RgpA-Kgp peptide-based immunogens provide protection against Porphyromonas gingivalis challenge in a murine lesion model

Porphyromonas gingivalis, a gram-negative bacterium, has been linked to the onset and progression of periodontitis, a chronic inflammatory disease of the supporting tissues of the teeth. A major virulence factor of P. gingivalis is an extracellular complex of Arg- and Lys-specific proteinases and adhesins designated the RgpA-Kgp complex (formerly the PrtR-PrtK complex). In this study we show that the RgpA-Kgp complex, when used as an immunogen with incomplete Freund adjuvant (IFA), protects against challenge with invasive and noninvasive strains of P. gingivalis in the murine lesion model. We identified a variety of peptide vaccine candidates from the RgpA and Kgp polyprotein sequences that involved the putative active site histidine of both proteinases and five repeat motifs in the adhesin domains of both polyproteins implicated in aggregation and binding to host substrates, designated adhesin-binding motif (ABM) peptides. These peptides were synthesized using standard, solid-phase protocols for 9-fluorenylmethoxy carbonyl chemistry with S-acetylmercaptoacetic acid (SAMA) as the N-terminal residue. The SAMA-peptides were then conjugated to diphtheria toxoid and used with IFA to immunize BALB/c mice. Both active-site peptides and three of the five ABM peptides gave protection (P < 0.005) against challenge with P. gingivalis in the murine lesion model. The three ABM peptide sequences that conferred protection exist within a 100-residue span in the RgpA44 and Kgp39 adhesins of the RgpA-Kgp complex. Protective anti-RgpA-Kgp complex mouse antisera recognized the RgpA27, Kgp39, and RgpA44 adhesins in an immunoblot. Epitope mapping of the RgpA27 adhesin using the protective anti-RgpA-Kgp antisera identified a major protective epitope that mapped immediately N terminal to one of the protective ABM peptides in the 100-residue span in RgpA44 and Kgp39. This identified protective epitope contains clusters of basic residues spatially surrounded by hydrophobic amino acids, a finding which is characteristic of a heparin binding motif.     

Porphyromonas gingivalis Outer Membrane Vesicles Induce Selective Tumor Necrosis Factor Tolerance in a Toll-Like Receptor 4- and mTOR-Dependent Manner

Porphyromonas gingivalis is an important member of the anaerobic oral flora. Its presence fosters growth of periodontal biofilm and development of periodontitis. In this study, we demonstrated that lipophilic outer membrane vesicles (OMV) shed from P. gingivalis promote monocyte unresponsiveness to live P. gingivalis but retain reactivity to stimulation with bacterial DNA isolated from P. gingivalis or AIM2 ligand poly(dA·dT). OMV-mediated tolerance of P. gingivalis is characterized by selective abrogation of tumor necrosis factor (TNF). Neutralization of interleukin-10 (IL-10) during OMV challenge partially restores monocyte responsiveness to P. gingivalis; full reactivity to P. gingivalis can be restored by inhibition of mTOR signaling, which we previously identified as the major signaling pathway promoting Toll-like receptor 2 and Toll-like receptor 4 (TLR2/4)-mediated tolerance in monocytes. However, despite previous reports emphasizing a central role of TLR2 in innate immune recognition of P. gingivalis, our current findings highlight a selective role of TLR4 in the promotion of OMV-mediated TNF tolerance: only blockade of TLR4—and not of TLR2—restores responsiveness to P. gingivalis. Of further note, OMV-mediated tolerance is preserved in the presence of cytochalasin B and chloroquine, indicating that triggering of surface TLR4 is sufficient for this effect. Taking the results together, we propose that P. gingivalis OMV contribute to local immune evasion of P. gingivalis by hampering the host response.     

