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The interplay among different cells in a tissue is essential for maintaining homeostasis. Although disease states have been traditionally attributed to individual cell types, increasing evidence and new therapeutic options have demonstrated the primary role of multicellular functions to understand health and disease, opening new avenues to understand pathogenesis and develop new treatment strategies. We recently described the cellular composition and dynamics of the human oral mucosa; however, the spatial arrangement of cells is needed to better understand a morphologically complex tissue. Here, we link single-cell RNA sequencing, spatial transcriptomics, and high-resolution multiplex fluorescence in situ hybridisation to characterise human oral mucosa in health and oral chronic inflammatory disease. We deconvolved expression for resolution enhancement of spatial transcriptomic data and defined highly specialised epithelial and stromal compartments describing location-specific immune programs. Furthermore, we spatially mapped a rare pathogenic fibroblast population localised in a highly immunogenic region, responsible for lymphocyte recruitment through CXCL8 and CXCL10 and with a possible role in pathological angiogenesis through ALOX5AP. Collectively, our study provides a comprehensive reference for the study of oral chronic disease pathogenesis.

The interplay among different cells in a tissue is essential for maintaining homeostasis. Although, disease states have been traditionally attributed to individual cell types, increasing evidence and new therapeutic options have demonstrated the primary role of multicellular functions to understand health and disease, opening new avenues to understand pathogenesis and develop new treatment strategies. We recently described the cellular composition and dynamics of the human oral mucosa; however, the spatial arrangement of cells is needed to better understand a morphologically complex tissue. Here, we link single-cell RNA sequencing, spatial transcriptomics, and high-resolution multiplex fluorescence in situ hybridisation to characterise human oral mucosa in health and oral chronic inflammatory disease. We deconvolved expression for resolution enhancement of spatial transcriptomic data and defined highly specialised epithelial and stromal compartments describing location-specific immune programs. Furthermore, we spatially mapped a rare pathogenic fibroblast population localised in a highly immunogenic region, responsible for lymphocyte recruitment through CXCL8 and CXCL10 and with a possible role in pathological angiogenesis through ALOX5AP. Collectively, our study provides a comprehensive reference for the study of oral chronic disease pathogenesis.

Human oral soft tissues provide the first barrier of defence against chronic inflammatory disease and hold a remarkable scarless wounding phenotype. Tissue homeostasis requires coordinated actions of epithelial, mesenchymal and immune cells. However, the extent of heterogeneity within the human oral mucosa and how tissue cell types are affected during the course of disease progression is unknown. Using single cell transcriptome profiling we reveal a striking remodelling of the epithelial and mesenchymal niches with a decrease in functional populations that are linked to the aetiology of the disease. Analysis of ligand-receptor interaction pairs identify potential intercellular hubs driving the inflammatory component of the disease. Our work establishes a reference map of the human oral mucosa in health and disease, and a framework for the development of new therapeutic strategies.

Introduction Tobacco smoking is one of the most important risk factors for periodontitis as it alters the host response to plaque. Although the prevalence of tobacco smoking has declined in recent years, the use of electronic-cigarettes (vaping) has increased. The effect of vaping on the gingiva is unknown and an evidence-base needs to be established before providing dental advice about the use of these products.Objective To compare the gingival health of a group of established smokers before and after substituting vaping for smoking tobacco.Design Pilot.Setting Guy's Dental Hospital (England) from April-December 2015.Materials and methods Twenty established smokers (all staff members at Guy's Hospital) with mild periodontal disease replaced their regular smoking habits with the use of e-cigarettes for two weeks.Main outcome measure The primary outcome measure of gingival inflammation was bleeding on probing. Levels of selected pro-inflammatory cytokines in GCF, saliva and serum samples were also determined.Results and conclusions There was a statistically significant increase in gingival inflammation when tobacco smokers switched from smoking to vaping for two weeks. However, this result must be interpreted with extreme caution since this is only a pilot study. Nonetheless, this study should provide a stepping stone to encourage further investigation of the effects of vaping on periodontal health.

