Subgingival biodiversity in subjects with uncontrolled type-2 diabetes and chronic periodontitis.

BACKGROUND AND OBJECTIVE: There is a bidirectional relationship between periodontal disease and type-2 diabetes mellitus (DM). Inflammatory mediators may negatively affect glycemic control, and increased glucose levels and resultant glycation end-products may alter the host response against bacterial infection. However, no agreement has been reached regarding the effect of DM on periodontal subgingival microbiota. Therefore, the purpose of the present study was to compare the subgingival biodiversity in deep periodontal pockets of subjects with chronic periodontitis and either uncontrolled type-2 diabetes or no diabetes using 16S rRNA gene cloning and sequencing. MATERIAL AND METHODS: Twelve subjects with uncontrolled type-2 diabetes (glycated hemoglobin > 8%) and eleven nondiabetic subjects presenting severe and generalized chronic periodontitis were selected. Subgingival biofilm from periodontal pockets > 5 mm were assessed using the 16S rRNA gene cloning and sequencing technique. RESULTS: Significant differences were observed in subgingival microbiota between diabetic and nondiabetic subjects. Diabetic subjects presented higher percentages of total clones of TM7, Aggregatibacter, Neisseria, Gemella, Eikenella, Selenomonas, Actinomyces, Capnocytophaga, Fusobacterium, Veillonella and Streptococcus genera, and lower percentages of Porphyromonas, Filifactor, Eubacterium, Synergistetes, Tannerella and Treponema genera than nondiabetic individuals (p < 0.05). Moreover, some phylotypes, such as Fusobacterium nucleatum, Veillonella parvula, V. dispar and Eikenella corrodens were detected significantly more often in diabetic subjects than in nondiabetic subjects (p < 0.05). CONCLUSION: Subjects with uncontrolled type-2 diabetes and chronic periodontitis presented significant dissimilarities in subgingival biodiversity compared with nondiabetic subjects.

Proteomic analysis of Porphyromonas gingivalis exposed to nicotine and cotinine.

BACKGROUND AND OBJECTIVE: Smokers are more predisposed than nonsmokers to infection with Porphyromonas gingivalis, one of the most important pathogens involved in the onset and development of periodontitis. It has also been observed that tobacco, and tobacco derivatives such as nicotine and cotinine, can induce modifications to P. gingivalis virulence. However, the effect of the major compounds derived from cigarettes on expression of protein by P. gingivalis is poorly understood. Therefore, this study aimed to evaluate and compare the effects of nicotine and cotinine on the P. gingivalis proteomic profile. MATERIAL AND METHODS: Total proteins of P. gingivalis exposed to nicotine and cotinine were extracted and separated by two-dimensional electrophoresis. Proteins differentially expressed were successfully identified through liquid chromatography-mass spectrometry and primary sequence databases using MASCOT search engine, and gene ontology was carried out using DAVID tools. RESULTS: Of the approximately 410 protein spots that were reproducibly detected on each gel, 23 were differentially expressed in at least one of the treatments. A particular increase was seen in proteins involved in metabolism, virulence and acquisition of peptides, protein synthesis and folding, transcription and oxidative stress. Few proteins showed significant decreases in expression; those that did are involved in cell envelope biosynthesis and proteolysis and also in metabolism. CONCLUSION: Our results characterized the changes in the proteome of P. gingivalis following exposure to nicotine and cotinine, suggesting that these substances may modulate, with minor changes, protein expression. The present study is, in part, a step toward understanding the potential smoke-pathogen interaction that may occur in smokers with periodontitis.

Healthcare-associated infections: a threat to the survival of patients with COVID-19 in intensive care units.