Serum immunoglobulin G (IgG) and IgG subclass responses to the RgpA-Kgp proteinase-adhesin complex of Porphyromonas gingivalis in adult periodontitis

Serum immunoglobulin G (IgG), IgM, and IgG subclass responses to the RgpA-Kgp proteinase-adhesin complex of Porphyromonas gingivalis were examined by enzyme-linked immunosorbent assay using adult periodontitis patients and age- and sex-matched controls. Twenty-five sera from subjects with adult periodontitis (diseased group) and 25 sera from healthy subjects (control group) were used for the study. Sera and subgingival plaque samples from 10 sites were collected from each patient at the time of clinical examination. The level of P. gingivalis in the plaque samples was determined using a DNA probe. Highly significant positive associations between the percentage of sites positive for P. gingivalis and measures of disease severity (mean pocket depth, mean attachment loss, and percentage of sites that bled on probing) were found. The diseased group had significantly higher specific IgG responses to the RgpA-Kgp complex than did the control group, and the responses were significantly associated with mean probing depths and percentage of sites positive for P. gingivalis. Analysis of the IgG subclass responses to the RgpA-Kgp complex revealed that the subclass distribution for both the diseased and control groups was IgG4 > IgG2 > IgG3 = IgG1. The IgG2 response to the complex was positively correlated with mean probing depth, whereas the IgG4 response was negatively correlated with this measure of disease severity. Immunoblot analysis of the RgpA-Kgp complex showed that sera from healthy subjects and those with low levels of disease, with high IgG4 and low IgG2 responses, reacted with the RgpA27, Kgp39, and RgpA44 adhesins; however, sera from diseased subjects with low IgG4 and high IgG2 responses reacted only with the RgpA44 and/or Kgp44 adhesins. Epitope mapping of the RgpA27 adhesin localized a major epitope recognized by IgG4 antibodies in sera from subjects with high IgG4 and low IgG2 responses to the RgpA-Kgp complex which was not recognized by sera from diseased subjects with low IgG4 and high IgG2 responses.     

In Vitro Invasion and Survival of Porphyromonas gingivalis in Gingival Fibroblasts; Role of the Capsule

Porphyromonas gingivalis is a Gram-negative, anaerobic bacterium involved in periodontitis and peri-implantitis that can invade and survive inside host cells in vitro. P. gingivalis can invade human gingival fibroblasts (GF), but no data are available about the role of P. gingivalis’ capsule in GF invasion. In the current study, we aimed to determine the ability of three strains of P. gingivalis (encapsulated wild type W83, non-encapsulated HG91 and the non-encapsulated insertional isogenic knockout mutant of W83, ΔEpsC) to invade GF and the ability of internalized P. gingivalis to survive in vitro antibiotic treatment. The ability of P. gingivalis strains to invade GF was tested using an antibiotic protection assay at multiplicity of infection (MOI) 100 and 1000. The survival of internalized P. gingivalis cells was further analyzed by subsequent in vitro treatment with either metronidazole or amoxicillin alone or a combination of metronidazole and amoxicillin and anaerobic culture viability counts. All strains of P. gingivalis used in this study were able to invade GFs. The non-encapsulated mutant of W83 (ΔEpsC mutant) was significantly more invasive than the wild type W83 at MOI 100 (p value 0.025) and MOI 1000 (p value 0.038). Furthermore, internalized P. gingivalis was able to resist in vitro antibiotic treatment. As demonstrated by the differences in invasion efficiencies of P. gingivalis strain W83 and its isogenic mutant ΔEpsC, the capsule of P. gingivalis makes it less efficient in invading gingival fibroblasts. Moreover, internalized P. gingivalis can survive antibiotic treatment in vitro.     

