had LEOP. P. intermedia was present in 58.8% of GEOP compared with 28% of LEOP subjects (p=0.046). At 82.4% of GEOP sites P. nigrescens was present while this bacteria was detected at 52% of LEOP (p=0.044). P. gingivalis was isolated from 22.6% of females but no male subjects (p=0.084). C. rectus was recovered from all female subjects compared to 72.7% of males (p=0.014). A. actinomycetemcomitans (37.5%) and C. rectus (86.5%) were more frequently identified in non-smokers compared to 7.6% and 68.8% of smokers, respectively (p <0.05). Microbial associations coincided with the clinical division of the cases into LEOP and GEOP in 83% of the subjects.

Quantitation of Bacteroides forsythus in subgingival plaque comparison of immunoassay and quantitative polymerase chain reaction.

Our objective was to compare three methods (enzyme-linked immunosorbent assay [ELISA], endpoint and quantitative polymerase chain reaction [E-PCR and Q-PCR]) for detection and quantitation of Bacteroides forsythus in 56 plaque samples from seven subjects with progressive periodontal disease. Samples collected in buffer were pelleted and resuspended in 500 microl of water. Fifty microl aliquots were removed for an ELISA performed on bacteria or plaque immobilized on 96-well plates and probed with B. forsythus specific antibody. An occurrence of 3.7+/-0.6 x 10(4) or more bacteria were detected by ELISA in pure culture; 26 of 54 plaque samples were positive, two samples could not be analyzed. Samples for PCR were autoclaved for 10 min prior to use. The detection level of E-PCR using primers specific for B. forsythus 16S rRNA was 200 cells and 42 out of 56 samples were positive based on ethidium bromide stained agarose gels. Q-PCR using the same primers combined with a nested fluorescent oligonucleotide probe detected 10+/-0.32 bacteria in pure culture; 43 of 56 plaque samples were positive. The ELISA and Q-PCR obtained identical results with 36 of the 54 samples assayed; there were one false positive and 17 false negative ELISA results using Q-PCR as standard. The positive proportions of plaque samples were almost the same for E-PCR and Q-PCR. We conclude that the PCR methods are more appropriate for a multicenter study because of greater sensitivity and convenience of sample transportation from clinics to a central laboratory.

Cellular localization of a Hsp90 homologue in Porphyromonas gingivalis.

We previously reported an association between elevated serum antibody titers to the 90-kDa human heat shock protein (Hsp90), periodontal health and colonization by Porphyromonas gingivalis. In this study, we examined the cellular localization of the Hsp90 homologue of P. gingivalis. Cultures of P. gingivalis were heat-stressed (45 degrees C) and examined for localization of the Hsp90 homologue. Heat stress induced a 4-5-fold increase in anti-Hsp90 antibody reactivity over that of the unstressed controls. Western blot analysis revealed two bands (44 and 68 kDa) that reacted with anti-Hsp90 antibodies. The 68-kDa band was heat-inducible, while the 44-kDa band was not. Immunogold staining revealed that the Hsp90 homologue localized principally to the membrane and extracellular vesicles. Subcellular fractionation confirmed that the Hsp90 homologue was primarily membrane-associated.

Humoral immunity to stress proteins and periodontal disease.

BACKGROUND: There is evidence that microbial heat shock (stress) proteins (Hsp) are immunodominant antigens of many microorganisms. Immunity to these proteins has been shown in non-oral infections to contribute to protection. This study was undertaken to assess the relationship(s) between immunity to human and microbial heat shock proteins, periodontal disease status, and colonization by periodontal disease-associated microorganisms. METHODS: Subgingival plaque and blood samples obtained from 198 patients during an earlier clinical study were examined for the presence of specific periodontal disease-associated microorganisms and antibodies to selected human and microbial heat shock proteins (Hsp70, Hsp90, DnaK, and GroEL). Particle concentration immunofluorescence assay (PCFIA) was used to detect anti-Hsp antibodies and slot immunoblot assay (SIB) was used to detect subgingival plaque species. Regression models were used to examine the contribution of age, gender, gingival index, probing depth, attachment loss, calculus index, plaque index, and microbial colonization to the anti-Hsp antibody concentrations. RESULTS: Our studies demonstrated that, when evaluated by ANOVA, patients with higher anti-Hsp (Hsp90, DnaK, and GroEL) antibody concentrations tended to have significantly (P< or =0.05) healthier periodontal tissues. This was most obvious when the relationship between mean probing depths and antibody concentrations were studied. For Hsp90 antibodies, 2 variables (probing depth and P. gingivalis concentration) were found to have significant contributions (R = 0.293, P<0.0002). The equation derived from the regression model was y = 12558-2070*PD +1842*PG. This confirmed the inverse relationship with probing depth and the positive relationship with colonization by P. gingivalis. Attempts to model the other stress protein antibodies were not successful. CONCLUSIONS: We believe that the present observations reflect the presence of protective anti-Hsp antibodies, rather than simply the presence of the microorganism in the gingival sulcus. The clinical significance of these observations lies in the potential of identifying patients who are at risk for developing periodontal disease based on their inability to mount an immune response to specific Hsp or Hsp epitopes, as well as the development of vaccines based on Hsp epitopes.