BACKGROUND: Wide variation in mortality rates among critically ill patients with coronavirus disease 2019 (COVID-19) has been reported. This study evaluated whether healthcare-associated infections (HAI) are a risk factor for death among patients with severe COVID-19 in the intensive care unit (ICU). METHODS: This retrospective cohort study included patients with severe COVID-19 hospitalized in the ICU of four hospitals in the city of Curitiba, Brazil. Patients with COVID-19 who died during ICU hospitalization were compared with those who were discharged. A second analysis compared patients who developed HAI in the ICU with those who did not. Multiple logistic regression models were used to control for confounders. RESULTS: In total, 400 patients were included, and 123 (31%) patients developed HAI. The most common HAI was lower respiratory tract infection (67%). Independent risk factors for death were: age [odds ratio (OR) 1.75, 95% confidence interval (CI) 1.43-2.15; P<0.0001]; clinical severity score (OR 2.21, 95% CI 1.70-2.87; P<0.0001); renal replacement therapy (OR 12.8, 95% CI 5.78-28.6; P<0.0001); and HAI (OR 5.9, 95% CI 3.31-10.5; P<0.0001). A longer interval between symptom onset and hospital admission was protective against death (OR 0.93, 95% CI 0.88-0.98; P=0.017). The only independent factors associated with HAI were high C-reactive protein and low PaO2/FiO2 ratio. CONCLUSIONS: No factors that could point to a high-risk group for HAI acquisition were identified. However, age, dialysis and HAI increased the risk of death in ICU patients with severe COVID-19; of these, HAI is the only preventable risk factor.

Impact of smoking on inflammation: overview of molecular mechanisms.

BACKGROUND: Inflammation is a critical component of normal tissue repair, as well as being fundamental to the body's defense against infection. Environmental factors, such as smoking, have been reported to modify the host response and hence modify inflammation progression, severity and outcome. Therefore, a comprehensive understanding of the molecular mechanisms by which smoking affects inflammation is vital for preventive and therapeutic strategies on a clinical level. AIM: The purpose of the present article is to review the potential biological mechanisms by which smoking affects inflammation, emphasizing recent developments. RESULTS: Smoking is reported to effect a number of biological mediators of inflammation through its effect on immune-inflammatory cells, leading to an immunosuppressant state. Recent evidence strongly suggests that the molecular mechanisms behind the modulation of inflammation by smoking mainly involve the nuclear factor-kappa B (NF-kB) family, through the activation of both an inhibitor of IkB kinase (IKK)-dependent and -independent pathway. In addition to NF-kB activation, a number of transcriptional factors including GATA, PAX5 and Smad 3/4, have also been implicated. CONCLUSION: Multiple mechanisms may be responsible for the association of smoking and inflammation, and the identification of potential therapeutic targets should guide future research.

Impact of Porphyromonas gingivalis inoculation on ligature-induced alveolar bone loss. A pilot study in rats.

BACKGROUND AND OBJECTIVE: Periodontitis is a polymicrobial infection characterized by the loss of connective tissue attachment, periodontal ligament and alveolar bone. The aim of this study was to evaluate the impact of Porphyromonas gingivalis inoculation on the ligature-induced alveolar bone loss (ABL) model in rats. MATERIAL AND METHODS: Forty male Wistar rats were randomly assigned to the following groups: G1, control (n = 10); G2, ligature-induced ABL (n = 15); and G3, ligature-induced ABL + P. gingivalis inoculation (n = 15). Rats in G2 and G3 were killed 15, 21 and 30 d after ligature placement, and the following parameters were assessed: microbiological load; ABL; and interleukin (IL)-1ß (Il1beta)/Il1ra, Il6/Il10 and Rankl/osteoprotegerin (Opg) mRNA ratios in the gingival tissues, as determined by quantitative PCR. RESULTS: Microbiological analyses demonstrated that rats in G1, G2 and G3 were positive for the presence of bacteria (determined using PCR amplification of the 16S gene), but that only the treatment sites of rats in G3 were positive for P. gingivalis at all time-points investigated. Histometrically, significant bone loss (p<0.001) was observed for both ligated groups (G2 and G3) compared with the nonligated group (G1), with higher ABL observed for G2 at all the experimental time-points. Furthermore, gene-expression analysis demonstrated that the presence of P. gingivalis in the dentogingival area significantly decreased the Il1ß/Il1ra, Il6/Il10 and Rankl/Opg mRNA ratios compared with ligature alone. CONCLUSION: Within the limits of this pilot study, it was concluded that inoculation of P. gingivalis affected the ligature-induced ABL model by the induction of an anti-inflammatory and antiresorptive host response.

