Recent advances in host defense mechanisms/therapies against oral infectious diseases and consequences for systemic disease.

The innate and adaptive immune systems are both crucial to oral disease mechanisms and their impact on systemic health status. Greater understanding of these interrelationships will yield opportunities to identify new therapeutic targets to modulate disease processes and/or increase host resistance to infectious or inflammatory insult. The topics addressed reflect the latest advances in our knowledge of the role of innate and adaptive immune systems and inflammatory mechanisms in infectious diseases affecting the oral cavity, including periodontitis and candidiasis. In addition, several potential links with systemic inflammatory conditions, such as cardiovascular disease, are explored. The findings elucidate some of the defense mechanisms utilized by host tissues, including the role of IL-17 in providing immunity to oral candidiasis, the antimicrobial defense of mucosal epithelial cells, and the pro-resolution effects of the natural inflammatory regulators, proresolvins and lipoxins. They also describe the role of immune cells in mediating pathologic bone resorption in periodontal disease. These insights highlight the potential for therapeutic benefit of immunomodulatory interventions that bolster or modulate host defense mechanisms in both oral and systemic disease. Among the promising new therapeutic approaches discussed here are epithelial cell gene therapy, passive immunization against immune cell targets, and the use of proresolvin agents.

Mechanism of adhesion maintenance by methionine sulphoxide reductase in Streptococcus gordonii.

Methionine sulphoxide reductase maintains adhesin function during oxidative stress. Using Streptococcus gordonii as a model, we now show the mechanistic basis of adhesin maintenance provided by MsrA. In biofilms, S. gordonii selectively expresses the msrA gene. When the wild-type strain was grown with exogenous hydrogen peroxide (H(2)O(2)), msrA-specific mRNA expression significantly increased, while acid production was unaffected. In the presence of H(2)O(2), a msrA-deletion mutant (ΔMsrA) showed a 6 h delay in lag phase growth, a 30% lower yield of H(2)O(2), significantly greater inhibition by H(2)O(2) on agar plates (reversed by complementation), 30% less adhesion to saliva-coated hydroxyapatite, 87% less biofilm formation and an altered electrophoretic pattern of SspAB protein adhesins. Using mass spectrometry, methionine residues in the Met-rich central region of SspB were shown to be oxidized by H(2)O(2) and reduced by MsrA. In intact wild-type cells, MsrA colocalized with a cell wall-staining dye, and MsrA was detected in both cell wall and cytosolic fractions. To maintain normal adhesion and biofilm function of S. gordonii in response to exogenous oxidants therefore msrA is upregulated, methionine oxidation of adhesins and perhaps other proteins is reversed, and adhesion and biofilm formation is maintained.

Plausibility of HIV-1 Infection of Oral Mucosal Epithelial Cells.

The AIDS pandemic continues. Little is understood about how HIV gains access to permissive cells across mucosal surfaces, yet such knowledge is crucial to the development of successful topical anti-HIV-1 agents and mucosal vaccines. HIV-1 rapidly internalizes and integrates into the mucosal keratinocyte genome, and integrated copies of HIV-1 persist upon cell passage. The virus does not appear to replicate, and the infection may become latent. Interactions between HIV-1 and oral keratinocytes have been modeled in the context of key environmental factors, including putative copathogens and saliva. In keratinocytes, HIV-1 internalizes within minutes; in saliva, an infectious fraction escapes inactivation and is harbored and transferable to permissive target cells for up to 48 hours. When incubated with the common oral pathogen Porphyromonas gingivalis, CCR5- oral keratinocytes signal through protease-activated receptors and Toll-like receptors to induce expression of CCR5, which increases selective uptake of infectious R5-tropic HIV-1 into oral keratinocytes and transfer to permissive cells. Hence, oral keratinocytes-like squamous keratinocytes of other tissues-may be targets for low-level HIV-1 internalization and subsequent dissemination by transfer to permissive cells.

Science for the Next Century: Deep Phenotyping.

