First Cultivation of Health-Associated Tannerella sp. HOT-286 (BU063).

Despite significant advances in recent years in culture-independent molecular microbiology methods, the detailed study of individual bacterial species still relies on having pure cultures in the laboratory. Yet, more than a third of the approximately 700 bacterial taxa found in the human oral cavity are as yet uncultivated in vitro. One such taxon, Tannerella sp. HOT-286 (phylotype BU063), is the focus of much interest since it is associated with periodontal health, while Tannerella forsythia, its closest phylogenetic neighbor, is strongly associated with periodontal disease. HOT-286, however, has remained uncultivated despite the efforts of several research groups, spanning over a decade. The aim of this study was to cultivate Tannerella sp. HOT-286. A heavily diluted sample of subgingival plaque was inoculated onto culture plates supplemented with siderophores (pyoverdines-Fe complex or desferricoprogen) or a neat plaque suspension. After 8 d of anaerobic incubation, microcolonies and colonies showing satellitism were passaged onto fresh culture plates cross-streaked with potential helper strains or onto cellulose-acetate membranes placed over lawn cultures of helper strains. Subcultured colonies were identified by 16S rRNA gene sequencing, and purity was confirmed by sequencing 20 clones per library prepared from a single colony. Three colonies of interest (derived from pyoverdines- and plaque-supplemented plates) were identified as Tannerella sp. HOT-286. The isolates were found to be incapable of independent growth, requiring helpers such as Propionibacterium acnes and Prevotella intermedia for stimulation, with best growth on membranes over "helper" lawns. A representative isolate was subjected to phenotypic characterization and found to produce a range of glycosidic and proteolytic enzymes. Further comparison of this novel "periodontal health-associated" taxon with T. forsythia will be valuable in investigating virulence factors of the latter and possible health benefits of the former.

Differential expression of suppressor of cytokine signalling proteins in Behçet's disease.

Behçet's disease (BD) is a chronic, multisystemic, recurrent vasculitis disease of unknown aetiology. Proinflammatory cytokines are a key feature of the disease, but the triggers for their induction are not well understood and/or controversial. Suppressor of cytokine signalling (SOCS) proteins which negatively regulate the JAK-STAT signalling pathway of cytokine induction may be dysregulated in BD. The expression of SOCS1 and 3 mRNA and protein was studied in peripheral blood mononuclear cells (PBMCs) and neutrophils of patients with BD and compared with healthy controls (HCs) and patients with recurrent aphthous stomatitis (RAS) using RT-PCR, Western blot and immunohistochemistry. SOCS1 and 3 mRNA was also measured in buccal mucosal cells (BMC) of patients with BD and HCs. SOCS1 and 3 mRNA was significantly upregulated in PBMCs of patients with BD compared with HCs (P = 0.0149; P = 0.0007). In addition, there were subtle differences between expression in active and symptom-free BD (quiescent BD). SOCS1 and SOCS 3 were also significantly upregulated in BMC from oral ulcers of BD compared with HCs (both at P = 0.0001). A differential expression of both SOCS1 and 3 was observed between PBMCs and neutrophils in patients with BD. Immunohistochemical analysis revealed differential expression of SOCS proteins in the buccal mucosa with an increased expression at the ulcer surface of ulcers than in the non-ulcerated tissue. These observations suggest a dysregulation of the expression of these important regulators not only between patients with BD and healthy controls but also between mucosal and systemic tissues, which may reflect the nature of the aetiopathology of the disease.

Dysregulation of the suppressor of cytokine signalling 3-signal transducer and activator of transcription-3 pathway in the aetiopathogenesis of Sjögren's syndrome.

The suppressor of cytokine signalling 3 (SOCS3) negatively regulates the Janus kinase (JAK)/signal transducer and activator of transcription-3 (STAT-3)/interleukin (IL)-17 pathway. The proinflammatory cytokine IL-17 is over-expressed in Sjögren's syndrome (SS) and is a key factor in its pathogenesis. We hypothesized that IL-17 over-expression in SS results from ineffective regulation by SOCS3. The expression of SOCS3 was analysed in peripheral blood mononuclear cells (PBMC) from SS cases, sicca controls (SC) and healthy controls (HC) and tissue samples from SS, SC and healthy salivary glands (HSG). PBMC and salivary gland tissue from SS and controls were dual-immunostained for SOCS3 and IL-17. IL-6-stimulated PBMC from SS and controls were evaluated for time-dependent STAT-3 activation and SOCS3 induction, and for IL-17 expression. Immunoblotting revealed greater levels of SOCS3 in PBMC from SS than SC (P = 0·017) or HC (P < 0·001). Similarly, the proportion of salivary-gland tissue cells staining for SOCS3 was significantly higher in SS than SC (P = 0·029) or HSG (P = 0·021). The cells in PBMC/salivary gland samples from controls predominantly expressed either SOCS3 or IL-17. However, there was a high frequency of SOCS3/IL-17 co-expression within cells of SS samples. IL-6-stimulation of PBMC from SS cases revealed prolonged activation of STAT-3 with reduced negative regulation by SOCS3, and enhanced expression of IL-17. This study showed that SOCS3 expression is up-regulated in SS. However, the absence in SS of the normal inverse relationship between SOCS3 and pSTAT-3/IL-17 indicates a functional disturbance in this signalling cascade. Consequently, a reduction in function, rather than a reduction in expression of SOCS3 accounts for the unregulated expression of IL-17 in SS, and may play a crucial role in aetiopathogenesis.