Inhibition of kit expression in P815 mouse mastocytoma cells by a hammerhead ribozyme.

Using P815 cells, we designed and tested an antisense hammerhead ribozyme for its ability to mediate cleavage of mouse c-kit mRNA at the transmembrane-encoding region. The ribozyme demonstrated in vitro cis cleavage of extraneous 5' sequence in the ribozyme construct as well as trans cleavage of a target c-kit mRNA sequence. The in vitro cleavage was effective at 37 degrees C. A subpopulation of P815 cells transfected with the pCDNA1 plasmid-containing the anti-c-kit ribozyme demonstrated inhibited Kit expression as indicated by flow cytometry, but the inhibition could not be maintained over time. The anti-c-kit ribozyme was put under the control of a tetracycline-inducible two-plasmid system of expression. Incomplete inhibition of Kit expression was observed when transcription of the ribozyme was allowed by the absence of tetracycline, but not when tetracycline was present. Finally, complete inhibition of Kit expression was observed when pCDNA1-ribozyme-transfected cells were analyzed immediately after enrichment by cotransfection with a plasmid expressing green fluorescent protein, followed by cell sorting. The results suggest that anti-c-kit ribozymes might be useful for inhibiting Kit expression in hyperplastic mast cells, if delivery of ribozymes can be optimized.

Inhibition of Kit expression by IL-4 and IL-10 in murine mast cells: role of STAT6 and phosphatidylinositol 3'-kinase.

The c-kit protooncogene encodes a receptor tyrosine kinase that is known to play a critical role in hemopoiesis and is essential for mast cell growth, differentiation, and cytokine production. Studies have shown that the Th2 cytokine IL-4 can down-regulate Kit expression on human and murine mast cells, but the mechanism of this down-regulation has remained unresolved. Using mouse bone marrow-derived mast cells, we demonstrate that IL-4-mediated Kit down-regulation requires STAT6 expression and phosphotidylinositide-3'-kinase activation. We also find that the Th2 cytokine IL-10 potently down-regulates Kit expression. IL-4 enhances IL-10-mediated inhibition in a manner that is STAT6 independent and phosphotidylinositide-3'-kinase dependent. Both IL-4- and IL-10-mediated Kit down-regulation were coupled with little or no change in c-kit mRNA levels, no significant change in Kit protein stability, but decreased total Kit protein expression. Inhibition of Kit expression by IL-4 and IL-10 resulted in a loss of Kit-mediated signaling, as evidenced by reduced IL-13 and TNF-alpha mRNA induction after stem cell factor stimulation. These data offer a role for STAT6 and phosphotidylinositide-3'-kinase in IL-4-mediated Kit down-regulation, coupled with the novel observation that IL-10 is a potent inhibitor of Kit expression and function. Regulating Kit expression and signaling may be essential to controlling mast cell-mediated inflammatory responses.

Cutting edge: effects of an allergy-associated mutation in the human IL-4R alpha (Q576R) on human IL-4-induced signal transduction.

A mutation in the human (hu) IL-4R alpha, Q576R, has been linked with allergy in humans. Increased sensitivity of patients cells with this mutation to IL-4 suggest that a Q576R change enhances IL-4 signaling. To directly test this hypothesis, we analyzed the ability of huIL-4R alpha cDNA bearing the Q576R and Y575F mutations to signal tyrosine phosphorylation, DNA-binding activity, proliferation, protection from apoptosis, and CD23 induction in response to huIL-4 in murine cells. Responses generated by the Q576R and Y575F mutants were similar to those of the wild-type receptor, using various concentrations of huIL-4 and times of stimulation. These results indicate that neither the Q576R nor the Y575F mutations have a significant direct effect on IL-4 signal transduction, and that hypersensitive induction of CD23 in cells derived from human allergy patients may be due to different and/or additional alterations in the IL-4 signaling pathway.

IL-4 inhibits mouse mast cell Fc epsilonRI expression through a STAT6-dependent mechanism.

Mast cell activation by IgE-mediated stimuli is a central event in atopic disease. The regulation of the mast cell high affinity receptor, Fc epsilonRI, is poorly understood. We show that IL-4 can inhibit Fc epsilonRI expression on mouse bone marrow-derived mast cells and fetal liver-derived mast cell progenitors. This effect could be observed at 2.5 ng/ml IL-4 and was dose dependent. IL-4-mediated inhibition of cultured BMMC required 4 days of stimulation and was sustained at maximum levels for at least 21 days. The inhibition of Fc epsilonRI expression resulted in decreased sensitivity to IgE-mediated stimulation, as measured by serotonin release, and the induction of mRNA for IL-4, IL-5, IL-6, and IL-13. Additionally, IL-4 could abrogate the IgE-mediated increase in Fc epsilonRI expression. Lastly, IL-4-mediated inhibition was dependent upon expression of the STAT6 transcription factor, as STAT6-deficient bone marrow-derived mast cells did not decrease Fc epsilonRI levels in response to IL-4. These data argue for a homeostatic role of IL-4 in the regulation of Fc epsilonRI expression, a role that could be critical to understanding atopic disease.