Our ability to unravel the mysteries of human health and disease have changed dramatically over the past 2 decades. Decoding health and disease has been facilitated by the recent availability of high-throughput genomics and multi-omics analyses and the companion tools of advanced informatics and computational science. Understanding of the human genome and its influence on phenotype continues to advance through genotyping large populations and using "light phenotyping" approaches in combination with smaller subsets of the population being evaluated using "deep phenotyping" approaches. Using our capability to integrate and jointly analyze genomic data with other multi-omic data, the knowledge of genotype-phenotype relationships and associated genetic pathways and functions is being advanced. Understanding genotype-phenotype relationships that discriminate human health from disease is speculated to facilitate predictive, precision health care and change modes of health care delivery. The American Association for Dental Research Fall Focused Symposium assembled experts to discuss how studies of genotype-phenotype relationships are illuminating the pathophysiology of craniofacial diseases and developmental biology. Although the breadth of the topic did not allow all areas of dental, oral, and craniofacial research to be addressed (e.g., cancer), the importance and power of integrating genomic, phenomic, and other -omic data are illustrated using a variety of examples. The 8 Fall Focused talks presented different methodological approaches for ascertaining study populations and evaluating population variance and phenotyping approaches. These advances are reviewed in this summary.

Shosaikoto increases calprotectin expression in human oral epithelial cells.

BACKGROUND AND OBJECTIVE: Oral epithelial cells help to prevent against bacterial infection in the oral cavity by producing antimicrobial peptides (AMPs). A broad-spectrum AMP, calprotectin (a complex of S100A8 and S100A9 proteins), is expressed by oral epithelial cells and is up-regulated by interleukin-1alpha (IL-1alpha). Shosaikoto (SST) is a traditional Japanese herbal medicine that has immunomodulatory effects and is reported to enhance the levels of IL-1alpha in epithelial cells. The purpose of this study was to investigate the effect of SST on the expression of calprotectin and other AMPs through the regulation of IL-1alpha in oral epithelial cells. MATERIAL AND METHODS: Human oral epithelial cells (TR146) were cultured with SST (at concentrations ranging from 10 to 250 microg/mL) in the presence or absence of anti-IL-1alpha or IL-1 receptor antagonist. The expression of S100A8- and S100A9-specific mRNAs was examined by northern blotting. Calprotectin expression and IL-1alpha secretion were investigated by immunofluorescent staining or ELISA. The expression of other AMPs and IL-1alpha was analyzed by RT-PCR and by quantitative real-time PCR. RESULTS: Shosaikoto (25 microg/mL) significantly increased the expression of S100A8- and S100A9-specific mRNAs and calprotectin protein. Shosaikoto increased S100A7 expression, but had no effect on the expression of other AMPs. The expression of IL-1alpha-specific mRNA and its protein were slightly increased by SST. A neutralizing antibody against IL-1alpha or IL-1 receptor antagonist inhibited SST up-regulated S100A8/S100A9 mRNA expression. CONCLUSION: These results suggest that SST increases the expression of calprotectin and S100A7 in oral epithelial cells. In response to SST, up-regulation of calprotectin may be partially induced via IL-1alpha.

Calprotectin and the Initiation and Progression of Head and Neck Cancer.