Isolation of Porphyromonas gingivalis and Detection of Immunoglobulin A Specific to Fimbrial Antigen in Gingival Crevicular Fluid

The present study evaluated the prevalence of Porphyromonas gingivalis and the correlation between the bacterial culture method and the detection of immunoglobulin A (IgA) specific to the P. gingivalis fimbrial antigen in gingival crevicular fluid (GCF). P. gingivalis was isolated from 78.3% of subgingival plaque samples obtained from active sites and 34.7% of those from inactive sites of periodontal patients. P. gingivalis was isolated from only 4.7% of healthy subjects (control group). Immunoglobulins specific to the P. gingivalis fimbrial antigen were detected by enzyme-linked immunosorbent assay (ELISA). The overall agreement between the results of the P. gingivalis culture method and the results of specific IgA detection in periodontal patients was 71.7% for active sites and 58.7% for inactive sites. IgA specific to P. gingivalis was absent in GCF from all of the sites of healthy subjects. The results suggest that P. gingivalis is associated with the local production of specific IgA. The detection of IgA antibodies specific to P. gingivalis in GCF by ELISA may be used as a predictive parameter to reveal the early phase of the activation of recurrent periodontal infections.     

Porphyromonas gingivalis virulence in a Drosophila melanogaster model

Porphyromonas gingivalis has been implicated in the etiology of adult periodontitis. In this study, we examined the viability of Drosophila melanogaster as a new model for examining P. gingivalis-host interactions. P. gingivalis (W83) infection of Drosophila resulted in a systemic infection that killed in a dose-dependent manner. Differences in the virulence of several clinically prevalent P. gingivalis strains were observed in the Drosophila killing model, and the results correlated well with studies in mammalian infection models and human epidemiologic studies. P. gingivalis pathobiology in Drosophila did not result from uncontrolled growth of the bacterium in the Drosophila hemolymph (blood) or overt damage to Drosophila tissues. P. gingivalis killing of Drosophila was multifactorial, involving several bacterial factors that are also involved in virulence in mammals. The results from this study suggest that many aspects of P. gingivalis pathogenesis in mammals are conserved in Drosophila, and thus the Drosophila killing model should be useful for characterizing P. gingivalis-host interactions and, potentially, polymicrobe-host interactions.     

Transformation and expression of a cloned fimA gene in Porphyromonas gingivalis

The Porphyromonas gingivalis fimbria is an important virulence factor involved in the adherence and colonization of the organism in the oral cavity. In this study, we transformed this organism with a gene, fimA381, encoding the fimbrial subunit of P. gingivalis 381 (fimbrillin) by using the host-vector system that we developed previously and examined expression of the cloned fimA381 gene. The recombinant plasmid pYHF2 was constructed by ligating a fragment containing the fimA381 gene into the plasmid vector pYH420 and transformed into the restriction-deficient P. gingivalis host YH522. pYHF2 was autonomously maintained in YH522 cells, and the fimbrillin polypeptide (recombinant fimbrillin) was fully expressed. The molecular mass of the recombinant fimbrillin was evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis as 41 kDa, which was identical to that of the native fimbrillin of strain 381. The amino acid sequences of the 20 amino-terminal residues of the recombinant fimbrillin and the native fimbrillin of the strain 381 were identical. In addition, characteristic long and thin fimbrial structures (recombinant fimbriae) that were distinguishable from the host's native fimbriae when examined by immunogold electron microscopy were observed around the cell surface of the transformants containing the fimA381 gene. These results suggested that transformation of fimA gene from a different strain of P. gingivalis followed by accumulation of the mature fimbrial subunit protein was sufficient for production of fimbrial structures that were observable by electron microscopy.     

Age and prevalence of Porphyromonas gingivalis in children.

The acquisition of Porphyromonas gingivalis was examined in a cross-sectional study of 198 subjects from 0 to 18 years of age using a PCR-based assay. P. gingivalis was detected in the oral cavities of 37% of subjects and at similar frequencies among subjects of all ages. These data indicate that P. gingivalis may be acquired in the first days of life.     

