Streptococcal H2O2 inhibits IgE-triggered degranulation of RBL-2H3 mast cell/basophil cell line by inducing cell death.

Mast cells and basophils are central players in allergic reactions triggered by immunoglobulin E (IgE). They have intracellular granules containing allergic mediators (e.g., histamine, serotonin, inflammatory cytokines, proteases and ß-hexosaminidase), and stimulation by IgE-allergen complex leads to the release of such allergic mediators from the granules, that is, degranulation. Mast cells are residents of mucosal surfaces, including those of nasal and oral cavities, and play an important role in the innate defense system. Members of the mitis group streptococci such as Streptococcus oralis, are primary colonizers of the human oral cavity. They produce hydrogen peroxide (H2O2) as a by-product of sugar metabolism. In this study, we investigated the effects of streptococcal infection on RBL-2H3 mast cell/basophil cell line. Infection by oral streptococci did not induce degranulation of the cells. Stimulation of the RBL-2H3 cells with anti-dinitrophenol (DNP) IgE and DNP-conjugated human serum albumin triggers degranulation with the release of ß-hexosaminidase. We found that S. oralis and other mitis group streptococci inhibited the IgE-triggered degranulation of RBL-2H3 cells. Since mitis group streptococci produce H2O2, we examined the effect of S. oralis mutant strain deficient in producing H2O2, and found that they lost the ability to suppress the degranulation. Moreover, H2O2 alone inhibited the IgE-induced degranulation. Subsequent analysis suggested that the inhibition of degranulation was related to the cytotoxicity of streptococcal H2O2. Activated RBL-2H3 cells produce interleukin-4 (IL-4); however, IL-4 production was not induced by streptococcal H2O2. Furthermore, an in vivo study using the murine pollen-induced allergic rhinitis model suggested that the streptococcal H2O2 reduces nasal allergic reaction. These findings reveal that H2O2 produced by oral mitis group streptococci inhibits IgE-stimulated degranulation by inducing cell death. Consequently, streptococcal H2O2 can be considered to modulate the allergic reaction in mucosal surfaces.

Hydrogen peroxide release by bacteria suppresses inflammasome-dependent innate immunity.

Hydrogen peroxide (H2O2) has a major function in host-microbial interactions. Although most studies have focused on the endogenous H2O2 produced by immune cells to kill microbes, bacteria can also produce H2O2. How microbial H2O2 influences the dynamics of host-microbial interactions is unclear. Here we show that H2O2 released by Streptococcus pneumoniae inhibits inflammasomes, key components of the innate immune system, contributing to the pathogen colonization of the host. We also show that the oral commensal H2O2-producing bacteria Streptococcus oralis can block inflammasome activation. This study uncovers an unexpected role of H2O2 in immune suppression and demonstrates how, through this mechanism, bacteria might restrain the immune system to co-exist with the host.

Bacteriotherapy for preventing recurrent upper respiratory infections in children: a real-world experience.

Background Recurrent upper respiratory infections (RURI) constitute a social problem for both their pharmaco-economic impact and the burden for the family. Bacteriotherapy could be an interesting preventive option. Objective The aim of this study was to evaluate the preventive effects of RURI in children. Design The study was designed as spontaneous, and was conducted in real-life seting. Globally, 80 children (40 males, mean age 5.26 (2.52) years) with RURI were enrolled. Children were treated with Streptococcus salivarius 24SMB and Streptococcus oralis 89a: nasal spray 2 puffs per nostril twice/day for a week for 3 monthly courses. Number of URI, and school and work absences were evaluated and compared with the past year. Results Bacteriotherapy significantly halved the mean number of URI episodes being 5.98 (2.30) in the past year and 2.75 (2.43) after the treatment (p<0.0001). Bacteriotherapy also induced an over 35% reduction both in the number of school days and in the number of working days missed per month from 4.50 (2.81) to 2.80 (3.42) and from 2.33 (2.36) to 1.48 (2.16) respectively (p<0.0001). Conclusions This and real-life study provides the first evidence that Streptococcus salivarius 24SMB and Streptococcus oralis 89a nasal spray could be effective in preventing RURI in children.

Identification of proteins in Streptococcus pneumoniae by reverse vaccinology and genetic diversity of these proteins in clinical isolates.