Calprotectin (S100A8/A9), a heterodimeric complex of calcium-binding proteins S100A8 and S100A9, is encoded by genes mapping to the chromosomal locus 1q21.3 of the epidermal differentiation complex. Whereas extracellular calprotectin shows proinflammatory and antimicrobial properties by signaling through RAGE and TLR4, intracytoplasmic S100A8/A9 appears to be important for cellular development, maintenance, and survival. S100A8/A9 is constitutively expressed in myeloid cells and the stratified mucosal epithelia lining the oropharyngeal and genitourinary mucosae. While upregulated in adenocarcinomas and other cancers, calprotectin mRNA and protein levels decline in head and neck squamous cell carcinoma (HNSCC). S100A8/A9 is also lost during head and neck preneoplasia (dysplasia). Calprotectin decrease does not correlate with the clinical stage (TNM) of HNSCC. When expressed in carcinoma cells, S100A8/A9 downregulates matrix metalloproteinase 2 expression and inhibits invasion and migration in vitro. S100A8/A9 regulates cell cycle progression and decelerates cancer cell proliferation by arresting at the G2/M checkpoint in a protein phosphatase 2α-dependent manner. In HNSCC, S100A8 and S100A9 coregulate with gene networks controlling cellular development and differentiation, cell-to-cell signaling, and cell morphology, while S100A8/A9 appears to downregulate expression of invasion- and tumorigenesis-associated genes. Indeed, tumor formation capacity is attenuated in S100A8/A9-expressing carcinoma cells in vivo. Hence, intracellular calprotectin appears to function as a tumor suppressor in head and neck carcinogenesis. When compared with S100A8/A9-low HNSCC based on analysis of TCGA, S100A8/A9-high HNSCC shows significant upregulation of apoptosis-related genes, including multiple caspases. Accordingly, S100A8/A9 facilitates DNA damage responses in HNSCC, promotes apoptotic cell death, and confers sensitivity to cisplatin and X-radiation in vitro. In the tumor milieu, loss of S100A8/A9 strongly associates with poor squamous differentiation and higher tumor grading, EGFR upregulation, increased DNA methylation, and, finally, poorer overall survival for patients with HNSCC. Hence, intracellular calprotectin shows a multifaceted protective role against the development of HNSCC.

(C3) The oral epithelial cell and first encounters with HIV-1.

The oral epithelium is the site of first exposure of HIV-1 to host tissues during oral sex with an infected partner or through breast-feeding by an infected mother. Although the oral epithelium is distinguishable by its apparent resistance, the mucosal surfaces represent a primary target of HIV-1. After oral exposure and swallowing, infection is detected prominently in the gastrointestinal tract, which becomes depleted of CD4+ T-cells. The oral cavity and palatine tonsils appear to resist infection and transfer to susceptible lymphoid cells in the lamina propria by local anti-HIV-1 mechanisms. In some cases, expression of these antiviral mechanisms increases after exposure to HIV-1. During primary exposure and before seroconversion, based on limited in vitro and primate data, a window of opportunity for capture of HIV-1 by the oral epithelium may exist. After seroconversion, the risk of infectious HIV-1 appearing in saliva is negligible. This report considers evidence that oral epithelium has the potential both to enable and to resist infection by HIV-1.

Analysis of polarization and orientation of human polymorphonuclear leukocytes by computer-interfaced video microscopy.

Chemotactic behavior is a complex cellular response to chemical environmental stimuli. For polymorphonuclear leukocytes (PMNs), such behavior involves net migration as well as changes in cell shape and cell orientation. Accordingly, we have applied computer-interfaced video microscopy to analyze cell shape and orientation in control and patient PMNs migrating under agarose. From a digitized tracing of the PMNs at the leading front of migration, cells were characterized in terms of area, circumference, and longest dimension. A shape factor and angle of orientation were computed. Numerical shape factors discriminated three PMN morphologies: polar, apolar, and hyperpolar. Only polar cells could be oriented. Orientation of polar cells was defined as toward, away, or disoriented with respect to the chemotactic gradient. Apolar cells were considered to be nonoriented. Of PMNs from healthy controls, 30 +/- 5% of the cells were oriented toward and 11 +/- 4% of the cells were oriented away from the gradient. For PMNs from patients with localized juvenile periodontitis, a 40% deficit in net migration was associated with reduced orientation toward (5 +/- 2%) and elevated orientation away from the gradient (33 +/- 9%). PMNs from a panel of patients with thermal injury showed reduced migration and orientation toward the gradient associated with elevated percentages of apolar cells. Such analysis of PMN polarization and orientation of the leading front permitted calculation of a chemotactic behavior index. Application of this multiparameter index to the analysis of the chemotactic response may identify PMNs that are defective, but not by evaluation of any single variable.

Concurrent estimation of the kinetics of adhesion and ingestion of Staphylococcus aureus by human polymorphonuclear leukocytes (PMNs).