Levels of Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, inflammatory cytokines and species-specific immunoglobulin G in generalized aggressive and chronic periodontitis.

BACKGROUND AND OBJECTIVE: Aggressive periodontitis pathogenesis still is not completely understood in the literature regarding the relationship between microbial and inflammatory aspects. So this study aimed to compare microbial and inflammatory patterns in the gingival crevicular fluid of generalized aggressive and chronic periodontitis patients. MATERIAL AND METHODS: Forty aggressive and 28 chronic periodontitis patients were selected. Biofilm and gingival crevicular fluid were collected from a deep pocket (periodontal probing depth >7 mm) and a moderate pocket (periodontal probing depth = 5 mm) of each patient, and microbiological and immunoenzymatic assays were performed. Real-time PCR was used to determine quantities of Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis. Enzyme-linked immunosorbent assay (ELISA) was employed to determine gingival crevicular fluid levels of interleukin-1beta, interferon-gamma, prostaglandin E(2) and interleukin-10. In addition, immunoglobulin G (IgG) levels against A. actinomycetemcomitans and P. gingivalis lipopolysaccharide were also determined by ELISA. Analysis of variance/Tukey test, Mann-Whitney U-test and the Pearson correlation test were used to determine differences and correlations between variables analysed (alpha = 5%). RESULTS: Patients suffering from generalized aggressive periodontitis had their mouth colonized by higher amounts of A. actinomycetemcomitans and P. gingivalis than chronic periodontitis patients. Conversely, the gingival crevicular fluid levels of IgG against both pathogens were statistically inferior in aggressive periodontitis patients (p < 0.05). With regard to gingival crevicular fluid levels of cytokines, aggressive periodontitis patients presented reduced levels of interleukin-10 (p < 0.05). CONCLUSION: In comparison to chronic periodontitis, generalized aggressive periodontitis patients have an imbalance in the host response, with reduced levels of interleukin-10 and IgG, and increased periodontal pathogens.

Assessment of intraradicular bacterial composition by terminal restriction fragment length polymorphism analysis.

BACKGROUND: The aim of the study was to assess the bacterial community structures associated with endodontic infections using terminal restriction fragment length polymorphism (T-RFLP), and to investigate the correlation of whole community profiles with the manifestation of particular clinical features. METHODS: Intraradicular samples were collected from 34 subjects and classified into three study groups based on the observed clinical symptoms: acute (n = 16), sub-acute (n = 8), and asymptomatic (n = 10). Genomic DNA was extracted from each sample, submitted to polymerase chain reaction using a fluorescently labeled 16S ribosomal DNA forward primer, and digested with two tetrameric endonucleases (HhaI and MspI). The terminal restriction fragments (T-RFs) were subsequently discriminated in an automated DNA sequencer, and the results were filtered using a statistics-based criterion. RESULTS: Totals of 138 (HhaI) and 145 (MspI) unique T-RFs were detected (means 13.1 and 11.9) and there was high inter-subject variability in the bacterial assemblages. Odds-ratio analysis unveiled the existence of higher order groups of positively associated T-RFs, restating the concept that intricate ecological relationships may take place in the root canal space. A significantly greater T-RF prevalence was detected in acute cases, suggesting a straight correlation between species richness and spontaneous pain. CONCLUSION: Overall, no T-RFLP profile representing a specific bacterial consortium could be associated with the manifestation of symptoms of endodontic origin.

Transcriptional analysis of gtfB, gtfC, and gbpB and their putative response regulators in several isolates of Streptococcus mutans.