Streptococcus pneumoniae is a major cause of morbidity and mortality worldwide. Virulence-associated proteins common and conserved among all capsular types now represent the best strategy to combat pneumococcal infections. Our aim was to identify conserved targets in pneumococci that showed positive prediction for lipoprotein and extracellular subcellular location using bioinformatics programs and verify the distribution and the degree of conservation of these targets in pneumococci. These targets can be considered potential vaccine candidate to be evaluated in the future. A set of 13 targets were analyzed and confirmed the presence in all pneumococci tested. These 13 genes were highly conserved showing around >96 % of amino acid and nucleotide identity, but they were also present and show high identity in the closely related species Streptococcus mitis, Streptococcus oralis, and Streptococcus pseudopneumoniae. S. oralis clusters away from S. pneumoniae, while S. pseudopneumoniae and S. mitis cluster closer. The divergence between the selected targets was too small to be observed consistently in phylogenetic groups between the analyzed genomes of S. pneumoniae. The proteins analyzed fulfill two of the initial criteria of a vaccine candidate: targets are present in a variety of different pneumococci strains including different serotypes and are conserved among the samples evaluated.

Association between polycystic ovary syndrome, oral microbiota and systemic antibody responses.

Polycystic ovary syndrome (PCOS) is a hormonal disorder of women that not only is the leading cause of infertility but also shows a reciprocal link with oral health. This study aimed to investigate the hypothesis that the levels of putative periodontal pathogens in saliva and their antibody response in serum are elevated in PCOS, compared to systemic health. A total of 125 women were included in four groups; 45 women with PCOS and healthy periodontium, 35 women with PCOS and gingivitis, 25 systemically and periodontally healthy women, 20 systemically healthy women with gingivitis. Salivary levels of seven putative periodontal pathogens were analyzed by quantitative real-time polymerase chain reaction and serum antibody levels were analyzed by ELISA. In women with PCOS, salivary Porphyromonas gingivalis, Fusobacterium nucleatum, Streptococcus oralis and Tannerella forsythia levels were higher than matched systemically healthy women, particularly in the case of gingivitis. Aggregatibacter actinomycetemcomitans and Treponema denticola levels were similar among study groups. The presence of PCOS also enhanced P. gingivalis, Prevotella intermedia and S. oralis serum antibody levels, when gingivitis was also present. Gingival inflammation correlated positively with levels of the studied taxa in saliva, particularly in PCOS. The presence of P. gingivalis and F. nucleatum in saliva also exhibited a strong positive correlation with the corresponding serum antibody levels. In conclusion, as an underlying systemic endocrine condition, PCOS may quantitatively affect the composition of oral microbiota and the raised systemic response to selective members of this microbial community, exerting a confounding role in resultant gingival inflammation and periodontal health. The most consistent effect appeared to be exerted on P. gingivalis.

Streptococcus oralis coaggregation receptor polysaccharides induce inflammatory responses in human aortic endothelial cells.

Streptococcus oralis, belonging to the oral viridans group streptococci, has been detected in human cardiovascular lesions including infective endocarditis and atheromatous plaques. The organism has coaggregation receptor polysaccharides (RPS) on the cell wall, which function as receptors for surface adhesins on other members of the oral biofilm community. The present study examined the capacity of S. oralis RPS to induce inflammatory responses in human aortic endothelial cells (HAECs). Purified RPS was used to stimulate HAECs, and the induction of cytokines, adhesion molecules and Toll-like receptors (TLRs) was examined. Involvement of RPS in HAEC invasion by S. oralis was also examined. RPS-stimulated HAECs produced more cytokines (interleukin-6, interleukin-8 and monocyte chemoattractant protein-1) and intercellular adhesion molecule-1 than non-stimulated HAECs. The messenger RNA (mRNA) expression of cytokines and adhesion molecules in RPS-stimulated HAECs increased markedly compared with that in non-stimulated HAECs. Upregulation of TLR-2 mRNA expression was demonstrated in RPS-stimulated HAECs. Moreover, TLR-2 mRNA expression and cytokine production were reduced by the incubation of HAECs with inhibitors against p38 mitogen-activated protein kinase and nuclear factor-κB. An RPS-defective mutant of S. oralis showed greater invasion into HAECs than an RPS-possessing strain. However, HAECs invaded by the RPS-defective mutant produced less cytokines than HAECs invaded by the RPS-possessing strain, indicating that RPS can stimulate HAECs intracellularly. These results suggest that S. oralis RPS may be an important contributor to the pathogenesis of cardiovascular diseases such as infective endocarditis and atherosclerosis.