Direct recording of the kinetics of early phagocytic events, recognition or adhesion and ingestion, may better characterize some forms of polymorphonuclear leukocyte (PMN) dysfunction. This report describes a method and criteria to discriminate concurrently between adhesion and ingestion of Staphylococcus aureus by human PMNs at the level of the light microscope. The criteria were confirmed by several lines of direct evidence: low temperature and cytochalasin b treatment of PMNs; lysostaphin digestion of target Staphylococcus aureus after PMN incubation; and differential ingestibility of colony types I and III N. gonorrheae. Concurrent determinations of the initial kinetics of adhesion and ingestion of bacteria by PMNs demonstrated generalized first order kinetics, sensitive to bacterial challenge ratio and total particle density. Most importantly, the method may be applied to determine if phagocytically defective PMNs actually fail to recognize opsonized bacteria.

Responses of platelets to strains of streptococcus sanguis: findings in healthy subjects, Bernard-Soulier, Glanzmann's, and collagen-unresponsive patients.

The ability of endocarditis and dental strains of Streptococcus sanguis to induce platelet aggregation in plasma (PRP) from normal subjects were examined and compared to responses of PRP with known platelet membrane glycoprotein (GP) and response defects. S. sanguis strains differed in their ability to induce normal PRPs to aggregate. Strains that induced PRP aggregation in more than 60% of donors were significantly faster agonists (mean lag times to onset of aggregation less than 6 min) than those strains inducing response in PRPs of fewer than 60% of donors. Platelets from patients with Bernard-Soulier syndrome aggregated in response to strains of S. sanguis. In contrast, platelets from patients with Glanzmann's thrombasthenia and from a patient with a specific defect in response to collagen were unresponsive to S. sanguis. These observations show that GPIb and V are not essential, but GPIIb-IIIa and GPIa are important in the platelet response mechanism to S. sanguis. Indeed, the data suggests that the platelet interaction mechanisms of S. sanguis and collagen may be similar.

Therapeutic advantage of recombinant human plasminogen activator in endocarditis: evidence from experiments in rabbits.

In infective endocarditis vegetations are stabilized by fibrin. To learn if fibrin digestion would be therapeutic, experimental endocarditis was induced in rabbits by inoculation with a platelet-aggregating strain (Agg+) of Streptococcus sanguis and treated with recombinant tissue plasminogen activator (rt-PA), rt-PA with penicillin, or penicillin alone. Control rabbits were inoculated with saline. All treatments of Agg+ endocarditis reduced the mass of valvular vegetations and clinical signs of endocarditis, including the frequency of left axis deviation and heart ischemia. rt-PA with penicillin was more effective than penicillin or rt-PA alone, reducing the mass of vegetations and clinical signs to that of saline controls. Within 50 min, rt-PA cleared 5-fold more 111Indium-labelled platelets from the heart than untreated rabbits and 1.4-fold more after 3 days. Combined with penicillin, thrombolytic therapy for human endocarditis should be reconsidered.

Nicotine-induced release of elastase and eicosanoids by human neutrophils.

We examined the direct effects of nicotine on a variety of neutrophil functions at concentrations achievable in lung and oral tissues from cigarette smoking. The results show dose-dependent suppression of chemotaxis and phagocytosis, and enhancement of degranulation and eicosanoid generation, but not superoxide production. Cell viability was not affected by the concentrations of nicotine used in these experiments, as shown by trypan blue dye exclusion and MTT assays. These results implicate nicotine as the ingredient in cigarette smoke responsible for inflammatory damage to lungs and oral tissues observed in cigarette smokers.

Serum and gingival crevicular fluid anti-desmosomal antibodies in periodontitis.