BACKGROUND: Streptococcus mutans, a major dental caries pathogen, expresses several virulence genes that mediate its growth, accumulation on tooth surfaces, and acid-mediated tooth demineralization. GtfB and GtfC catalyze the extracellular synthesis of water-insoluble glucan matrix from sucrose, and are essential for accumulation of bacteria in the dental biofilm. GbpB, an essential protein of S. mutans, might also mediate cell-surface interaction with glucan. AIM/METHODS: In this study, we determined the transcription levels of gtfB, gtfC, and gbpB, and several putative transcriptional response regulators (rr) at different phases of planktonic growth in 11 S. mutans strains. RESULTS: Activities of gtfB and gtfC were growth-phase dependent and assumed divergent patterns in several strains during specific phases of growth, while gbpB activities appeared to be under modest influence of the growth phase. Transcription patterns of the rr vicR, covR, comE, ciaR, and rr1 were growth-phase dependent and some of these genes were expressed in a highly coordinated way. Each rr, except comE, was expressed by all the strains. Patterns of virulence and regulatory genes were, however, strain-specific. CONCLUSIONS: The findings suggest that mechanisms controlling virulence gene expression are variable among genotypes, providing the notion that the genetic diversity of S. mutans may have important implications for understanding mechanisms that regulate the expression of virulence genes in this species.

Bacteroides fragilis isolates compared by AP-PCR.

Bacteroides fragilis is a component of the normal intestinal flora and an important pathogen in nonintestinal endogenous infections. It has been associated with enteric infections and has already been detected in polluted water. In order to evaluate the genetic diversity of B. fragilis, a total of 31 isolates and two reference strains were examined. This collection included strains from nonintestinal infections [12], intestinal infections [5], intestinal microflora [10], aquatic environments [4], and the reference strains ATCC 25285 and ATCC 23745. DNA fingerprints were detected using two separate PCR reactions with different arbitrary primers. The computer-assisted system Taxotron (Institut Pasteur, Dr P. Grimont) was used to analyze the profiles obtained and dendrograms were generated. By using a distance of 0.65 as the threshold, two clusters (hereafter referred to as genotypes I and II) were defined. Strains of differents origins could be distributed into both genotypes. We were unable to detect any obvious correlation between a given genotype and the specific disease or the source of the corresponding strains.

Genetic relatedness between oral and intestinal isolates of Porphyromonas endodontalis by analysis of random amplified polymorphic DNA.

Genomic fingerprints from the DNA of 27 strains of Porphyromonas endodontalis from diverse clinical and geographic origins were generated as random amplified polymorphic DNA (RAPD) using the technique of PCR amplification with a single primer of arbitrary sequence. Cluster analysis of the combined RAPD data obtained with three selected 9- or 10-mer-long primers identified 25 distinct RAPD types which clustered as three main groups identifying three genogroups. Genogroups I and II included exclusively P. endodontalis isolates of oral origin, while 7/9 human intestinal strains of genogroup III which linked at a similarity level of 52% constituted the most homogeneous group in our study. Genotypic diversity within P. endodontalis, as shown by RAPD analysis, suggests that the taxon is composed of two oral genogroups and one intestinal genogroup. This hypothesis remains to be confirmed.

Use of rep-PCR to define genetic relatedness among Bacteroides fragilis strains.

Bacteroides fragilis, a component of the normal flora and an important anaerobic pathogen in non-intestinal endogenous infections, has recently been associated with enteric diseases. In this study, 41 B. fragilis strains were analysed in relation to their genetic diversity. This collection included two reference strains (ATCC 23745 and 25285), 20 isolates from non-intestinal infections, six from intestinal infections, five from intestinal microflora and eight from an aquatic environment. The fingerprints were generated by using two repetitive sequences (REP and ERIC) as primers to PCR (rep-PCR). A dendrogram was obtained with the Taxotron Program. Three clusters (threshold genotypes I, II and III) were observed when the genetic distance was 0.30. These results confirm previous data found regarding the genotypical diversity of B. fragilis.

Molecular detection of Bacteroides forsythus in infected root canals.

The purpose of the present study was to investigate the occurrence of Bacteroides forsythus in infections of dental root canals. Eleven samples from infected root canals were analyzed by four different molecular methods. The prevalence of the monitored species varied as a function of the detection method. The polymerase chain reaction-DNA probe method after immunocapture yielded the highest prevalence value (6/11), whereas the lowest value was observed with the slot-blot (3/11). Of the 11 canal samples, 5 were positive by ELISA and 4 were positive by immunofluorescence. The presence of B. forsythus was detected by all four methods in 3/11 canals, whereas 4/11 appeared to be free of B. forsythus. Our data indicate that B. forsythus can be part of the endodontic microflora. The procedure consisting of immunomagnetic capture and a polymerase chain reaction-DNA probe assay can be useful as an alternative to culture for clinical studies of the species infecting human dental pulp.

