This PhD consisted of a series of studies aiming at detecting genetic risk factors for Aggressive Periodontitis (AgP). AgP is a destructive disease of the periodontium affecting around 1% of the population and leading to early tooth loss. Microbiological and environmental factors are thought to act on a genetically susceptible host to determine AgP. We conducted a case-control association study on 224 AgP patients (both Generalised AgP and Localised AgP) and 231 healthy controls to detect differences in genotype distributions of 13 single nucleotide polymorphisms (SNPs). The selected SNPs included FcR and FPR, NADPH oxidase, IL-6, TNF-a and VDR polymorphisms. Further studies on subsets of patients were conducted to detect associations between these SNPs and classical features of AgP: disease severity, familial aggregation, presence of periodontopathogenic bacteria and neutrophil hyperactivity. The NADPH p22phox 242 polymorphism was associated with the AgP trait and with disease severity. The IL-6 -174 SNP was associated with LAgP and with increased detection of periodontopathogenic bacteria. The FcyRIIIb NA polymorphism was associated with GAgP, while FcyR haplotypes were linked with AgP in Blacks and FcyRIIa was associated with familial aggregation of the AgP phenotype. The VDR Taq-I polymorphism showed a trend for association with AgP in smokers. The overall results of the study provide two possible pathogenic pathways leading to AgP: one is mediated through an excessive inflammatory response triggered by the presence of specific bacteria in individuals with hyper-responsive genotypes (NADPH p22phox 242 T allele, FcyRIIIb NA1 homozygosity, IL-6 -174 G homozygosity) the second is initiated by an increased susceptibility to bacterial colonization (FcyRIIa R homozygosity). In conclusion, this study supports the importance of genetic factors in Aggressive Periodontitis and hypothesizes possible pathogenic mechanisms.

Healthy children, aged between 5 and 9 years who had not taken antibiotics in the preceding three months were recruited. Plaque was sampled from the gingival crevice of either the lower left or lower right first permanent molar to estimate the prevalence of Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans and Tannerella forsythensis which have been implicated as main etiological agents of periodontal disease. Analysis of plaque from subjects without gingivitis (n = 65) and those with gingivitis (n = 53) by PCR targeting of the 16S rRNA gene demonstrated negligible differences in the prevalence of these pathogens with the exception of T. forsythensis. This pathogen was detected more frequently in children with no gingivitis (P = 0.03). Community analysis of plaque was carried out to determine (i) any significant differences in the microbiota of both cohorts and (ii) if specific taxa influenced the prevalence of the three periodontal pathogens. Community analysis was attempted by using both culture dependent and culture independent techniques. The culture dependent technique involved community level physiological profiling (CLPP) and attempted to measure metabolic differences between cohorts. Preliminary studies demonstrated that this technique was not suitable for small sample volumes, such as plaque from single sites and was abandoned. The culture independent technique, denaturing gradient gel electrophoresis (DGGE), is a rapid and cost effective method for analysing bacterial communities. Statistical analysis of the DGGE data for both cohorts suggested that bacterial diversity was lower in subjects with gingivitis (P = 0.009). Logistic regression analysis of the DGGE banding patterns demonstrated that specific bands were significantly associated with gingival health and with gingivitis. Similarly, other bands were significantly associated with the prevalence of the three periodontal pathogens. These bands were excised and PCR-cloned. The 16S rRNA sequence data for these clones demonstrated that these DGGE bands were mixed with DNA of multiple taxa. Further attempts to separate individual excised bands over shorter denaturing gradients proved unsuccessful. This work has demonstrated that P. gingivalis, A. actinomycetemcomitans and T. forsythensis are present in the dental plaque of pre-pubertal children both with and without gingivitis. It has also provided valuable insights into the applications and limitations of fingerprinting techniques such as CLPP and DGGE.