SDS polyacrylamide gel electrophoresis separates desmosomal proteins and glycoproteins of bovine tongue epithelium by their relative molecular mass. The Western immunoblot technique was used to reveal the reactions of desmosomal proteins and glycoproteins with naturally occurring antibodies in serum and gingival crevicular fluid (GCF). Naturally-occurring serum and GCF antibodies (IgG and IgM) from periodontitis patients and healthy, unaffected controls reacted with desmosomal proteins (desmoplakins) and glycoproteins (desmogleins). Sera from 90% of subjects with periodontitis showed increased reactions of IgG with desmoplakins (240 and 210 kDa) and 80% with desmogleins (165, 130, and 115 kDa), when compared with unaffected controls. Patients' IgG reacted with desmosomal components more strongly than IgM. IgG antibodies against desmosomal antigens in GCF showed similar specificities from patients and healthy subjects and to their serums. When GCF within individuals with periodontitis was compared, anti-desmosomal IgG from diseased sites showed greater reactivity than healthy controls. These data suggest that anti-desmosomal antibodies are a normal part of the immune repertoire. The presence of elevated titers of anti-desmosomal antibodies appear to distinguish periodontitis from unaffected sites. When detected, elevated titers of anti-desmosomal antibodies may contribute to the pathogenesis and indicate increased risk of periodontitis.

Effects of oral flora on platelets: possible consequences in cardiovascular disease.

During episodes of dental bacteremia, viridans group streptococci encounter platelets. Among these microorganisms, certain Streptococcus sanguis induce human and rabbit platelets to aggregate in vitro. In experimental rabbits, circulating streptococci induced platelets to aggregate, triggering the accumulation of platelets and fibrin into the heart valve vegetations of endocarditis. At necropsy, affected rabbit hearts showed ischemic areas. We therefore hypothesized that circulating S. sanguis might cause coronary thrombosis and signs of myocardial infarction (MI). Signs of MI were monitored in rabbits after infusion with platelet-aggregating doses of 4 to 40 x 10(9) cells of S. sanguis 133-79. Infusion resulted in dose-dependent changes in electrocardiograms, blood pressure, heart rate, and cardiac contractility. These changes were consistent with the occurrence of MI. Platelets isolated from hyperlipidemic rabbits showed an accelerated in vitro aggregation response to strain 133-79. Cultured from immunosuppressed children with septic shock and signs of disseminated intravascular coagulation, more than 60% of isolates of viridans streptococci induced platelet aggregation when tested in vitro. The data are consistent with a thrombogenic role for S. sanguis in human disease, contributing to the development of the vegetative lesion in infective endocarditis and a thrombotic mechanism to explain the additional contributed risk of periodontitis to MI.

Human neutrophil migration under agarose to bacteria associated with the development of gingivitis.

In clinically healthy gingiva and increasingly with the development of inflammation, neutrophils are found in the gingival tissues and sulcus. This study evaluated the relative ability of bacteria associated with gingival health and developing inflammation to stimulate this increase in neutrophil accumulation. Dialyzed bacterial sonic extracts (BE) in buffer and pooled human serum (PHS) from pure cultures of Streptococcus sanguis. Actinomyces viscosus, A naeslundii, Bacteroides intermedius, Fusobacterium sp and Veillonella sp were tested for stimulation of human neutrophil migration under agarose. In addition, fractions of S sanguis culture fluids (CFs) from Sephadex G-10 chromatography were evaluated. All BE solutions were incubated for 1 hour at 37 degrees C and heat-inactivated prior to testing. All BEs in buffer attracted neutrophils, with the greatest responses seen to S sanguis and B intermedius followed by A viscosus. Migration to all BEs in PHS was greater than in buffer, suggesting that all BEs are capable of generating serum chemoattractants. A viscosus BE activated serum attractants to the greatest degree. CFs of S sanguis, A viscosus, and to a lesser degree, Fusobacterium sp, also attracted neutrophils. Evidence from [3H]FMLP competitive ligand-binding assays indicated that S sanguis CFs contained low molecular weight (less than 700) chemoattractants, which were probably formylmethionyl oligopeptide-like materials. Of the bacteria associated with health, S sanguis and A viscosus appeared at least as able to generate chemoattractants during growth or with exposure to serum as bacteria associated with gingivitis. This observation suggests that these "healthy" bacteria, which are found in greater numbers with developing inflammation, may mediate increased neutrophil transmigration in early disease.