A new exhaust emission inventory of ocean-going vessels (OGVs) was compiled for Hong Kong by using Automatic Identification System (AIS) data for the first time to determine typical main engine load factors, through vessel speed and operation mode characterization. It was found that in 2007, container vessel was the top emitting vessel type, contributing 9,886, 11,480, 1,173, 521 and 1166 tonnes of SO2, NOx, PM10, VOC and CO, respectively, or about 80%–82% of the emissions. The top five, which also included ocean cruise, oil tanker, conventional cargo vessel and dry bulk carrier, accounted for about 98% of emissions. Emission maps, which add a new spatial dimension to the inventory, show the key emission hot spots in Hong Kong and suggest that a significant portion of emissions were emitted at berth. Scientific evidence about the scale and distribution of ship emissions has contributed in raising public awareness and facilitating stakeholder engagement about the issue. Fair Winds Charter, the world's first industry-led voluntary emissions reduction initiative, is a perfect example of how careful scientific research can be used in public engagement and policy deliberation to help drive voluntary industry actions and then government proposals to control and regulate marine emissions in Hong Kong and the Pearl River Delta region.

Three strains of anaerobic Gram-negative bacilli isolated from human oral sites were subjected to a comprehensive range of phenotypic and genotypic tests and were found to comprise a homogeneous group. 16S rRNA gene sequence analysis revealed the strains to constitute a novel group within the genus Prevotella, most closely related to Prevotella oris and Prevotella salivae. A novel species, Prevotella maculosa sp. nov., is proposed to accommodate these strains. Prevotella maculosa is saccharolytic and produces acetic and succinic acids as end products of fermentation. The G+C content of the DNA of the type strain is 48 mol%. The type strain of Prevotella maculosa is W1609T (=DSM 19339T =CCUG 54766T).

Background and Objective: Elastase is carried into the oral cavity by gingival crevicular fluid (GCF) from periodontal lesions. Our study investigated the regulation of elastase activity by secretory leukocyte protease inhibitor (SLPI) and the possible action of another GCF protease on this protective salivary component. Material and Methods: Whole-mouth saliva (WMS), parotid saliva (PS) and GCF were obtained from 19 patients with periodontitis. The concentrations of active elastase and cathepsin B were determined using peptide substrates. SLPI and α1-proteinase inhibitor (α1PI) concentrations were determined using enzyme-linked immunosorbent assays (ELISAs). The molecular forms of SLPI were examined by immunoblotting. Results: The molar concentrations of elastase, cathepsin B and α1PI were higher in GCF than in WMS and especially PS (p

Metagenomic techniques are used to analyse bacterial communities allowing both culturable and unculturable species to be represented. However, the screening of oral metagenomic samples can be hindered by high animal host DNA content. This study evaluated methods for the reduction of human DNA concentrations within oral metagenomic samples. Plaque samples were collected from 27 patients presenting with periodontal disease and treated to remove human DNA using either selective lysis of eukaryotic cells at several buffer concentrations or differential centrifugation after treatment with trypsin and/or detergents. Human and bacterial DNA levels were determined by quantitative polymerase chain reaction (qPCR). The human DNA content of plaque extracts was significantly reduced by all treatments compared with an untreated control (P < 0.05). However, differential centrifugation simultaneously reduced the bacterial DNA content unless samples were pretreated with a detergent. Observations of Gram stained samples that were processed using differential centrifugation without detergent suggest that many bacteria remain adhered to human cells. An approach that uses differential centrifugation in parallel with selective lysis is recommended to fully represent the oral microbiota in metagenomic samples, including those tightly adhered to human cells and more delicate bacteria such as Mycoplasma.

