Janet M. Guthmiller

Discovery of new human β-defensins using a genomics-based approach

Abstract Epithelial β-defensins are broad-spectrum cationic antimicrobial peptides that also act as chemokines for adaptive immune cells. In the human genome, all known defensin genes cluster to a

Human β-Defensins 2 and 3 Demonstrate Strain-Selective Activity against Oral Microorganisms

ABSTRACT Human β-defensins 2 and 3 (HBD-2 and HBD-3) are inducible peptides present at sites of infection in the oral cavity. A few studies have reported broad-spectrum antimicrobial activity for both peptides. However, no comprehensive study has thoroughly investigated their potential against oral pathogens. The purpose of this study was to test the effectiveness of HBD-2 and HBD-3 against a collection of oral organisms (Actinobacillus actinomycetemcomitans, Fusobacterium nucleatum, Porphyromonas gingivalis, Peptostreptococcus micros, Actinomyces naeslundii, Actinomyces israelii, Streptococcus sanguis, Streptococcus mutans, Candida tropicalis, Candida parapsilosis, Candida krusei, Candida glabrata, and Candida albicans). Radial diffusion assays were used to test HBD-2 and HBD-3 activities against at least three strains of each species. There was significant variability in MICs, which was strain specific rather than species specific. MICs ranged from 3.9 to >250 μg/ml for HBD-2 and from 1.4 to >250 μg/ml for HBD-3. HBD-3 demonstrated greater antimicrobial activity and was effective against a broader array of organisms. Overall, aerobes were 100% susceptible to HBD-2 and HBD-3, whereas only 21.4 and 50% of the anaerobes were susceptible to HBD-2 and HBD-3, respectively. HBD-2 and HBD-3 also demonstrated strain-specific activity against the Candida species evaluated. Interestingly, an association between HBD-2 and HBD-3 activities was noted. This suggests that the two peptides may have similar mechanisms yet utilize distinct pathways. The lack of activity against specific anaerobic strains and Candida warrants further investigation of the potential resistance mechanisms of these organisms. Finally, the significant variability between strains underlies the importance of testing multiple strains when evaluating activities of antimicrobial peptides.

Human polymicrobial infections

Summary Context Polymicrobial diseases, caused by combinations of viruses, bacteria, fungi, and parasites, are being recognised with increasing frequency. In these infections, the presence of one micro-organism generates a niche for other pathogenic micro-organisms to colonise, one micro-organism predisposes the host to colonisation by other micro-organisms, or two or more non-pathogenic micro-organisms together cause disease. Starting point Recently, Gili Regev-Yochay (JAMA 2004; 292: 716–20) and Debby Bogaert (Lancet 2004; 363: 1871–72), and their colleagues, suggested another interaction: microbial interference—the ability of Streptococcus pneumoniae carriage to protect against Staphylococcus aureus carriage, and the inverse effect of pneumococcal conjugate vaccination on the increased carriage of Staph aureus and Staph-aureus-related disease. Strep pneumoniae carriage protected against Staph aureus carriage, and the bacterial interference could be disrupted by vaccinating children with pneumococcal conjugate vaccines that reduced nasopharyngeal carriage of vaccine-type Strep pneumoniae. Where next The medical community is recognising the significance of polymicrobial diseases and the major types of microbial community interactions associated with human health and disease. Many traditional therapies are just starting to take into account the polymicrobial cause of diseases and the repercussions of treatment and prevention.

Influence of smoking on gingival crevicular fluid cytokines in severe chronic periodontitis.

AIM The aim of this study was to compare the expression of 22 chemokines and cytokines in gingival crevicular fluid (GCF) from smokers and non-smokers with periodontitis and periodontally healthy control subjects. MATERIALS AND METHODS Forty subjects with generalized severe chronic periodontitis (20 smokers and 20 non-smokers) and 12 periodontally healthy control subjects participated in this study. Four diseased and two healthy sites were selected from each of the periodontitis subjects. GCF samples were collected and cytokines analysed utilizing a multiplexed immunoassay (Luminex(®) ). Statistical analyses employed non-parametric tests including the Mann-Whitney and Wilcoxon matched-pairs signed-rank tests. RESULTS Compared with healthy control subjects, GCF in subjects with chronic periodontitis contained significantly higher amounts of interleukin (IL)-1α, IL-1β, IL-6, IL-12(p40) (pro-inflammatory cytokines); IL-8, macrophage chemotactic protein (MCP)-1, macrophage inflammatory protein (MIP)-1α, regulated on activation normal T-cell expressed and secreted (RANTES) (chemokines); IL-2, IFN-γ, IL-3, IL-4 (Th1/Th2 cytokines); IL-15 [regulator of T-cells and natural killer (NK) cells]. Smokers displayed decreased amounts of pro-inflammatory cytokines [IL-1α, IL-6, IL-12(p40)], chemokines (IL-8, MCP-1, MIP-1, RANTES), and regulators of T-cells and NK cells (IL-7, IL-15). CONCLUSIONS Periodontitis subjects had significantly elevated cytokine and chemokine profiles. Smokers exhibited a decrease in several pro-inflammatory cytokines and chemokines and certain regulators of T-cells and NK-cells. This reflects the immunosuppressant effects of smoking which may contribute to an enhanced susceptibility to periodontitis.