Tissue destruction induced by Porphyromonas gingivalis infection in a mouse chamber model is associated with host tumor necrosis factor generation

Intrachamber challenge with Porphyromonas gingivalis strain 381 in a mouse subcutaneous chamber model results in a local infection that progresses to exfoliation of the chambers within 15 days. This study was designed to elucidate the contribution of host reactions to tissue destruction manifested by chamber exfoliation in animals infected with P. gingivalis. Chamber fluids showed increasing levels of prostaglandin E(2) with infection, and the levels of tumor necrosis factor (TNF) in chamber fluids peaked just before chamber exfoliation. Intraperitoneal injection of a TNF inhibitor, thalidomide (TH), reduced the number of exfoliated chambers, while indomethacin had no effect. Exogenous TNF in chambers without bacterial infection did not cause chamber exfoliation but induced neutrophil infiltration. In a dual-chamber model, two chambers were implanted in the same mouse. One chamber was infected with P. gingivalis, and 9 days later exogenous TNF was added to the other chamber. Altogether, 66.67% of uninfected chambers were exfoliated between day 11 and day 16, although no bacteria were recovered from uninfected chambers. TH treatment alleviated both infected and uninfected chamber exfoliation. In this study, tissue destruction caused by P. gingivalis 381 infection was due to the elevation of the TNF levels and not due to local bacterial activities. Our results further indicate that local infection by P. gingivalis 381, a nondisseminating strain, actually has systemic effects on the host pathological outcome.     

Separation and structural analysis of lipoprotein in a lipopolysaccharide preparation from Porphyromonas gingivalis

Lipopolysaccharide (LPS) preparations from the periodontopathic bacterium Porphyromonas gingivalis (Pg-LPS) are thought to require Toll-like receptor (TLR)2 rather than TLR4, a receptor of Escherichia coli LPS (Ec-LPS), for activation of immune cells. However, we previously reported that P. gingivalis lipid A, an immunostimulatory principal component of LPS, and its synthetic counterpart activate cells through a TLR4-dependent pathway but not via TLR2. In the present study, a lipoprotein from Pg-LPS (Pg-LP) was shown to be a principal component for TLR2-mediated cell activation. Pg-LP was separated by hydrophobic interaction chromatography followed by preparative electrophoresis and identified by internal peptide sequencing as PG1828, a putative lipoprotein encoded in the P. gingivalis genome. The N-terminal structure was characterized as a triacylated lipopeptide using mass spectrometry. Pg-LP, as well as Ec-LPS, was potent in inducing IL-8 production in human gingival fibroblasts. From our results, we propose that Pg-LP is a powerful inflammatory factor of P. gingivalis.     

Porins and Lipopolysaccharide Induce Apoptosis in Human Spermatozoa

Treatment of human spermatozoa with porins or lipopolysaccharide (LPS) increases spontaneous apoptosis in these cells. Porins and LPS were extracted from Salmonella enterica serovar Typhimurium and Pasteurella multocida and were mixed with human spermatozoa for detection of levels of apoptosis.     

Effect of Porphyromonas gingivalis and Lactobacillus acidophilus on Secretion of IL1B, IL6, and IL8 by Gingival Epithelial Cells

Porphyromonas gingivalis alters cytokine expression in gingival epithelial cells, stimulating inflammatory responses that may lead to periodontal disease. This study explored the effect of Lactobacillus acidophilus on the specific expressions of the interleukins (ILs) IL1B, IL6, and IL8 induced by the pathogen. Human gingival epithelial cells were co-cultured with P. gingivalis, L. acidophilus, or L. acidophilus + P. gingivalis; the control group consisted of the cells alone. Protein and gene expression levels of the ILs were detected using ELISA and qRT-PCR, respectively. The supernatant from the P. gingivalis group held significantly higher protein and mRNA levels of IL1B, IL6, and IL8, compared to the control group. In the mixed bacterial group (L. acidophilus + P. gingivalis), the levels of all three ILs decreased with increasing concentrations of L. acidophilus and were significantly different from the P. gingivalis group. This suggests that in gingival cells, L. acidophilus offsets the P. gingivalis-induced secretion of these ILs in a dose-dependent manner.     