Background/aims: Protein and carbohydrate antigens of Porphyromonas gingivalis interact with the host to produce antibody of different subclasses. IgG1 and IgG2 antibodies frequently account for approximately 90% of the total serum IgG. This work aimed to investigate serum IgG1 and IgG2 antibody responses of periodontitis patients to protein and carbohydrate-rich antigens of P. gingivalis. Methods: Sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blots of P. gingivalis antigens and proteinase K digested antigens rich in carbohydrates were used to investigate the molecular weight of antigen recognised by serum IgG1 and IgG2. Enzyme-linked immunosorbent assay was used to measure levels of IgG1 and IgG2 antibody to P. gingivalis and radial immunodiffusion was used to estimate the total concentration of IgG1 and IgG2 in serum. Results: Serum IgG antibodies bound to antigens of molecular weights 47, 39 and 32 kDa. Antigen most frequently recognised by both IgG1 and IgG2 antibody had a molecular weight of 47 kDa. Serum IgG2 antibody bound to carbohydrate antigen with a molecular weight of 32 kDa but there was no recognition of carbohydrate antigens by IgG1 antibodies. There was no correlation between the titre of anti-P. gingivalis IgG1 or IgG2 antibody and the total concentration of serum IgG1 or IgG2 antibodies of all specificities. Conclusion: Both IgG1 and IgG2 antibodies recognised a dominant antigen of 47 kDa, probably Arg-gingipain. Much of the response to carbohydrate antigen is of the IgG2 subclass. Neither the level of IgG1 nor the IgG2 antibody specific to P. gingivalis was related to the total serum concentration of that antibody.

Objective: The uncertain taxonomy of oral anaerobic gram-positive bacilli and their generally slow growing nature has limited the understanding of their role in periodontal disease. The current objective was to design and use species-specific oligonucleotide probes to investigate the relationship of selected gram-positive anaerobic bacilli to periodontal disease. Methods: Plaque and clinical measurements were collected from 40 patients with periodontitis and from 40 matched controls. Oligonucleotide probes were designed for Bulleidia extructa, Eubacterium nodatum, Mogibacterium timidum and Slackia exigua and used to probe nucleic acids extracted from the samples with a chemiluminescent detection method. Species were quantified as absent or present at low (approximately 103−104 cells), medium (approximately 104−105 cells) or high levels (approximately 105−106 cells). Results: M. timidum and B. extructa were detected in only three and four samples, respectively. The level of both E. nodatum and S. exigua was significantly higher in deep than shallow pockets (Wilcoxon, p

Vascular endothelial growth factor (VEGF) is a multifunctional angiogenic cytokine of importance in inflammation and wound healing but its presence in chronic inflammatory periodontal disease has never been reported. The aims of this study were to investigate the presence of VEGF in human periodontal tissue and gingival crevicular fluid (GCF) in periodontal health and disease. VEGF in tissue was localized by immunohistochemistry. GCF and unstimulated saliva were collected from patients and clinically healthy subjects and VEGF was assessed by using an ELISA. VEGF was detected within vascular endothelial cells, neutrophils, plasma cells and junctional, pocket and gingival epithelium. In periodontitis patients, the volume of GCF and total amount of VEGF collected from diseased sites were both greater than from clinically healthy sites (Wilcoxon test p < 0.01). However, the concentration of VEGF per unit volume of GCF was higher at healthy sites compared with diseased sites (Wilcoxon test p < 0.05). Higher concentrations of VEGF were detected in healthy sites in patients compared with similar sites in clinically healthy subjects (Mann-Whitney U-test p < 0.05). A logistic regression approach indicated that there was variation in VEGF between subjects (p < 0.01), and that age (p < 0.05), plaque (p < 0.05) and pocket depth (p < 0.07) were explanatory variables. VEGF was also detected in all saliva samples and was significantly higher in patients than in healthy controls (p < 0.05). This study suggests that VEGF could be relevant to angiogenic processes in healthy as well as diseased periodontal tissue and that the periodontal status influences the salivary level of VEGF.