Evaluation of the antibacterial activities of calcium hydroxide, chlorhexidine, and camphorated paramonochlorophenol as intracanal medicament. A clinical and laboratory study.

The antibacterial activities of camphorated paramonochlorophenol, chlorhexidine, and calcium hydroxide were compared using a clinical and laboratory evaluation. In the clinical experiment, root canals that yielded positive cultures a week after complete chemomechanical preparation and camphorated paramonochlorophenol dressing were medicated with one of the three substances tested. Postmedication samples were taken from the canal 1 week later. In the laboratory experiment, the agar diffusion test was used to evaluate the inhibitory activity of the medicaments against bacteria commonly found in endodontic infections. The results of the clinical evaluation showed that all medicaments were effective in reducing or eliminating the endodontic microbiota, as demonstrated by the incidence of negative cultures. There was no statistically significant difference among the medicaments tested. In the laboratory evaluation, camphorated paramonochlorophenol showed the largest zones of bacterial inhibition against all bacterial strains tested.

Isolation of the lectin and an L4 isolectin from Phaseolus vulgaris by affinity chromatography on insoluble ovomucoid.

Lectins from extracts of Phaseolus vulgaris seeds have potent cell-agglutinating and lymphocyte-stimulating activity. An affinity adsorbent for lectins with specificity for the oligosaccharide structure was prepared by transforming ovomucoid, an oligosaccharide-rich glycoprotein, into an insoluble and stable gel. The ovomucoid was made insoluble by boiling a 20% solution (200 mg/ml) in 0.1 M Tris-HCl, pH 8.9, for 20 min. This insoluble gel was desialylated by treatment with 50 mN sulfuric acid for 1 h at 90 degrees C and fixed with 1% glutaraldehyde, pH 7.4, for 10 min. The Phaseolus lectin and the L4 isolectin could be isolated essentially in a single-step procedure, using different eluting conditions: 50 mM sodium formate buffer, pH 3.0, was used for PHA elution; a different column was eluted with 15 mM sodium tetraborate, pH 8.0, for desorbed L4 isolectin. Polyacrylamide gel electrophoresis of the lectin showed five distinct bands, whereas the L4 isolectin only presented one band. From 250 mg of saturated column, 8.25 mg of PHA was isolated. This adsorbent could be used several times with little change in binding capacity or selectivity.

Evaluation of biochemical and serological methods to identify and clustering yeast cells of oral Candida species by CHROMagar test, SDS-PAGE and ELISA.

The purpose of this work was to evaluate biochemical and serological methods to characterize and identify Candida species from the oral cavity. The strains used were five Candida species previously identified: C. albicans, C. guilliermondii, C. parapsilosis, C. krusei, C. tropicalis, and Kluyveromyces marxianus, as a negative control. The analyses were conducted through the SDS-PAGE associated with statistical analysis using software, chromogenic medium, and CHROMagar Candida (CA), as a differential medium for the isolation and presumptive identification of clinically important yeasts and an enzyme-linked immunoabsorbent assay (ELISA), using antisera produced against antigens from two C. albicans strains. This method enabled the screening of the three Candida species: C. albicans, C. tropicalis, and C. krusei, with 100% of specificity. The ELISA using purified immunoglobulin G showed a high level of cross-reaction against protein extracts of Candida species. The SDS-PAGE method allowed the clustering of species-specific isolates using the Simple Matching coefficient, S(SM) = 1.0. The protein profile analysis by SDS-PAGE increases what is known about the taxonomic relationships among oral yeasts. This methodology showed good reproducibility and allows collection of useful information for numerical analysis on information relevant to clinical application, and epidemiological and systematical studies.