Streptococcus cristatus attenuates Fusobacterium nucleatum-induced interleukin-8 expression in oral epithelial cells.

BACKGROUND AND OBJECTIVE: Oral epithelial cells may be invaded by a polymicrobial intracellular flora, including pathogens together with commensals. Various oral pathogens can induce the production of interleukin-8, a potent neutrophil chemotractant, in oral epithelial cells. Evidence from the gut suggests that commensal species may modulate inflammatory responses to pathogens. The aim of this study was to examine the interleukin-8 responses of oral epithelial cells to an oral pro-inflammatory species, Fusobacterium nucleatum, in combination with an oral commensal, Streptococcus cristatus. MATERIAL AND METHODS: KB, TERT-2, TR146 and SCC15 cells were cocultured with F. nucleatum and S. cristatus, either alone or in combination, at 37 degrees C in 5% CO2 under various conditions. The mRNA expression of interleukin-8 was analyzed by reverse transcription-polymerase chain reaction and protein secretion was measured by enzyme-linked immunosorbent assay. RESULTS: F. nucleatum alone evoked a potent interleukin-8 response, whereas S. cristatus alone did not induce significant interleukin-8 expression in oral epithelial cells. When present together, S. cristatus attenuated the F. nucleatum-induced interleukin-8 production in the four oral epithelial cell lines to varying degrees. The inhibitory effect of S. cristatus was independent of its viability and its co-aggregation with F. nucleatum, was not related to soluble bacterial products and appeared to require bacterial contact with epithelial cells. Similar effects were seen with several other species of oral streptococci. CONCLUSION: Our data suggest that S. cristatus may exert immunomodulatory effects on the interleukin-8 response of oral epithelial cells to F. nucleatum challenge.

Physiological and serological variation in Streptococcus mitis biovar 1 from the human oral cavity during the first year of life.

OBJECTIVE: The purpose of the study was to explore the physiological and antigenic diversity of a large number of Streptococcus mitis biovar 1 isolates in order to begin to determine whether these properties contribute to species persistence. DESIGN: S. mitis biovar 1 was collected from four infants from birth to the first year of age. At each of eight to nine visits, 60 isolates each were obtained from the cheeks, tongue and incisors (once erupted) yielding 4440 in total. These were tested for production of neuraminidase, beta1-N-acetylglucosaminidase, beta1-N-acetylgalactosaminidase, IgA1 protease and amylase-binding. Antigenic diversity was examined by ELISA and Western immunoblotting using antisera raised against S. mitis biovar 1 NCTC 12261(T) and SK145. RESULTS: Three thousand three hundred and thirty (75%) of the isolates were identified as S. mitis biovar 1 and 3144 (94.4%) could be divided into four large phenotypic groups based on glycosidase production. Fifty-four percent of the isolates produced IgA1 protease, but production was disproportionate among the phenotypes. Between one-third and one-half of the strains of each phenotype bound salivary alpha-amylase. Antisera against strains NCTC 12261(T) and SK145 displayed different patterns of reactivity with randomly selected representatives of the four phenotypes. CONCLUSIONS: S. mitis biovar 1 is physiologically and antigenically diverse, properties which could aid strains in avoiding host immunity and promote re-colonization of a habitat or transfer to a new habitat.

The influences of hinge length and composition on the susceptibility of human IgA to cleavage by diverse bacterial IgA1 proteases.

The influences of IgA hinge length and composition on its susceptibility to cleavage by bacterial IgA1 proteases were examined using a panel of IgA hinge mutants. The IgA1 proteases of Streptococcus pneumoniae, Streptococcus sanguis strains SK4 and SK49, Neisseria meningitidis, Neisseria gonorrhoeae, and Haemophilus influenzae cleaved IgA2-IgA1 half hinge, an Ab featuring half of the IgA1 hinge incorporated into the equivalent site in IgA1 protease-resistant IgA2, whereas those of Streptococcus mitis, Streptococcus oralis, and S. sanguis strain SK1 did not. Hinge length reduction by removal of two of the four C-terminal proline residues rendered IgA2-IgA1 half hinge resistant to all streptococcal IgA1 metalloproteinases but it remained sensitive to cleavage by the serine-type IgA1 proteases of Neisseria and Haemophilus spp. The four C-terminal proline residues could be substituted by alanine residues or transferred to the N-terminal extremity of the hinge without affect on the susceptibility of the Ab to cleavage by serine-type IgA1 proteases. However, their removal rendered the Ab resistant to cleavage by all the IgA1 proteases. We conclude that the serine-type IgA1 proteases of Neisseria and Haemophilus require the Fab and Fc regions to be separated by at least ten (or in the case of N. gonorrhoeae type I protease, nine) amino acids between Val(222) and Cys(241) (IgA1 numbering) for efficient access and cleavage. By contrast, the streptococcal IgA1 metalloproteinases require 12 or more appropriate amino acids between the Fab and Fc to maintain a minimum critical distance between the scissile bond and the start of the Fc.