Fibrinogen-Neutrophil Interactions in Response to fMLP and Porphyromonas gingivalis Fimbrial Peptides

Porphyromonas gingivalis (P.g) is the primary bacterial agent in many forms of chronic periodontitis. Since polymorphonuclear leukocytes (PMNs) are first-line responders to P.g.- induced inflammation, and fibrinogen is important for in vivo PMN in this disease, we have studied the effect of N-formyl-methionyl-leucyl-phenylalanine (fMLP) (an inflammatory stimulus), P.g. fimbriae and fimbrial peptides (based on FimA, the main structural protein of P.g. fimbriae) on PMN-fibrinogen interactions. Freshly isolated human PMNs were allowed to react with FITC-Fibrinogen and various fimbrial peptides (denoted as FimA followed by amino acid number within whole FimA protein), and FITC-Fibrinogen binding was measured using flow cytometry. Freshly isolated neutrophils were also challenged with Fibrinogen and/or fimbrial peptides to measure IL-8 secretion using ELISA. Our studies show that fibrinogen binding to PMNs is enhanced (p < 0.01) in response to fMLP as well as fimbrial peptides (FimA 61–80) containing the motif LTTE (p < 0.01) in a dose dependent manner but not in response to peptides without that motif. We also observed that fMLP and FimA 61–80 have an additive effect on fibrinogen binding to PMNs (p < 0.05), and fMLP and FimA 171–185 significantly inhibit fMLP-induced fibrinogen binding (p < 0.01). To determine of the role of inflammatory cytokines, we examined IL-8 release from PMNs in response to combinations of P. gingivalis fimbriae, fMLP and fibrinogen. In all cases, IL-8 release increased in a dose-dependent manner (p < 0.05). fMLP-fibrinogen effect on IL-8 release from PMNs was synergistic while fimbriae-fibrinogen effect was additive. In summary, PMN priming by fimbrial peptides facilitates fibrinogen-PMN interaction and may increase inflammation.     

Porphyromonas gingivalis and adverse pregnancy outcomes: a review on its intricate pathogenic mechanisms

Abstract

Porphyromonas gingivalis (P. gingivalis), a Gram-negative facultative anaerobe of the oral cavity, is associated with the onset of various adverse pregnancy outcomes. P. gingivalis is linked with the development of preeclampsia, preterm labour, spontaneous abortion, gestational diabetes, foetal growth restriction, and misconception. The unique virulence factors, surface adhesions, enzymes of P. gingivalis can directly injure and alter the morphology, microbiome the foetal and maternal tissues. P. gingivalis can even exaggerate the production of cytokines, free radicals and acute-phase proteins in the uterine compartment that increases the risk of myometrial contraction and onset of preterm labour. Although evidence confirms the presence of P. gingivalis in the amniotic fluid and placenta of women with poor pregnancy outcomes, the intricate molecular mechanisms by which P. gingivalis initiates various antenatal and postnatal maternal and foetal complications are not well explained in the literature. Therefore, the present review aims to comprehensively summarise and highlight the recent and unique molecular pathogenic mechanisms of P. gingivalis associated with adverse pregnancy outcomes.     

Effects of a Porphyromonas gingivalis infection on inflammatory mediator response and pregnancy outcome in hamsters.

This study examines the effects of various localized, nondissemination challenges of Porphyromonas gingivalis on inflammatory mediator production and pregnancy outcome in the golden hamster. Live or heat-killed (HK) organisms were inoculated into a previously implanted subcutaneous tissue chamber on the 8th day of gestation to determine the effects on fetal weight, viability, and resorption. In one group of animals, HK organisms were inoculated prior to mating to determine the effects of previous exposure on day-8 gestational challenges. Chamber contents were assayed at 1 and 5 days after challenge for prostaglandin E2 (PGE2) and tumor necrosis factor alpha (TNF-alpha). All P. gingivalis challenges caused a significant increase in chamber PGE2 and TNF-alpha at P < 0.01 in the following order of potency: HK < Live < HK+Live. For example, following the HK+Live challenge, PGE2 levels increased from 4.7 pg/ml at baseline to 362 pg/ml at day 5 and TNF-alpha increased from 26.4 pg/ml to 724 pg/ml at day 5. The same order of potency of the various challenges was maintained with regard to the toxic effects of P. gingivalis on pregnancy outcome. For the HK+Live challenge, fetal weight was decreased 24%; embryolethality increased to 26.5% and the percent fetal resorption increased to 10.6% compared with control animal levels. There was a statistically significant association between increasing levels of both PGE2 and TNF-alpha and fetal growth retardation and embryolethality at P < 0.001. These data suggest that infections with gram-negative periodontal pathogens can elicit adverse pregnancy outcomes and that the levels of PGE2 and TNF-alpha produced as a result of challenge are associated with the severity of fetal effect.     

