BACKGROUND AND OBJECTIVE:Triclosan/copolymer toothpaste is effective in controlling plaque and gingivitis and in slowing the progression of periodontitis. This study describes its influence on microbiological and clinical outcomes, over a 5-year period, in patients with established cardiovascular disease (CVD).MATERIAL AND METHODS:Four-hundred and thirty-eight patients were recruited from the Cardiovascular Unit at The Prince Charles Hospital, Brisbane, Australia, and randomized to triclosan or placebo groups. Six sites per tooth were examined annually for probing pocket depth and loss of attachment. These outcomes were analysed, using generalized linear modelling, in 381 patients who had measurements from consecutive examinations. Concurrent load of the periodontal pathogens Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum, Tannerella forsythia and Porphyromonas gingivalis was determined, using quantitative real-time PCR, in 437 patients with baseline plaque samples. Group comparisons were expressed as geometric means. The chi-square test was used to test for differences between the two groups of patients with regard to the proportion of patients with different numbers of bacterial species.RESULTS:There was no difference in general health or periodontal status between the groups at baseline. There was a significant reduction in the number of interproximal sites showing loss of attachment between examinations, by 21% on average (p < 0.01), in the triclosan group compared with the placebo group. The prevalence of patients with F. nucleatum and A. actinomycetemcomitans was high and remained relatively constant throughout the 5 years of the study. In contrast, the prevalence of T. forsythia and P. gingivalis showed more variability; however, there was no significant difference between the groups, at any time point, in the prevalence of any organism. A significant difference in the geometric means for P. gingivalis (p = 0.01) was seen at years 1 and 4, and for F. nucleatum (p = 0.01) and in the total bacterial load (p = 0.03) at year 2; however, these differences were not statistically significant following a Bonferroni correction for multiple comparisons. There was no difference between the groups in the geometric means for each organism at year 5.CONCLUSION:Within the limitations of the study, these data suggest that the use of triclosan/copolymer toothpaste significantly slowed the progression of periodontitis in patients with CVD but that it had little influence on key subgingival periodontopathic bacteria in these patients over the 5 years of the study.

BackgroundThe growing awareness of transfusion‐associated morbidity and mortality necessitates investigations into the underlying mechanisms. Small animals have been the dominant transfusion model but have associated limitations. This study aimed to develop a comprehensive large animal (ovine) model of transfusion encompassing: blood collection, processing and storage, compatibility testing right through to post‐transfusion outcomes.Materials and methodsTwo units of blood were collected from each of 12 adult male Merino sheep and processed into 24 ovine‐packed red blood cell (PRBC) units. Baseline haematological parameters of ovine blood and PRBC cells were analysed. Biochemical changes in ovine PRBCs were characterized during the 42‐day storage period. Immunological compatibility of the blood was confirmed with sera from potential recipient sheep, using a saline and albumin agglutination cross‐match. Following confirmation of compatibility, each recipient sheep (n = 12) was transfused with two units of ovine PRBC.ResultsProcedures for collecting, processing, cross‐matching and transfusing ovine blood were established. Although ovine red blood cells are smaller and higher in number, their mean cell haemoglobin concentration is similar to human red blood cells. Ovine PRBC showed improved storage properties in saline–adenine–glucose–mannitol (SAG‐M) compared with previous human PRBC studies. Seventy‐six compatibility tests were performed and 17·1% were incompatible. Only cross‐match compatible ovine PRBC were transfused and no adverse reactions were observed.ConclusionThese findings demonstrate the utility of the ovine model for future blood transfusion studies and highlight the importance of compatibility testing in animal models involving homologous transfusions.

AIM To determine the relationship between periodontal pathogen load and anti-human heat shock protein 60 (hHSP60) antibodies in patients with established cardiovascular disease (CVD). MATERIALS AND METHODS Participants were cardiovascular patients (n = 74) with a previous hospital admission for myocardial infarction. Concurrent periodontal pathogen load of Porphyromonas gingivalis, Fusobacterium nucleatum, Tannerella forsythia and Aggregatibacter actinomycetemcomitans was determined using quantitative real-time PCR. Serum antibodies to these pathogens, GroEL and hHSP60 were determined using an ELISA. RESULTS There was a trend for increasing anti-hHSP60 antibody as the number of bacterial species increased. The strongest positive correlations were found between anti-hHSP60 levels and numbers of T. forsythia (r = 0.43; p < 0.001) and between anti-hHSP60 and anti-GroEL levels (r = 0.39; p = 0.001). Patients with extensive periodontal pocketing (≥4 mm) had higher numbers of P. gingivalis and T. forsythia (p < 0.05) and a higher subgingival pathogen load (p < 0.05) than patients with minimal pocketing (≤1 site ≥ 4 mm). They also had significantly elevated anti-hHSP60 levels (p < 0.05). Overall, the highest anti-hHSP60 levels were seen in patients with extensive periodontal pocketing and all four bacterial species. CONCLUSIONS In cardiovascular patients, a greater burden of subgingival infection with increased levels of P. gingivalis and T. forsythia is associated with modestly higher anti-hHSP60 levels.