Phosphorylcholine-dependent cross-reactivity between dental plaque bacteria and oxidized low-density lipoproteins.

Antibodies reactive with phosphorylcholine (PC) are ubiquitous in human sera, but the antigens stimulating their production and their function are not clear. Previous studies have shown that a significant proportion of dental plaque bacteria contain PC as determined by reactivity with PC-specific mouse myeloma proteins and monoclonal antibodies. Additionally, serum antibody concentrations of immunoglobulin (IgG) G anti-PC are higher in sera of individuals who have experienced periodontal attachment loss than those who are periodontally healthy. These data implicate the oral microflora as a source of antigen-stimulating anti-PC responses. Recent data also indicate that antibodies with specificity for PC are elevated in ApoE-deficient mice, a model for studies of athersclerosis, and that such antibodies bound oxidized low-density lipoproteins (LDL) (oxLDL) in atherosclerotic plaques. These data prompted the hypothesis that human anti-PC could bind to both oral bacteria and human oxLDL, and that these antigens are cross-reactive. We therefore examined the ability of human anti-PC to bind to PC-bearing strains of oral bacteria using enzyme-linked immunosorbent inhibition assays and by assessment of direct binding of affinity-purified human anti-PC to PC-bearing Actinobacillus actinomycetemcomitans. Our results indicated that PC-bearing strains of Streptococcus oralis, Streptococcus sanguis, Haemophilus aphrophilus, Actinomyces naeslundii, Fusobacterium nucleatum, and A. actinomycetemcomitans, as well as a strain of Streptococcus pneumoniae, absorbed up to 80% of anti-PC IgG antibody from human sera. Furthermore, purified anti-PC bound to a PC-bearing strain of A. actinomycetemcomitans but only poorly to a PC-negative strain. OxLDL also absorbed anti-PC from human sera, and oxLDL but not LDL reacted with up to 80% of the anti-PC in human sera. Furthermore, purified anti-PC bound directly to oxLDL but not to LDL. The data indicate that PC-containing antigens on a variety of common oral bacteria are cross-reactive with neoantigens expressed in oxLDL. We propose that PC-bearing dental plaque microorganisms may induce an antibody response to PC that could influence the inflammatory response associated with atherosclerosis.

Crohn disease arthropathy: antigens in synovial fluid share epitopes with strains of two species of viridans streptococci.

BACKGROUND: There is evidence to suggest that Crohn disease is caused by an immunologic response to an unknown intestinal luminal antigen, probably of bacterial origin. The reported demonstration of yersinia antigen in the synovial fluid of patients with yersinosis therefore prompted a search for bacterial antigens in the synovial fluid of patients with Crohn arthropathy. METHODS: Antisera were raised in rabbits to synovial fluids obtained from seven patients with Crohn arthropathy and from seven 'control' subjects with other forms of arthropathy. These antisera were used to probe sonicates of the bacteria cultured from the gastric juice of patients with gastric Crohn disease. RESULTS: The antisera made from the Crohn synovial fluids, but none of those made from the controls, reacted uniquely with antigens in sonicates of strains of two species of viridans streptococci (Streptococcus parasanguis and an atypical S. oralis) isolated from four of the five patients with gastric Crohn disease. CONCLUSIONS: These findings suggest that the arthropathy of Crohn disease and, possibly, the intestinal disease itself may involve an immunologically mediated inflammatory response to these antigens.

The use of immunoliposomes for specific delivery of antimicrobial agents to oral bacteria immobilized on polystyrene.