Antimicrobial peptides and nitric oxide production by neutrophils from periodontitis subjects.

Neutrophils play an important role in periodontitis by producing nitric oxide (NO) and antimicrobial peptides, molecules with microbicidal activity via oxygen-dependent and -independent mechanisms, respectively. It is unknown whether variation in the production of antimicrobial peptides such as LL-37, human neutrophil peptides (HNP) 1-3, and NO by neutrophils influences the pathogenesis of periodontal diseases. We compared the production of these peptides and NO by lipopolysaccharide (LPS)-stimulated neutrophils isolated from healthy subjects and from patients with periodontitis. Peripheral blood neutrophils were cultured with or without Aggregatibacter actinomycetemcomitans-LPS (Aa-LPS), Porphyromonas gingivalis-LPS (Pg-LPS) and Escherichia coli-LPS (Ec-LPS). qRT-PCR was used to determine quantities of HNP 1-3 and LL-37 mRNA in neutrophils. Amounts of HNP 1-3 and LL-37 proteins in the cell culture supernatants were also determined by ELISA. In addition, NO levels in neutrophil culture supernatants were quantitated by the Griess reaction. Neutrophils from periodontitis patients cultured with Aa-LPS, Pg-LPS and Ec-LPS expressed higher HNP 1-3 mRNA than neutrophils from healthy subjects. LL-37 mRNA expression was higher in neutrophils from patients stimulated with Aa-LPS. Neutrophils from periodontitis patients produced significantly higher LL-37 protein levels than neutrophils from healthy subjects when stimulated with Pg-LPS and Ec-LPS, but no difference was observed in HNP 1-3 production. Neutrophils from periodontitis patients cultured or not with Pg-LPS and Ec-LPS produced significantly lower NO levels than neutrophils from healthy subjects. The significant differences in the production of LL-37 and NO between neutrophils from healthy and periodontitis subjects indicate that production of these molecules might influence individual susceptibility to important periodontal pathogens.

Antimicrobial potential of some plant extracts against Candida species.

The increase in the resistance to antimicrobial drugs in use has attracted the attention of the scientific community, and medicinal plants have been extensively studied as alternative agents for the prevention of infections. The Candida genus yeast can become an opportunistic pathogen causing disease in immunosuppressive hosts. The purpose of this study was to evaluate dichloromethane and methanol extracts from Mentha piperita, Rosmarinus officinalis, Arrabidaea chica, Tabebuia avellanedae, Punica granatum and Syzygium cumini against Candida species through the analysis of Minimum Inhibitory Concentration (MIC). Results presented activity of these extracts against Candida species, especially the methanol extract.

Antimicrobial peptides and nitric oxide production by neutrophils from periodontitis subjects

Neutrophils play an important role in periodontitis by producing nitric oxide (NO) and antimicrobial peptides, molecules with microbicidal activity via oxygen-dependent and -independent mechanisms, respectively. It is unknown whether variation in the production of antimicrobial peptides such as LL-37, human neutrophil peptides (HNP) 1-3, and NO by neutrophils influences the pathogenesis of periodontal diseases. We compared the production of these peptides and NO by lipopolysaccharide (LPS)-stimulated neutrophils isolated from healthy subjects and from patients with periodontitis. Peripheral blood neutrophils were cultured with or without Aggregatibacter actinomycetemcomitans-LPS (Aa-LPS), Porphyromonas gingivalis-LPS (Pg-LPS) and Escherichia coli-LPS (Ec-LPS). qRT-PCR was used to determine quantities of HNP 1-3 and LL-37 mRNA in neutrophils. Amounts of HNP 1-3 and LL-37 proteins in the cell culture supernatants were also determined by ELISA. In addition, NO levels in neutrophil culture supernatants were quantitated by the Griess reaction. Neutrophils from periodontitis patients cultured with Aa-LPS, Pg-LPS and Ec-LPS expressed higher HNP 1-3 mRNA than neutrophils from healthy subjects. LL-37 mRNA expression was higher in neutrophils from patients stimulated with Aa-LPS. Neutrophils from periodontitis patients produced significantly higher LL-37 protein levels than neutrophils from healthy subjects when stimulated with Pg-LPS and Ec-LPS, but no difference was observed in HNP 1-3 production. Neutrophils from periodontitis patients cultured or not with Pg-LPS and Ec-LPS produced significantly lower NO levels than neutrophils from healthy subjects. The significant differences in the production of LL-37 and NO between neutrophils from healthy and periodontitis subjects indicate that production of these molecules might influence individual susceptibility to important periodontal pathogens.