Use of insertion sequence element IS1126 in a genotyping and transmission study of Porphyromonas gingivalis

Porphyromonas gingivalis is strongly associated with periodontal diseases and is regarded as one of the risk factors for periodontitis. Insertion sequence element IS1126-based PCR was used to investigate the genetic heterogeneity of P. gingivalis from periodontitis patients and to examine the frequency of the parent-child and spouse-spouse transmission. Two sets of IS1126-specific primers were used for the PCR. The inward primer set (PI1 and PI2), which amplifies the IS1126 fragment of approximately 690 bp, was used to identify P. gingivalis. The outward primer set (PI1RC and PI2RC), which is reverse complementary to PI1 and PI2, respectively, and amplifies the gene fragments between the adjacent IS1126 elements was used to characterize the genotypes of the P. gingivalis strains. PCR of P. gingivalis with PI1RC and PI2RC resulted in the production of two to seven amplicons, which showed a unique electrophoretic pattern in each strain (4 laboratory strains and 37 clinical isolates cultured from 12 patients with aggressive periodontitis). The usefulness of the method for transmission study was confirmed by detecting identical genotypes between the isolates and the plaque samples from which the isolates were cultured and between the plaque samples from different tooth sites in the same patient. Thirty probands with periodontal diseases and their thirty immediate family members were included in the transmission study. In 11 of 14 parent-child pairs (78.6%), P. gingivalis revealed an identical or similar band pattern, whereas 5 of 16 spouse pairs (31.25%) had this similarity. These results show that IS1126-based PCR for genotyping P. gingivalis has a highly discriminating potential with reproducible data and is a simple and reliable method for a transmission study.     

Short fimbriae of Porphyromonas gingivalis and their role in coadhesion with Streptococcus gordonii

Porphyromonas gingivalis, one of the causative agents of adult periodontitis, attaches and forms biofilms on substrata of Streptococcus gordonii. Coadhesion and biofilm development between these organisms requires the interaction of the short fimbriae of P. gingivalis with the SspB streptococcal surface polypeptide. In this study we investigated the structure and binding activities of the short fimbriae of P. gingivalis. Electron microscopy showed that isolated short fimbriae have an average length of 103 nm and exhibit a helical structure with a pitch of ca. 27 nm. Mfa1, the major protein subunit of the short fimbriae, bound to SspB protein, and this reaction was inhibited by purified recombinant Mfa1 and monospecifc anti-Mfa1 serum in a dose-dependent manner. Complementation of a polar Mfa1 mutant with the mfa1 gene restored the coadhesion phenotype of P. gingivalis. Hence, the Mfa1 structural fimbrial subunit does not require accessory proteins for binding to SspB. Furthermore, the interaction of Mfa1 with SspB is necessary for optimal coadhesion between P. gingivalis and S. gordonii.     