Refractory periodontitis associated with abnormal polymorphonuclear leukocyte phagocytosis and cigarette smoking.

To learn if refractory periodontitis may be associated with defects in peripheral blood polymorphonuclear leukocyte (PMN) function, phagocytosis and chemotaxis were analyzed in 31 otherwise healthy patients and 12 unaffected controls. When compared to controls, no chemotactic defects to 10 nM f-Met-Leu-Phe (fMLP) were detected. In contrast, phagocytosis was significantly impaired (P < 0.001). The mean rates of adhesion and ingestion of opsonized Staphylococcus aureus by PMNs were 7.1 +/- 1.7 (+/- SD) and 1.4 +/- 0.5 bacteria/100 PMNs/minute respectively for patients, and 11.0 +/- 2.4 and 3.1 +/- 0.6 for unaffected, healthy controls. While the quality of oral hygiene and access to dental care were high, a retrospective search for associated environmental variables showed that 90% (28 of 31) of the refractory patients were smokers. The frequency of smokers is particularly striking, since only 21% of adults in Minnesota use tobacco regularly. These data suggest that there is a strong association between a peripheral blood PMN defect and refractory periodontitis. Furthermore, these studies suggest that tobacco use may contribute to this association.

Association between cigarette smoking, bacterial pathogens, and periodontal status.

The purposes of this study were to determine if: 1) an association exists between cigarette smoking and signs of periodontal disease after controlling for the confounding variables of age, sex, plaque, and calculus; 2) the prevalence of 5 bacteria commonly associated with periodontal disease differs between smokers and non-smokers; and 3) the presence of any of these bacteria or smoking are associated with a mean proximal posterior probing depth > or = 3.5 mm. Plaque, calculus, gingivitis, and probing depth were measured at the proximal surfaces of all teeth in one randomly selected posterior dental sextant in 615 adults. Subgingival plaque was sampled from the same sites and assayed for the presence of Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans, Prevotella intermedia, Eikenella corrodens, and Fusobacterium nucleatum. A subsample of non-smokers (n = 126), who were similar to smokers (n = 63) with respect to age, sex, plaque, and calculus, was randomly drawn from the original sample. These two groups were then compared on the basis of clinical and microbial parameters. The results indicated that the odds of having a mean probing depth > or = 3.5 mm were 5 times greater for smokers than the non-smoker subsample (odds ratio = 5.3; 95% CI = 2.0 to 13.8). No statistically significant difference in the prevalence of any of the bacteria was found between smokers and the non-smoker subsample. Based on logistic regression analyses of each of the 5 bacteria and smoking, mean probing depth > or = 3.5 mm was significantly associated with the presence of A. actinomycetemcomitans, P. intermedia, E. corrodens, and smoking (P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Emergence of antibiotic resistant Streptococcus sanguis in dental plaque of children after frequent antibiotic therapy.

PURPOSE: In the pediatric population, several different antibiotic regimens are currently recommended for the treatment of otitis media. This study investigated whether therapy for otitis media was associated with the emergence of antibiotic-resistant oral bacteria. METHODS: Streptococcus sanguis (S. sanguis) was isolated from supragingival dental plaque of children after a recent course of antibiotic. The isolated strains were tested for resistance to penicillin, amoxicillin, trimethoprim-sulfamethoxazole, and erythromycin and compared to isolated strains from age- and sex-matched control subjects, who had received no antibiotics within two years before sampling. RESULTS: While control subjects harbored no resistant strains of S. sanguis, about 60% of children who had received antibiotics harbored S. sanguis which were resistant to at least one of the tested antibiotics. Nearly half of these strains were resistant to two or more antibiotics. Resistance to penicillin and amoxicillin decreased with the age of the child and with the length of time since exposure to the antibiotic. However, resistance to trimethoprim-sulfamethoxazole or erythromycin showed no relationship to the age of the child or the length of time since exposure to the antibiotic. CONCLUSION: The data show that children who had been treated for otitis media with common antibiotic protocols do harbor antibiotic-resistant oral streptococci which may complicate prophylactic and therapeutic regimens for bacterial endocarditis.