Cross-reactive adaptive immune response to oral commensal bacteria results in an induction of receptor activator of nuclear factor-kappaB ligand (RANKL)-dependent periodontal bone resorption in a mouse model.

INTRODUCTION: The present study examined whether induction of an adaptive immune response to orally colonizing non-pathogenic Pasteurella pneumotropica by immunization with the phylogenetically closely related bacterium, Actinobacillus actinomycetemcomitans, can result in periodontal bone loss in mice. METHODS: BALB/c mice harboring P. pneumotropica (P. pneumotropica(+) mice) in the oral cavity or control P. pneumotropica-free mice were immunized with fixed A. actinomycetemcomitans. The animals were sacrificed on day 30, and the following measurements were carried out: (i) serum immunoglobulin G and gingival T-cell responses to A. actinomycetemcomitans and P. pneumotropica; (ii) periodontal bone loss; and (iii) identification of receptor activator of nuclear factor-kappaB ligand (RANKL) -positive T cells in gingival tissue. RESULTS: Immunization with A. actinomycetemcomitans induced a significantly elevated serum immunoglobulin G response to the 29-kDa A. actinomycetemcomitans outer membrane protein (Omp29), which showed strong cross-reactivity with P. pneumotropica OmpA compared to results in the control non-immunized mice. The A. actinomycetemcomitans-immunized P. pneumotropica(+) mice developed remarkable periodontal bone loss in a RANKL-dependent manner, as determined by the abrogation of bone loss by treatment with osteoprotegerin-Fc. The T cells isolated from the gingival tissue of A. actinomycetemcomitans-immunized P. pneumotropica(+) mice showed an in vitro proliferative response to both A. actinomycetemcomitans and P. pneumotropica antigen presentation, as well as production of soluble(s)RANKL in the culture supernatant. Double-color confocal microscopy demonstrated that the frequency of RANKL(+) T cells in the gingival tissue of A. actinomycetemcomitans-immunized P. pneumotropica(+) mice was remarkably elevated compared to control mice. CONCLUSION: The induction of an adaptive immune response to orally colonizing non-pathogenic P. pneumotropica results in RANKL-dependent periodontal bone loss in mice.

In vitro inhibition of oral streptococci binding to the acquired pellicle by algal lectins.

AIMS: The initial colonization of the tooth by streptococci involves their attachment to adsorbed components of the acquired pellicle. Avoiding this adhesion may be successful in preventing caries at early stages. Salivary mucins are glycoproteins that when absorbed onto hydroxyapatite may provide binding sites for certain bacteria. Algal lectins may be especially interesting for oral antiadhesion trials because of their great stability and high specificity for mucins. This work aimed to evaluate the potential of two algal lectins to inhibit the adherence of five streptococci species to the acquired pellicle in vitro. METHODS AND RESULTS: The lectins used were extracted from Bryothamnion triquetrum (BTL) and Bryothamnion seaforthii (BSL). Fluorescence microscopy was applied to visualize the ability of fluorescein isothiocyanate-labelled lectins to attach to the pellicle and revealed a similar capability for both lectins. Streptococcal adherence assays were performed using saliva-coated microtitre plates. BSL inhibited more than 75% of Streptococcus sanguis, Streptococcus mitis, Streptococcus sobrinus and Streptococcus mutans adherence, achieving 92% to the latter. BTL only obtained statistically significant results on S. mitis and S. sobrinus, whose adherence was decreased by 32.5% and 54.4%, respectively. CONCLUSION: Algal lectins are able to inhibit streptococcal adherence. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results support the proposed application of lectins in antiadhesion therapeutics.