Cultivation of a Synergistetes strain representing a previously uncultivated lineage.

Subgingival plaque samples obtained from human subjects with periodontitis, shown to include previously uncultivable members of the phylum Synergistetes, were used to inoculate Cooked Meat Medium (CMM). The presence of Cluster A (uncultivable) Synergistetes was monitored by fluorescent in situ hybridization (FISH) and quantitative PCR (Q-PCR). Cluster A Synergistetes were found to grow in CMM in co-culture with other plaque bacteria and growth was stimulated by the addition of mucin and serum. Plaque samples were also used to inoculate Blood Agar (BA) plates and growth of Cluster A Synergistetes was revealed after anaerobic incubation, by colony hybridization with specific probes. Surface growth on the plates in regions identified by colony hybridization was harvested and used to inoculate fresh plates, thus enriching for Cluster A Synergistetes. Cross-streaks of other plaque bacteria were also used to stimulate Synergistetes growth. In the early passages, no discrete Synergistetes colonies were seen, but after eight passages and the use of cross-streaks of other bacteria present in the enriched community, colonies arose, which consisted solely of Cluster A Synergistetes cells, as determined by 16S rRNA gene PCR and cloning. This is the first report of the successful culture of a member of the uncultivable branch of this phylum.

Diversity and morphology of members of the phylum "synergistetes" in periodontal health and disease.

Members of the phylum "Synergistetes" have frequently been detected in the human oral cavity at sites of dental disease, but they have rarely been detected in studies of oral health. Only two oral "Synergistetes" taxa are cultivable. The aims of this study were to investigate the diversity of "Synergistetes" in the oral cavity, to establish whether "Synergistetes" taxa are more strongly associated with periodontitis than with oral health, and to visualize unculturable "Synergistetes" in situ. Sixty samples (saliva, dental plaque, and mucosal swabs) were collected from five subjects with periodontitis and five periodontally healthy controls. Using phylum-specific 16S rRNA gene primers, "Synergistetes" were identified by PCR, cloning, and sequencing of 48 clones per PCR-positive sample. Subgingival plaque samples were labeled with probes targeting rRNA of unculturable oral "Synergistetes" using fluorescent in situ hybridization (FISH). Analysis of 1,664 clones revealed 12 "Synergistetes" operational taxonomic units (OTUs) at the 99% sequence identity level, 5 of which were novel. "Synergistetes" OTU 4.2 was found in significantly more subjects with periodontitis than controls (P = 0.048) and was more abundant in subgingival plaque at diseased sites than at healthy sites in subjects with periodontitis (P = 0.019) or controls (P = 0.019). FISH analysis revealed that unculturable oral "Synergistetes" cells were large curved bacilli. The human oral cavity harbors a diverse population of "Synergistetes." "Synergistetes" OTU 4.2 is associated with periodontitis and may have a pathogenic role.

Evaluation of a new periodontal probe tip design. A clinical and in vitro study.

AIMS: In the search for an accurate periodontal probe which does not frequently penetrate the pocket base, a new tip has been designed which is flattened, and of 1 mm width and 0.45 mm thickness. This study aimed to evaluate the physico-mechanical and clinical properties of this probe (test) in comparison to a conventional 0.5 mm circular probe (control). METHODS: Photoelastic stress analysis was undertaken for test and control probe tips at 3.15 and 5 N loads. To assess probing validity, the clinical probing depth with each probe (0.25 N force) at 125 sites on 27 teeth (27 subjects), was compared with the post-extraction connective tissue level measurement. Also evaluated were probing reproducibility (1200 sites in 25 subjects) and patient comfort (30 subjects). RESULTS: Using photoelastic stress analysis, the test probe demonstrated lower stresses and less local stress concentration than the control. Clinically, the test probe measured close to the post-extraction gold standard in greater frequency than the control - 26 versus 11 readings (21% versus 9%) exactly matched, and 90 versus 67 (72% versus 54%) were within +/-0.5 mm of the laboratory measurement. The test probe was, on average, 0.13 mm coronal to the connective tissue attachment level, whereas the control penetrated 0.27 mm past this level. The intraclass correlation between clinical and laboratory readings was greater for the test than the control (r=0.81 and 0.74, respectively). Although the control probe overestimated probing depth more markedly at bleeding (0.41 mm) than at non-bleeding (0.15 mm) sites, the relative position of the test probe hardly differed with inflammatory status (-0.11 and -0.14 mm, respectively). Each probe demonstrated good clinical reproducibility. However, the test probe examination was more comfortable for the patient. CONCLUSION: This new periodontal probe tip appears to have greater validity, good reproducibility and produces less patient discomfort.