Structural dissection and in vivo effectiveness of a peptide inhibitor of Porphyromonas gingivalis adherence to Streptococcus gordonii

The interaction of the minor fimbrial antigen (Mfa) with streptococcal antigen I/II (e.g., SspB) facilitates colonization of the dental biofilm by Porphyromonas gingivalis. We previously showed that a 27-mer peptide derived from SspB (designated BAR) resembles the nuclear receptor (NR) box protein-protein interacting domain and potently inhibits this interaction in vitro. Here, we show that the EXXP motif upstream of the NR core α-helix contributes to the Mfa-SspB interaction and that BAR reduces P. gingivalis colonization and alveolar bone loss in vivo in a murine model of periodontitis. Substitution of Gln for Pro(1171) or Glu(1168) increased the α-helicity of BAR and reduced its inhibitory activity in vitro by 10-fold and 2-fold, respectively. To determine if BAR prevents P. gingivalis infection in vivo, mice were first infected with Streptococcus gordonii and then challenged with P. gingivalis in the absence and presence of BAR. Animals that were infected with either 10(9) CFU of S. gordonii DL-1 or 10(7) CFU of P. gingivalis 33277 did not show a statistically significant increase in alveolar bone resorption over sham-infected controls. However, infection with 10(9) CFU of S. gordonii followed by 10(7) CFU of P. gingivalis induced significantly greater bone loss (P < 0.01) than sham infection or infection of mice with either organism alone. S. gordonii-infected mice that were subsequently challenged with 10(7) CFU of P. gingivalis in the presence of BAR exhibited levels of bone resorption similar to those of sham-infected animals. Together, these results indicate that both EXXP and the NR box are important for the Mfa-SspB interaction and that BAR peptide represents a potential therapeutic that may limit colonization of the oral cavity by P. gingivalis.     

Effect of Fusobacterium nucleatum on the T and B cell responses to Porphyromonas gingivalis in a mouse model

T cell cytokine profiles and specific serum antibody levels in five groups of BALB/c mice immunized with saline alone, viable Fusobacterium nucleatum ATCC 25586, viable Porphyromonas gingivalis ATCC 33277, F. nucleatum followed by P. gingivalis and P. gingivalis followed by F. nucleatum were determined. Splenic CD4 and CD8 cells were examined for intracytoplasmic interleukin (IL)-4, interferon (IFN)-gamma and IL-10 by dual colour flow cytometry and the levels of serum anti-F. nucleatum and anti-P. gingivalis antibodies determined by an ELISA. Both Th1 and Th2 responses were demonstrated by all groups, and while there were slightly lower percentages of cytokine positive T cells in mice injected with F. nucleatum alone compared with the other groups immunized with bacteria, F. nucleatum had no effect on the T cell production of cytokines induced by P. gingivalis in the two groups immunized with both organisms. However, the percentages of cytokine positive CD8 cells were generally significantly higher than those of the CD4 cells. Mice immunized with F. nucleatum alone had high levels of serum anti-F. nucleatum antibodies with very low levels of P. gingivalis antibodies, whereas mice injected with P. gingivalis alone produced anti-P. gingivalis antibodies predominantly. Although the levels of anti-F. nucleatum antibodies in mice injected with F. nucleatum followed by P. gingivalis were the same as in mice immunized with F. nucleatum alone, antibody levels to P. gingivalis were very low. In contrast, mice injected with P. gingivalis followed by F. nucleatum produced equal levels of both anti-P. gingivalis and anti-F. nucleatum antibodies, although at lower levels than the other three groups immunized with bacteria, respectively. Anti-Actinobacillus actinomycetemcomitans, Bacteroides forsythus and Prevotella intermedia serum antibody levels were also determined and found to be negligible. In conclusion, F. nucleatum immunization does not affect the splenic T cell cytokine response to P. gingivalis. However, F. nucleatum immunization prior to that of P. gingivalis almost completely inhibited the production of anti-P. gingivalis antibodies while P. gingivalis injection before F. nucleatum demonstrated a partial inhibitory effect by P. gingivalis on antibody production to F. nucleatum. The significance of these results with respect to human periodontal disease is difficult to determine. However, they may explain in part differing responses to P. gingivalis in different individuals who may or may not have had prior exposure to F. nucleatum. Finally, the results suggested that P. gingivalis and F. nucleatum do not induce the production of cross-reactive antibodies to other oral microorganisms.