This research work has investigated the molecular characterization of oral Candida of individuals (with no signs of candidosis) from different dental clinical settings in the United Kingdom and patients from diverse geographical regions of the world (UK, Greece, Italy and Brazil) who have undergone bone marrow transplantation (BMT). Subjects from the different dental settings were classified into healthy (subjects from a Private Dental Clinic, Prosthodontic Clinic and Orthodontic Clinic) or diseased subjects (patients from The Oral Medicine Clinic with mucosal diseases and Special Needs Clinic). The oral carriage of Candida and the presence of C. dubliniensis from the diseased subjects were significantly greater than that of the healthy subjects (p = 0.025 and p = 0.008 respectively). The healthy subjects had more non-albicans Candida (other than C. dubliniensis) than the diseased subjects (p = 0.024). Recipients of bone marrow transplantation (UK) had more non-albicans Candida (p = 0.024) than the healthy subjects. There was no difference in Candida diversity between isolates from BMT recipients from diverse geographic locales, although genotypic diversity among BMT recipients was observed from the Italian isolates (p = 0.041). PCR finger-printing with subsequent phylogenetic analysis revealed that there were significant differences between C. albicans strains from the UK BMT recipients with time and the strains from the UK BMT recipients were significantly more diverse than strains from the control subjects and the recipients of BMT from other geographical locales (p = 0.0001). Virulence attributes of Candida species were studied in-vitro utilizing adherence and proteinase assays. Candida isolates from BMT recipients in the UK had a trend of less in-vitro adherence (p = 0.076) than those from the healthy subjects, but there was no difference in adhesion between isolates from BMT recipients from different geographic regions. There was no difference in proteolytic activity of isolates from BMT recipients and control subjects, but there were significant differences in proteolytic activity between isolates from BMT recipients from different geographic regions (p = 0.001). Candida species from the Brazilian BMT recipients had very low extracellular proteinase activity. In-vitro fluconazole susceptibility test utilizing the National Committee for Clinical Laboratory Standards (NCCLS) M27-A showed that the isolates from BMT recipients from the UK were significantly more resistance to fluconazole than those from the control subjects (p = 0.027) or isolates from BMT recipients from other geographical regions (p = 0.009). There were no associations of Candida carriage, isolation of more non-albicans Candida, virulence attributes and developing of fluconazole resistance from subsequent sampling from the UK and Greek BMT recipients. Finally, the expression of secreted aspartyl proteinase (SAP) genes and agglutinin-like sequence (ALS) genes by strains of C. albicans utilizing RT-PCR was found to be influenced by the growth medium. It is concluded that bone marrow transplantation can have a significant impact upon the carriage of non-albicans Candida, virulence attributes and in-vitro fluconazole resistance of the oral Candida.

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Periodontitis has been associated with elevated inflammatory markers in otherwise healthy populations. However the nature of this association has not been determined. The aim of this work was to establish whether or not periodontitis causes systemic inflammation. A first pilot intervention trial indicated that standard periodontal therapy, in a cohort of 94 individuals suffering from severe generalized periodontitis, produced a 0,5 mg/L decrease in serum CRP concentration 6 months after therapy. This decrease was affected by the degree of clinical periodontal response and carriage of specific polymorphisms in inflammatory genes (IL-1 A, IL-6). Periodontal treatment, on the other hand, produced a moderate acute phase response of one week duration 10-fold increase in CRP (P<0.001) . Carriage of rare alleles in the CRP gene was associated with a greater acute CRP response to periodontal therapy after correcting for conventional cardiovascular and inflammatory factors. Standard (SPT) and an intensive periodontal therapy (IPT, including local delivery of antimicrobials) resulted in a significant reduction in serum CRP compared to an untreated control 2 months after treatment (0.5 0.2 mg/L, P=0.030 and 0.8 0.2 mg/L, P=0.001 respectively) in a randomized controlled trial involving 65 healthy subjects with severe generalized periodontitis. The IPT group showed also a decrease in total cholesterol and LDL-cholesterol. These data were confirmed by the results of a second randomized trial where a cluster of inflammatory and metabolic parameters were evaluated at baseline, 1, 2 and 6 months after either a SPT or IPT regimen. IPT patients showed significant reductions in inflammatory markers at one (p=O.04O6) and two (p=0.0060) months together with an improved metabolic state (2-6 months reduction in lipid markers p=0.0320 and p=0.0432 respectively). Periodontitis causes an increased reversible systemic inflammatory burden and an intensive therapy regimen is more effective in re-establishing a more favourable systemic homeostasis.

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Type 2 diabetes (T2DM) is a risk factor for periodontal disease, however the pathogenic links between the two disease are not completely understood. Both diseases are considered to be inflammatory conditions and, therefore, cytokines are likely to play a role in the shared susceptibility between the two diseases. Therefore, the study evaluated, longitudinally over 12 months, the impact of periodontal therapy on clinical outcomes, glycaemic control, hsCRP, lipids and local and systemic levels of IL-6, TNF- α, IL-1β and IFN-γ in patients with T2DM. 101 T2DM and 83 non-diabetic subjects were recruited and, of these, periodontitis was diagnosed in 47 T2DM and 48 non-diabetic subjects. Pre-treatment, subjects with T2DM had significantly higher BMI and significantly lower systolic BP and cholesterol compared to non-diabetic subjects. Serum levels of TNF-α, IL-1β and IFN-γ were significantly higher in subjects with T2DM compared to non-diabetic subjects. Regardless of diabetic status, GCF and salivary levels of IL-1β were significant predictors of the clinical periodontal condition. In T2DM and non-diabetic subjects, all clinical periodontal outcomes were significantly improved at 3, 6 and 12 months following NSM and both groups demonstrated significant reductions in GCF IL-1β levels at 3, 6 and 12 months. In nondiabetic subjects, a significant reduction in non-HDL and cholesterol was seen 6 months after NSM. In subjects with T2DM, serum TNF-α was significantly reduced 3 and 6 months after NSM. In subjects with T2DM, HbA1c showed 0.45% and 0.40% reductions at 3 and 12 months after NSM, although these reductions did not achieve statistical significance. Abstract XVII In conclusion, periodontal therapy led to significant reductions in GCF IL-1β in all subjects, and also produced a significant reduction in circulating levels of TNF-α in subjects with T2DM. Furthermore, IL-1β in saliva and GCF appear to be good prognostic markers for periodontitis regardless of diabetes status.