Endotoxemia and the host systemic response during experimental gingivitis.

AIM: To assess endotoxemia episodes and subsequent changes in serum inflammatory biomarkers using the experimental gingivitis model. MATERIALS AND METHODS: Data from 50 healthy black and white adult males and females were compared for serum concentrations of endotoxin, and serum biomarkers [neutrophil oxidative activity, interleukin (IL)-1ß, IL-6, IL-8, C-reactive protein (CRP), and fibrinogen] at baseline, at 3 weeks of experimental gingivitis, and after 2 weeks of recovery. Means were compared using repeated measures analysis of variance. RESULTS: Endotoxemia was reported in 56% of the serum samples at 3 weeks of induced gingivitis. At 2 weeks of recovery, endotoxin levels decreased to levels similar to those reported at baseline. Neutrophil oxidative activity increased significantly following 3 weeks of gingivitis versus baseline (p<0.05). In the endotoxin-negative group this increase was associated with the black subjects whereas in the endotoxin-positive group change in neutrophil activity was driven by the female subpopulation. Serum cytokines, CRP, and fibrinogen levels did not change during the study. CONCLUSIONS: Experimental gingivitis was associated with endotoxemia and hyperactivity of circulating neutrophils, but not with changes in systemic levels of cytokines and acute-phase proteins. This may be attributed to the mild nature and the short duration of the induced gingivitis.

Mapping of the nick site on conjugative plasmid pVT745 by interrupted mating.

Conjugal transfer of plasmid DNA initiates and terminates at a specific non-coding site called the origin of transfer (oriT). Previous analysis of conjugative plasmid pVT745 from Aggregatibacter actinomycetemcomitans suggested that oriT was located adjacent to the operon responsible for initiation of ssDNA transfer. The location of oriT was confirmed by assaying both subclones of the region as well as a pVT745 deletion derivative for mobilization. The precise DNA nick site (nic) and polarity of DNA transfer were identified by use of interrupted mating assays, a technique originally used for the mapping of bacterial chromosomes. Nucleotide sequence analysis revealed that the pVT745-specific nick region was similar to the consensus nick sequence of the IncP family albeit the actual cleavage site differed. Functionality of nic was confirmed by point mutations.

Is dentistry at risk? A case for interprofessional education.

The goal of interprofessional education (IPE) is to bring various professional groups together in the educational environment to promote collaborative practice and improve the health care of patients. Interest in IPE has been sparked by several factors in the health care system, including the increased awareness of oral-systemic connections, an aging population, the shift of the burden of illness from acute to chronic care, and lack of access to basic oral care. Increasingly, since the publication of the U.S. surgeon general's report in 2000, the dialogue surrounding IPE in dentistry has escalated. But how has dentistry changed regarding IPE since the report was released? This position paper argues that little has changed in the way dental students are taught and prepared to participate in IPE. The authors contend that academic dentistry and organized dentistry must take the lead in initiating and demanding IPE if dental students are to be prepared to work in the health care environment of the twenty-first century. Included are reasons why IPE is necessary and why dentistry must lead the conversation and participate in the solution to the oral health care crisis. It explores existing models and alternate approaches to IPE, barriers to implementation, and proposed strategies for academic institutions.

Entry exclusion activity on conjugative plasmid pVT745.

Conjugative plasmid transfer into a recipient cell containing the same or a closely related plasmid is inhibited by a mechanism called entry or surface exclusion. The function of entry exclusion is to reduce unproductive conjugation. The current study assessed the exclusion activity on conjugal plasmid pVT745 by conducting mating experiments with genetically distinguishable derivatives of this plasmid. Our results demonstrate that a single gene, magB05, that is located in a gene cluster associated with mating pore formation, is responsible for the entry exclusion phenotype of pVT745.

Resistance of fluorescent-labelled Actinobacillus actinomycetemcomitans strains to phagocytosis and killing by human neutrophils.

Neutrophils are initially the predominant cells involved in the host defence of bacterial infections, including periodontal disease. Aggressive periodontitis is associated with Actinobacillus actinomycetemcomitans, a Gram-negative capnophilic microorganism. Infections caused by A. actinomycetemcomitans are not resolved by the host immune response despite the accumulation of neutrophils at the site of inflammation. To better understand the role of natural host defence mechanisms in A. actinomycetemcomitans infections, the interaction of phenotypically diverse strains of this pathogen with human neutrophils was assessed directly using techniques such as genetic labelling with the gene for green fluorescent protein, fluorescence-activated cell sorting and fluorescence imaging. The study included clinical isolates of A. actinomycetemcomitans represented by self-aggregating, biofilm-associated and isogenic planktonic variants. Data obtained showed that complement-mediated phagocytosis of A. actinomycetemcomitans was generally inefficient regardless of strain-specific serotype or leukotoxin production. Furthermore, the majority of ingested bacteria remained viable after exposure to neutrophils for 1 h. Interestingly, uptake of antibody-opsonized bacteria resulted in the rapid cell death of neutrophils. This was in contrast to ingestion of complement-opsonized bacteria, which did not affect neutrophil viability. The methods used in this study provided reliable and reproducible results with respect to adherence, phagocytosis and killing of A. actinomycetemcomitans when encountering human neutrophils.

DNA inversion on conjugative plasmid pVT745.

Plasmid pVT745 from Actinobacillus actinomycetemcomitans strain VT745 can be transferred to other A. actinomycetemcomitans strains at a frequency of 10(-6). Screening of transconjugants revealed that the DNA of pDMG21A, a pVT745 derivative containing a kanamycin resistance gene, displayed a structural rearrangement after transfer. A 9-kb segment on the plasmid had switched orientation. The inversion was independent of RecA and required the activity of the pVT745-encoded site-specific recombinase. This recombinase, termed Inv, was highly homologous to invertases of the Din family. Two recombination sites of 22 bp, which are arranged in opposite orientation and which function as DNA crossover sequences, were identified on pVT745. One of the sites was located adjacent to the 5' end of the invertase gene, inv. Inversion of the 9-kb segment on pVT745 derivatives has been observed in all A. actinomycetemcomitans strains tested except for the original host, VT745. This would suggest that a host factor that is either inactive or absent in VT745 is required for efficient recombination. Inactivation of the invertase in the donor strain resulted in a 1,000-fold increase in the number of transconjugants upon plasmid transfer. It is proposed that an activated invertase causes the immediate loss of the plasmid in most recipient cells after mating. No biological role has been associated with the invertase as of yet.

Parameters associated with cloning in Actinobacillus actinomycetemcomitans.

Characterization of virulence traits in Actinobacillus actinomycetemcomitans requires the application of recombinant DNA techniques. To develop appropriate genetic tools it is necessary to identify suitable host-vector systems. The current study assessed cloning parameters in A. actinomycetemcomitans for two previously described vectors, pDMG4 and pMMB67. It was determined that the maximum size of recombinant molecules that could be transferred to A. actinomycetemcomitans strain ATCC29522 via electroporation was 33 kb. The size limit for transformation of the same strain with ligation mixtures (direct cloning), however, was limited to 23-24 kb. Additional experiments included electroporation of various A. actinomycetemcomitans strains with plasmid DNA isolated from Escherichia coli and different A. actinomycetemcomitans sources. Differences in transformation efficiencies suggested the presence of a restriction modification system for pDMG4 in some strains of A. actinomycetemcomitans. Cloning of portions of the enterococcal plasmid pJH1 into A. actinomycetemcomitans resulted in the insertion of the intact vector into the chromosome.