Cellular proliferation and cytokine responses of murine macrophage cell line J774A.1 to polymethylmethacrylate and cobalt-chrome alloy particles.

Wear debris from orthopedic joint implants have been postulated to initiate a cascade of complex cellular events that results in aseptic loosening of the prosthesis and eventually in loss of function of the device. The impact of biomaterials used in these devices on host inflammatory response has not been examined extensively. Polymethylmethacrylate (PMMA) and cobalt-chrome alloy (CoCr) are biomaterials widely used in orthopedic implant devices. Macrophages are an important component of the host inflammatory response, and we have examined the effect of PMMA and CoCr particles on the murine macrophage cell line J774A.1. Our objective was to obtain a comprehensive analysis of the particle-macrophage interaction, and we examined a number of basic biological responses of the J774A.1 cell line, including cell proliferation, apoptosis, cytokines secreted into the culture supernatant (TNFalpha, IL-1alpha, IL-6, and IL-12) and mRNA expression of the cytokines (TNFalpha, IL-1alpha, IL-6, IFN-alpha, M-CSF, and TGF-beta) in response to PMMA and CoCr particles. Our results indicate that the relative contribution of PMMA and CoCr particles in J774A.1 activation is negligible, and we observed a change in metabolic activity of J774A.1 cells only at higher concentrations of CoCr particles.

Stem cell factor activates STAT-5 DNA binding in IL-3-derived bone marrow mast cells.

The Kit tyrosine kinase regulates growth and differentiation of many hematopoietic cells through a signal transduction process that remains to be fully elucidated. Kit has been shown to associate with the receptor for erythropoietin (Epo), which is known to activate the signal transducer and activator of transcription, STAT-5. To determine if Kit signal transduction activated latent DNA-binding factors, including STAT-5, we performed electrophoretic mobility shift assays on stem cell factor (SCF)-stimulated mouse bone marrow-derived mast cells (BMMCs). SCF led to the rapid and transient activation of a DNA-binding factor that was identified by supershift analysis as STAT-5. STAT-5 DNA binding was shown to be specific for the oligonucleotide of the correct sequence and was dose-responsive. Epo stimulation of BMMCs led to the activation of a DNA-binding activity that comigrated with the SCF-induced band, but peaked and was maintained at later time points than SCF-induced activation. These data indicate that SCF stimulation of Kit leads to activation of STAT-5 DNA binding with kinetics distinct from Epo-mediated stimulation.

Genetic study of oxygen resistance and melanization in Cryptococcus neoformans.

Genetic analysis of oxygen-sensitive mutants of Cryptococcus neoformans revealed two loci (oxy1 and oxy2) linking hyperoxia sensitivity to production of melanin, a known virulence factor. Hyperoxia-sensitive strain 562 (oxy1 oxy2) is albino and avirulent. oxy2-defective strains lacking the oxy1 defect are melanin deficient but show normal hyperoxia resistance. Mutants defective at three additional mapped melanin loci fail to show hyperoxia sensitivity in the oxy1 background. Revertants of strain 562, which regain the ability to synthesize melanin by mutation at suppressor sites unlinked to oxy2, retain the oxygen sensitivity conferred by their oxy1 and oxy2 defects. These data identify the melanin gene oxy2 as unique in its association of hyperoxia resistance and melanization.

Natural distribution of 5 bacteria associated with periodontal disease.

The purpose of this study was to determine the prevalence and distribution of 5 bacterial pathogens in subgingival plaque, their relationship with each other and probing depth. Plaque was collected from 6905 sites in 938 subjects. A bacterial concentration fluorescence immunoassay and bacterial specific monoclonal antibodies were used to determine the presence and level of P. gingivalis (Pg), A. actinomycetemcomitans (Aa), P. intermedia (Pi), E. corrodens (Ec) and F. nucleatum (Fn) in each plaque sample. The prevalence in subjects was lowest for Pg (32%) and highest for Ec (49%). The site-based frequency distribution of these bacterial species ranged from 10.3% for Pg to 18.7% for Ec. Pi and Ec were the bacterial combination most often found together in a subject (27.2%). While 64.0% of the sites were without any of the 5 bacterial species evaluated, 20.2% had only 1 of the 5 bacterial species evaluated. The remaining 15.8% of sites had at least 2 bacteria species present. There was a general linear association of the detection level of bacterial species and probing depth. The odds ratios were 3.9 (Pg), 3.0 (Aa), 4.0 (Pi), 2.7 (Ec) and 2.8 (Fn) of finding high levels of these bacterial pathogens at > 5 mm probing depth (p < or = 0.01). Mean probing depth at molar sites without a specific bacteria was greater (p < or = 0.01) in subjects with a specific bacterium compared to molar sites in subjects without the bacteria.(ABSTRACT TRUNCATED AT 250 WORDS)