Antibacterial immunoliposomes have been prepared using covalently bound antibody, raised to the cell surface of the bacterium Streptococcus oralis (S. oralis), and incorporating the bactericides chlorhexidine and Triclosan. A regrowth assay, in which the ability of a bacterial biofilm immobilised on polystyrene to grow after exposure to a test solution, was undertaken to study the action of the antibacterial immunoliposomes. The antibacterial anti-oralis immunoliposomes show enhanced growth inhibition of S. oralis, compared to free bactericide, using low bactericide concentrations. For short exposure times to the biofilms, antibacterial anti-oralis immunoliposomes can show several times enhanced growth inhibition of S. oralis compared to free bactericide. Antibacterial anti-oralis immunoliposomes inhibit the growth of S. oralis more than that of other oral bacteria. The extent of growth inhibition by antibacterial anti-oralis immunoliposomes is linearly related to the number of immunoliposomes targeted to the biofilm surface.

The pattern of change in salivary immunoglobulins and antibodies to S. mitis and S. oralis in children undergoing bone marrow transplantation: use of an indirect method of assessment.

The objective of this study was to assess the pattern of change in salivary immunoglobulins and antibodies to S. mitis and S. oralis in 23 children following allogeneic bone marrow transplantation and their matched controls. To overcome the difficulty of obtaining a sufficient quantity of whole saliva from very young, sick children saliva was collected in a 5-ml oral rinse of sterile normal saline. It was not possible to measure the volume of whole saliva in each rinse and the concentration of the salivary immunoglobulins and bacterial antibodies were estimated from 1 ml of oral rinse. Despite these shortcomings a pattern of change in the mean concentrations of total salivary IgA, secretory IgA, antibodies to S. mitis and S. oralis and total IgG at specific event- related times during the transplantation period has been demonstrated. There was a significant increase in the concentration of salivary IgG 7 days post-transplantation, followed by significant decreases in total salivary IgA, secretory IgA and antibodies to S. mitis after recovery of the peripheral neutrophil count above 0.5 x 10(9). The concentrations of total IgA and antibodies to S. oralis was significantly greater in the transplant group 119 days post-transplantation.

Humoral immunity to commensal oral bacteria in human infants: salivary secretory immunoglobulin A antibodies reactive with Streptococcus mitis biovar 1, Streptococcus oralis, Streptococcus mutans, and Enterococcus faecalis during the first two years of life.

Secretory immunoglobulin A (SIgA) antibodies reactive with the pioneer oral streptococci Streptococcus mitis biovar 1 and Streptococcus oralis, the late oral colonizer Streptococcus mutans, and the pioneer enteric bacterium Enterococcus faecalis in saliva samples from 10 human infants from birth to age 2 years were analyzed. Low levels of salivary SIgA1 and SIgA2 antibodies reactive with whole cells of all four species were detected within the first month after birth, even though S. mutans and E. faecalis were not recovered from the mouths of the infants during the study period. Although there was a fivefold increase in the concentration of SIgA between birth and age 2 years, there were no differences between the concentrations of SIgA1 and SIgA2 antibodies reactive with the four species over this time period. When the concentrations of SIgA1 and SIgA2 antibodies reactive with all four species were normalized to the concentrations of SIgA1 and SIgA2 in saliva, SIgA1 and SIgA2 antibodies reactive with these bacteria showed a significant decrease from birth to 2 years of age. Adsorption of each infant's saliva with cells of one species produced a dramatic reduction of antibodies recognizing the other three species. Sequential adsorption of saliva samples removed all SIgA antibody to the bacteria, indicating that the SIgA antibodies were directed to antigens shared by all four species. The induction by the host of a limited immune response to common antigens that are likely not involved in adherence may be among the mechanisms that commensal streptococci employ to persist in the oral cavity.

Periodontal findings and systemic antibody responses to oral microorganisms in Behçet's disease.

BACKGROUND: Behçet's disease is a multisystem disorder of unknown etiology, affecting predominantly the oral mucosa, skin, and eyes. Recurrent and painful episodes of oral ulcerations interfere with regular oral hygiene leading to rapid bacterial plaque accumulation. The aims of this study were to evaluate the periodontal status of patients with Behçet's disease and determine serum antibody responses to selected oral microorganisms, including major periodontopathogens in these patients. METHODS: Thirty-three patients with Behçet's disease and 15 healthy subjects were included in the study. Plaque, sulcular bleeding, periodontal index scores, probing depths, and total number of teeth were recorded. Serum IgG antibody levels to a panel of 13 oral microorganisms were determined. RESULTS: Significantly higher values for each of the clinical measures were observed in patients with Behçet's disease compared to healthy subjects (P <0.0001). Antibody levels to selected members of plaque, including Actinomyces viscosus, Streptococcus mutans, Streptococcus sanguis, Streptococcus oralis, Eikenella corrodens, Campylobacter rectus, and Prevotella intermedia were significantly lower in patients with Behçet's disease than in controls (P <0.001-0.05). In contrast, these patients exhibited significantly elevated antibody levels to Actinobacillus actinomycetemcomitans Y4 compared to controls (P <0.01). CONCLUSIONS: Our data indicate that the patients with Behçet's disease generally exhibit clinical findings of established periodontal disease. Decreased antibody responses to early colonizers of both supra- and subgingival plaque were observed along with the elevation in antibody levels to A. actinomycetemcomitans. These results suggest that the bacterial plaque ecology and/or immune responses to these microorganisms may be affected in Behçet's disease which could lead to changes in the expression of periodontal disease.