BACKGROUND:Previous studies have demonstrated variable effects on systemic inflammatory and immune responses following improved periodontal health. This study examined changes in serum levels of the inflammatory mediators IL-1β, IL-6, TNF-α and sICAM-1, and antibodies to Porphyromonas gingivalis, human heat shock protein (hHSP) 60 and P. gingivalis GroEL following improvement in periodontal health in high cardiovascular (CV) risk and low CV-risk patients.METHODS:Patients retrospectively selected from a longitudinal study, had undergone yearly periodontal examinations and peripheral blood collections. They had demonstrated a quantifiable improvement in periodontal health (>60% reduction in number of sites with probing depth ≥ 4 mm from the baseline visit) and could be classified as either high CV-risk (≥ 6 classical risk factors, n = 13) or low CV-risk (≤ 1 classical risk factor, n = 14). Serum levels of the cytokines and antibodies were measured using ELISA.RESULTS:For sICAM-1 and anti-P. gingivalis GroEL and anti-hHSP60 antibodies, most patients recorded decreased levels. Reductions in serum sICAM-1 levels were more notable in low CV-risk patients (p = 0.006); and reductions in levels of anti-P. gingivalis GroEL and anti-hHSP60 antibodies (p = 0.001 and 0.009 respectively) were more notable in high CV-risk patients.CONCLUSIONS:This study found that subsequent to improved periodontal health, the anti-HSP (HSP60 and GroEL) antibody response was reduced, particularly for high CV-risk patients. sICAM-1 levels were also lowered, more so for low CV-risk patients.

Objectives: Tissue from Marfan syndrome (MFS) or bicuspid aortic valve (BAV) aneurysm is characterized by vascular smooth muscle cell (VSMC) loss, cystic medial necrosis and elastic tissue destruction. We examined morphological changes in aneurysm tissue using light microscopy (LM) and transmission electron microscopy (TEM). Conclusions: The findings suggest that morphological abnormalities in VSMC precede recognizable alterations of EL, consistent with the hypothesis that the primary defect in the pathogenesis of MFS and BAV aneurysm arises within VSMCs.

In terms of the pathogenesis of cardiovascular disease (CVD) the focus has traditionally been on dyslipidemia. Over the decades our understanding of the pathogenesis of CVD has increased, and infections, including those caused by oral bacteria, are more likely involved in CVD progression than previously thought. While many studies have now shown an association between periodontal disease and CVD, the mechanisms underpinning this relationship remain unclear. This review gives a brief overview of the host-bacterial interactions in periodontal disease and virulence factors of oral bacteria before discussing the proposed mechanisms by which oral bacterial may facilitate the progression of CVD.

Objectives: Porphyromonas gingivalis has been associated with both periodontitis and cardiovascular disease. Filamentous components on P. gingivalis cell surfaces (fimA) play an important role in the colonization and invasion of host cells. In this study, P.gingivalis fimA genotypic distribution was analysed in dental plaque specimens from high and low cardiovascular (CV) risk patients in an Australian population. Methods: Patients were retrospectively selected from an existing longitudinal study. A total of 90 patients with P.gingivalis positive plaque samples were selected according to CV-risk (≥6 classical risk factors=high CV-risk, n=48; ≤1 classical risk factor=low CV-risk, n=42) and periodontal status (≥6 sites with ≥4mm periodontal probing depth (PPD)=periodontitis, n=42; ≤1 site with ≥4mm PPD=periodontal health, n=48). The plaque specimens were analysed to discriminate the fimA genotype using conventional polymerase chain reaction with fimA type-specific primer sets. Results: The distribution of fimA genotypes was similar in periodontally healthy individuals irrespective of their CV-risk group. In contrast, the prevalence of the type II, type IV and type 1b virulent fimA genotypes was greater in periodontitis patients with high CV-risk than in periodontitis patients with low CV-risk. The virulent type II fimA genotype had the highest prevalence in high CV-risk periodontitis patients (40%) and was 8 times greater than in low CV-risk periodontitis patients. The non-virulent type III fimA genotype was most prevalent in low CV-risk periodontitis patients (27%). Conclusions: The results of this study indicate that virulent fimA genotypes occurred more frequently in patients with an increased risk of developing CVD, suggesting that the presence of specific fimA genotypes in dental plaque is associated with an increased risk of developing CVD. Acknowledgements: This study was supported by unrestricted grants from National Health and Medical Research Council, HCF Health and Medical Research Foundation, Australian Dental Research Foundation and Queensland Health.