BACTERIAL PENETRATION THROUGH CANALS OF ENDODONTICALLY TREATED TEETH IN THE PRESENCE OR ABSENCE OF THE SMEAR LAYER

OBJECTIVES The goal of this study is to determine if the smear layer affects the passage of bacteria through or around obturating material as evidenced by penetration of bacteria through and out the canal. Specifically, this study focused on determining the effect of the smear layer on the magnitude of bacterial penetration through the apical foramen. METHODS Thirty extracted, maxillary central or lateral incisors were collected. Teeth were randomly assigned (10 teeth per group) to three groups: (1) smear layer removed, (2) smear layer present, and (3) negative control. Canal preparation and obturation using lateral condensation, gutta percha, and AH 26 sealer was performed on all of the teeth. Removal of the smear layer was accomplished by rinsing with 17% EDTA. The model systems consisted of an upper chamber attached to the cemento-enamel junction and a lower chamber at the apices of the teeth. Standardized bacterial suspensions containing Fusobacterium nucleatum, Campylobacter rectus, and Peptostreptococcus micros were inoculated into the upper chambers. Models were incubated anaerobically at 37 degrees C. At various times over a 60-day period, samples were taken from the lower chamber and spiral-plated on selective-differential media to determine numbers and types of bacteria. RESULTS Leakage results were as follows: (1) smear layer present-6/10 leaked; (2) smear layer removed-0/10 leaked; (3) negative control-0/10 leaked. Profiles of bacterial leakage were similar among the groups. F. nucleatum was the predominant microorganism. CONCLUSIONS This study indicated that removal of the smear layer reduced the leakage of bacteria through the root canal system.

A multiplex immunoassay demonstrates reductions in gingival crevicular fluid cytokines following initial periodontal therapy

BACKGROUND AND OBJECTIVE Cytokines and chemokines play an important role in the pathogenesis of periodontal diseases. The objective of this study was to quantitatively assess the effect of initial periodontal therapy on gingival crevicular fluid levels of a comprehensive panel of cytokines and chemokines, including several less extensively studied mediators. MATERIAL AND METHODS Clinical examinations were performed and gingival crevicular fluid samples obtained from six subjects with generalized severe chronic periodontitis prior to initial periodontal therapy and at re-evaluation (6-8 weeks). Four diseased and two healthy sites were sampled in each subject. Twenty-two gingival crevicular fluid mediators were examined using a multiplex antibody capture and detection platform. Statistical analyses were performed by fitting mixed effects linear models to log-transformed gingival crevicular fluid values. RESULTS Gingival crevicular fluid interleukin (IL)-1alpha and IL-1beta were the only cytokines to differ in initially diseased vs. initially healthy sites. Following initial therapy, 13 of the 16 detectable cytokines and chemokines decreased significantly in diseased sites, including IL-1alpha, IL-1beta, IL-2, IL-3, IL-6, IL-7, IL-8, IL-12 (p40), CCL5/regulated on activation, normally T cell expressed and secreted (RANTES), eotaxin, macrophage chemotactic protein-1, macrophage inflammatory protein-1alpha and interferon-gamma. At healthy sites, only three of the 16 mediators were significantly altered following therapy. CONCLUSION This is the first study, to our knowledge, to evaluate such an extensive panel of gingival crevicular fluid mediators within the same sample prior to and following initial therapy. The results confirm that periodontal therapy effectively reduces pro-inflammatory cytokines and chemokines, including less well-described mediators that may be important in initiation and progression of periodontitis. The multiplex assay will prove useful for future gingival crevicular fluid studies.