Serum antibodies reacting with subgingival species in refractory periodontitis subjects.

The purpose of this investigation was to compare the levels of serum IgG antibody to 85 subgingival species in 32 refractory periodontitis, 56 successfully treated, and 33 periodontally healthy subjects. Refractory subjects showed mean full mouth attachment loss and/or >3 sites showing attachment loss >2.5 mm within 1 year after 2 treatment modalities, scaling and root planing and surgery plus systemically administered tetracycline. Successfully-treated subjects showed mean attachment level gain and no sites with attachment loss >2.5 mm, 1 year post-therapy. Periodontally healthy subjects exhibited no pocket or attachment level >3 mm, and no evidence of progressing attachment loss during 1 year of monitoring. Baseline serum was obtained from each subject and tested against 85 subgingival species, including reference strains and strains isolated from refractory subjects, using checkerboard immunoblotting. Significance of differences in levels of serum antibody among groups were sought using the Kruskal-Wallis test. Refractory subjects constituted a heterogeneous group based on their serum antibody response to subgingival species. Some individuals had antibody reactions to many subgingival species, while other subjects showed fewer or low numbers of responses. On average, refractory subjects exhibited higher numbers and levels of serum antibody reactions to a wide range of subgingival species than successfully treated or periodontally healthy subjects. Differences in serum antibody among clinical groups were more striking at higher threshold levels of antibody (>50 microg/ml and > 100 microg/ml). The data showed that a subject was 10.1 x more likely to be refractory if the subject exhibited antibody reactions with >9 subgingival species at >50 microg/ml (p<0.001, after adjusting for multiple comparisons). Serum antibody to a subset of the test species differed among the clinical groups. Porphyromonas gingivalis, Bacteroidesforsythus, and some strains isolated from refractory subjects (a novel Neisseria sp., Enterococcus faecalis, Prevotella loescheii and Prevotella oulora) elicited high serum antibody in the successfully treated and refractory subjects. High levels of serum antibody to a Microbacterium lacticum-like organism, Streptococcus oralis, Streptococcus constellatus, Actinobacillus actinonmycetemcomitans serotype c and Haemophilus aphrophilus significantly increased the likelihood of a subject being refractory to conventional periodontal therapy.

Periodontal status and serum antibody responses to oral microorganisms in Sjögren's syndrome.

Sjögren's syndrome is an autoimmune disease characterized by keratoconjunctivitis sicca and xerostomia. Rapid bacterial plaque accumulation occurs in Sjögren's syndrome patients due to decreases in salivary flow rate. The purpose of this study was to evaluate the periodontal status of patients with Sjögren's syndrome and evaluate serum antibody responses to selected oral microorganisms, including major periodontopathogens, compared to healthy controls. Seventeen Sjögren's syndrome patients and 14 healthy subjects were included in the study. Plaque (PL), sulcular bleeding (SBI), periodontal index scores (PI), probing depths (PD), and total number of teeth were recorded. An ELISA was used to determine the serum IgG antibody level to a panel of 13 oral microorganisms. Significantly higher PL, SBI, PD, and PI scores, as well as an increased number of lost teeth were observed in patients with Sjögren's syndrome compared to healthy subjects (P <0.0001). Antibody levels to Streptococcus oralis were significantly lower in Sjögren's syndrome patients than controls (P <0.0002). These patients exhibited significantly elevated antibody levels to Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis compared to controls (P <0.006 to 0.0004). Our findings indicate that Sjögren's syndrome patients have established periodontal disease and serum antibody responses to oral microorganisms previously identified as periodontopathogens in systemically healthy subjects. These results suggest that Sjögren's syndrome may affect bacterial colonization in plaque and contribute to increased periodontal disease in this compromised population.