There is evidence that periodontal disease may be associated with atherosclerosis due to cross-reactivity of bacterial GroEL immunity with human heat shock protein 60 (hHSP60). Objective: To examine changes in serum antibody responses to Porphyromonas gingivalis, hHSP60, and P. gingivalis GroEL following improvement in periodontal health in patients with cardiovascular disease (CVD) and in patients with high cardiovascular (CV) risk and low CV risk. Methods: Patients were selected from two large longitudinal studies and had undergone yearly periodontal examinations and peripheral blood collections. CVD patients (n=15) selected from one study had experienced a significant CV event while patients derived from the other study had not experienced a CV event. These latter patients were further classified according to CV risk (≥6 classical risk factors=high CV risk n=13; ≤1 classical risk factor=low CV risk n=14). Patients demonstrating a quantifiable improvement in periodontal health (≥62% reduction in number of sites with probing depth≥4mm) from the baseline visit were selected. Serum IgG antibody levels to P.gingivalis, hHSP60, and P.gingivalis GroEL were measured using ELISA. Results: Median reductions, after improvement in periodontal health, in antibody levels to P.gingivalis GroEL differed significantly across the three groups (Kruskal-Wallis p<0.001). These reductions were greatest for high CV-risk patients (908.38ng/mL) compared with 125.18ng/mL for CVD patients, whilst low CV-risk patients showed a slight median increase of 13.57ng/mL. All patients with high CV-risk (13/13=100%) showed reductions in antibody levels to P.gingivalis GroEL compared with CVD (10/15=66.7%) and low-CV risk patients (6/14=42.9%). The difference in these proportions was significant at the 5% level (2 p=0.006). Changes in anti-HSP60 and anti-P.gingivalis antibody levels were not significant. Conclusions: Improved periodontal health reduced levels of the potentially host-reactive antibody, anti- P.gingivalis GroEL, particularly in high CV-risk patients and therefore may be an effective therapy to reduce CV risk for these patients.

Thoracic aortic aneurysm associated with Marfan syndrome (MFS) or bicuspid aortic valve (BAV) is characterized by over-expression of matrix metalloproteinases (especially MMP-2 and MMP-9). Up-regulated MMP-10 has been linked to reduced cell–matrix interaction, cell detachment, dilation and rupture of blood vessels. We investigated MMP-10 expression in aortic aneurysm tissue and cultured VSMCs and its relation to VSMC apoptosis. Aortic tissue and cultured VSMCs were derived from subjects with MFS (five males, two females; 40 ± 22 years, mean ± S.D.) and BAV (five males, two females; 60 ± 16 years), and normal subjects (three males, six females; 44 ± 14 years). Caspase-3 was used as a marker of cells undergoing apoptosis. Specific staining of aortic tissue showed increased expression of MMP-10 in MFS and BAV (P < 0.05). Similar results were obtained in cultured VSMCs. In VSMCs characterized by cell shrinkage of cell and nuclear condensation there was enhanced staining of MMP-10. The same VSMCs showed caspase-3 labelling in alternate serial sections. The proportion of apoptotic VSMCs in aneurysm tissue was significantly increased in the aneurysm wall of patient groups compared to normal subjects (P < 0.05). Gelatin zymography showed no active form of MMP-10 and Western blot indicated the presence of the MMP-10 pro-form only with higher levels of expression in VSMC conditioned media from patients compared to normals (P < 0.05). The study suggests that expression of MMP-10 is related to progression of aortic wall aneurysm in MFS and BAV.