Human β-defensin 3 binds to hemagglutinin B (rHagB), a non-fimbrial adhesin from Porphyromonas gingivalis, and attenuates a pro-inflammatory cytokine response

Regulatory mechanisms in mucosal secretions and tissues recognize antigens and attenuate pro‐inflammatory cytokine responses. Here, we asked whether human β‐defensin 3 (HBD3) serves as an upstream suppressor of cytokine signaling that binds and attenuates pro‐inflammatory cytokine responses to recombinant hemagglutinin B (rHagB), a non‐fimbrial adhesin from Porphyromonas gingivalis strain 381. We found that HBD3 binds to immobilized rHagB and produces a significantly higher resonance unit signal in surface plasmon resonance spectroscopic analysis, than HBD2 and HBD1 that are used as control defensins. Furthermore, we found that HBD3 significantly attenuates (P<0.05) the interleukin (IL)‐6, IL‐10, granulocyte macrophage colony stimulating factor (GM‐CSF) and tumor‐necrosis factor‐α (TNF‐α) responses induced by rHagB in human myeloid dendritic cell culture supernatants and the extracellular signal‐regulated kinases (ERK 1/2) response in human myeloid dendritic cell lysates. Thus, HBD3 binds rHagB and this interaction may be an important initial step to attenuate a pro‐inflammatory cytokine response and an ERK 1/2 response.

Susceptibilities of Oral Bacteria and Yeast to Mammalian Cathelicidins

ABSTRACT The effects of cathelicidins against oral bacteria and clinically important oral yeasts are not known. We tested the susceptibilities of Actinobacillus actinomycetemcomitans, Fusobacterium nucleatum,Porphyromonas gingivalis, Streptococcus sanguis, Candida krusei, Candida tropicalis and Candida albicans to the following cathelicidins: FALL39, SMAP29, and CAP18. SMAP29 and CAP18 were antimicrobial, whereas FALL39 did not exhibit antimicrobial activity. Future studies are needed to determine the potential use of these antimicrobial peptides in prevention and treatment of oral infections.

The impact of oral health literacy on periodontal health status

OBJECTIVE The objective of this study was to describe oral health literacy (OHL) among periodontal patients and to examine its association with periodontal health status. METHODS This cross-sectional study included new and referred patients presenting to the University of North Carolina Graduate Periodontology Clinic. Sociodemographic and dental history information were collected. OHL was measured using a dental word recognition instrument, Rapid Estimate of Adult Literacy-30 (REALD-30). Clinical periodontal examinations were completed. RESULTS One hundred and twenty-eight participants enrolled and 121 completed all study examinations and instruments. Despite a high level of education among participants in our study, low levels of OHL were found in one-third (33 percent) of the study population. Thirty-one percent had moderate OHL (score of 22-25), 37 percent had high OHL (score ≥ 26). The mean REALD-30 score was 23. Fifty-three percent of participants had severe periodontitis, 29 percent had moderate periodontitis, and 18 percent had mild or no periodontitis. Bivariate analysis showed a significant association between OHL and periodontal status (P < 0.05). The effect of OHL on periodontal health status remained statistically significant (P < 0.002) even after controlling for smoking, race, and dental insurance. CONCLUSION Lower OHL was associated with more severe periodontal disease among new and referred patients presenting to the University of North Carolina Graduate Periodontology Clinics.

Interleukin-1 and interleukin-8 in nicotine- and lipopolysaccharide-exposed gingival keratinocyte cultures

BACKGROUND AND OBJECTIVE Tobacco use is associated with increased periodontal destruction in both cigarette smokers and smokeless tobacco users. Gingival keratinocytes are the first cells in contact with microbial and tobacco components and play a key role in the innate immune response to these agents. The objective of this study was to evaluate the effect of nicotine and bacterial lipopolysaccharide (LPS) alone and in combination on gingival keratinocyte production of interleukin-1 alpha (IL-1 alpha) and interleukin-8 (IL-8). MATERIAL AND METHODS Gingival keratinocyte cultures were established from 10 healthy, non-tobacco-using subjects. The cells were stimulated for 24 h with 1 mum or 1 mm nicotine and/or 10 microg/mL Escherichia coli or Porphyromonas gingivalis LPS. Interleukin-1 alpha and IL-8 proteins were quantified using ELISAs. RESULTS Compared with untreated cultures, 1 mm nicotine stimulated production of IL-1 alpha (p < 0.001); E. coli and P. gingivalis LPS increased IL-8 production (p = 0.0014 and p = 0.0232, respectively). A combination of nicotine and LPS produced the highest cytokine quantities. Amounts of IL-1 alpha and IL-8 following 1 mm nicotine and LPS exposure were significantly greater than in untreated cultures (p < 0.001). Interleukin-8 was also responsive to 0.1 mum nicotine combined with E. coli or P. gingivalis LPS compared with control cultures (p < 0.0001 and p = 0.0029, respectively). Both cytokines tended to be elevated following the combined treatment relative to nicotine or LPS treatment alone. CONCLUSION These results demonstrate that nicotine and LPS differentially regulate IL-1 and IL-8 production by gingival keratinocytes. Combined treatment tended to elevate cytokine production further, which may have implications for the progression of periodontitis in tobacco users.