The specificity and affinity of immunoliposome targeting to oral bacteria.

Immunoliposomes have been prepared using antibodies raised to an antigenic determinant on the cell surface of the oral bacterium Streptococcus oralis (S. oralis) in an investigation of their potential to reduce dental plaque. The N-succinimidyl-S-acetylthioacetate (SATA) derivative of the antibodies were conjugated through the reactive m-maleimidobenzoyl-N-hydroxysuccinimide (MBS) derivative of dipalmitoyl-phosphatidylethanolamine (DPPE) incorporated into liposomes. The degree of antibody conjugation to the liposomes was controlled by the percentage of DPPEMBS incorporated into the liposomes. The chemical modification of the antibodies did not affect the ability of the antibodies to bind to a S. oralis biofilm. However, the affinity of the immunoliposomes for S. oralis was much lower than that of the free antibody. The anti-oralis antibodies were highly specific for S. oralis. The anti-oralis immunoliposomes showed the greatest affinity for S. oralis, when targeted to a range of different oral bacterial biofilms. The immunoliposome targeting affinity for S. oralis was largely unaffected by the number of antibodies conjugated to the liposomal surface or by the net charge of the liposomal lipid bilayer. The immunoliposomes showed a greater affinity for S. oralis than 'naked' (bearing no antibody) liposomes. However, positively charged liposomes, incorporating stearylamine, adsorbed to S. oralis with greater affinities than the immunoliposomes. The immunoliposomes appeared to be physically stable over a period of 18 months, as judged by particle-size measurements.

Comparison of the indigenous oral microbiota and immunoglobulin responses of athymic (nu/nu) and euthymic (nu/+) mice.

The role of the immune system in the homeostasis of indigenous oral bacterial populations is poorly understood. In this study, we compared the evolution of the indigenous oral microbiota of specific pathogen-free athymic nude (nu/nu) BALB/c mice with that of their corresponding phenotypically normal (nu/-) littermates. We also evaluated corresponding salivary and serum antibody activities (IgA and IgG) against the predominant indigenous oral bacteria. The bacterial species recovered from the two mouse strains were Lactobacillus murinus, Enterococcus faecalis, Streptococcus oralis and Staphylococcus epidermidis. From 27 days of age, nu/+ and nu/nu mice had significantly different proportions of oral bacterial populations. When the microbiota stabilized (at 40 days of age), the total cultivable microbiota of nu/+ mice was dominated by L. murinus (65-85%), while that of nu/nu mice was dominated by E. faecalis (40-60%). The precise factors that alter the oral resident microbiota in nu/nu mice are unknown. We found that total salivary IgA levels were significantly lower in nu/nu mice, but no association were observed between the level of salivary IgA antibody against indigenous bacteria and the proportion of these indigenous bacteria in the oral microbiota. The change in the microbiota of nude mice may have been caused by other factors such as defects in other immune functions or cold stress.

Effect of antibodies on the chain length and growth of Streptococcus sobrinus.

Passive immunization has been suggested as a method to prevent colonization of teeth by mutans streptococci. However, the mechanism of action of antibodies, both polyclonal and monoclonal, is not clear. In this study we investigated the effect of polyclonal antibodies (pAbs) and a monoclonal antibody (OMVU10) on the chain length and growth of Streptococcus sobrinus. During growth in broth S. sobrinus formed significantly longer chains in the presence of pAbs in comparison to pre-immune serum (p < 0.01), but pAbs did not influence the growth rate of S. sobrinus in broth. OMVU10 did not influence the growth rate nor the chain length. In order to study the effect of the antibodies on adhesion and growth on a surface, S. sobrinus was grown on hydroxyapatite discs in the presence of two other bacteria, Streptococcus oralis and Actinomyces viscosus. No effect of the antibodies was found on the total cultivable count of the three bacteria after growth on hydroxyapatite discs. However, the morphology of the S. sobrinus microcolonies was different after growth in the presence of both pAbs and OMVU10. The colonies were less dense and chains of S. sobrinus could be seen using a confocal laser microscope. After growth in the presence of the control antibodies, the colonies were dense and no long chains could be observed. It was concluded that pAbs influenced the chain length, in broth and on hydroxyapatite discs, and the colony morphology of S. sobrinus on hydroxyapatite discs, whereas OMVU10 influenced the colony morphology of S. sobrinus on hydroxyapatite discs.