In Vivo Regulation of Active Matrix Metalloproteinase-8 (aMMP-8) in Periodontitis: From Transcriptomics to Real-Time Online Diagnostics and Treatment Monitoring.

BACKGROUND: This study investigated in vivo regulation and levels of active matrix metalloproteinase-8 (aMMP-8), a major collagenolytic protease, in periodontitis. METHODS: Twenty-seven adults with chronic periodontitis (CP) and 30 periodontally healthy controls (HC) were enrolled in immunohistochemistry and transcriptomics analytics in order to assess Treponema denticola (Td) dentilisin and MMP-8 immunoexpression, mRNA expression of MMP-8 and its regulators (IL-1ß, MMP-2, MMP-7, TIMP-1). Furthermore, the periodontal anti-infective treatment effect was monitored by four different MMP-8 assays (aMMP-8-IFMA, aMMP-8-Oralyzer, MMP-8-activity [RFU/minute], and total MMP-8 by ELISA) among 12 CP (compared to 25 HC). RESULTS: Immunohistochemistry revealed significantly more Td-dentilisin and MMP-8 immunoreactivities in CP vs. HC. Transcriptomics revealed significantly elevated IL-1ß and MMP-7 RNA expressions, and MMP-2 RNA was slightly reduced. No significant differences were recorded in the relatively low or barely detectable levels of MMP-8 mRNAs. Periodontal treatment significantly decreased all MMP-8 assay levels accompanied by the assessed clinical indices (periodontal probing depths, bleeding-on-probing, and visual plaque levels). However, active but not total MMP-8 levels persisted higher in CP than in periodontally healthy controls. CONCLUSION: In periodontal health, there are low aMMP-8 levels. The presence of Td-dentilisin in CP gingivae is associated with elevated aMMP-8 levels, potentially contributing to a higher risk of active periodontal tissue collagenolysis and progression of periodontitis. This can be detected by aMMP-8-specific assays and online/real-time aMMP-8 chair-side testing.

Composition of subgingival microbiota associated with periodontitis and diagnosis of malignancy-a cross-sectional study.

Periodontitis is one of the world's most prevalent infectious conditions, affecting between 25 and 40% of the adult population. It is a consequence of the complex interactions between periodontal pathogens and their products, which trigger the host inflammatory response, chronic inflammation, and tissue destruction. Chronic systemic low-grade inflammation is involved in numerous diseases, and it is also known that long-lasting inflammation and chronic infections predispose one to cancer. Here, we characterized and compared the subgingival microbiota associated with periodontitis and diagnosis of malignancy in a longitudinal 10-year follow-up study. The study was conducted on 50 patients with periodontitis and 40 periodontally healthy individuals. The recorded clinical oral health parameters were periodontal attachment loss (AL), bleeding on probing (BOP), gingival index (GI), probing depth (PD), and plaque index (PI). Subgingival plaque was collected from each participant, from which DNA was extracted, and 16S rRNA gene amplicon sequencing performed. Cancer diagnoses data were collected between the years 2008-2018 from the Swedish Cancer Registry. The participants were categorized based on having cancer at the time of sample collection (CSC), having developed cancer later (DCL), and controls without any cancer. The most abundant phyla across all 90 samples were Actinobacteria, Proteobacteria, Firmicutes, Bacteroidetes, and Fusobacteria. At the genus level, Treponema, Fretibacterium, and Prevotella were significantly more abundant in samples of periodontitis patients compared to non-periodontitis individuals. With regard to samples of cancer patients, Corynebacterium and Streptococcus were more abundant in the CSC group; Prevotella were more abundant in the DCL group; and Rothia, Neisseria, and Capnocytophaga were more abundant in the control group. In the CSC group, we also found that the presence of periodontal inflammation, in terms of BOP, GI, and PLI, significantly correlated with species belonging to the genera Prevotella, Treponema, and Mycoplasma. Our results revealed that several subgingival genera were differentially enriched among the studied groups. These findings underscore the need for further research to fully understand the role that oral pathogens may play in the development of cancer.

Salivary Microbiota and Host-Inflammatory Responses in Periodontitis Affected Individuals With and Without Rheumatoid Arthritis.

Objectives: Periodontitis and rheumatoid arthritis (RA) are two widespread chronic inflammatory diseases with a previously suggested association. The objective of the current study was to compare the oral microbial composition and host´s inflammatory mediator profile of saliva samples obtained from subjects with periodontitis, with and without RA, as well as to predict biomarkers, of bacterial pathogens and/or inflammatory mediators, for classification of samples associated with periodontitis and RA. Methods: Salivary samples were obtained from 53 patients with periodontitis and RA and 48 non-RA with chronic periodontitis. The microbial composition was identified using 16S rRNA gene sequencing and compared across periodontitis patients with and without RA. Levels of inflammatory mediators were determined using a multiplex bead assay, compared between the groups and correlated to the microbial profile. The achieved data was analysed using PCoA, DESeq2 and two machine learning algorithms, OPLS-DA and sPLS-DA. Results: Differential abundance DESeq2 analyses showed that the four most highly enriched (log2 FC >20) amplicon sequence variants (ASVs) in the non-RA periodontitis group included Alloprevotella sp., Prevotella sp., Haemophilus sp., and Actinomyces sp. whereas Granulicatella sp., Veillonella sp., Megasphaera sp., and Fusobacterium nucleatum were the most highly enriched ASVs (log2 FC >20) in the RA group. OPLS-DA with log2 FC analyses demonstrated that the top ASVs with the highest importance included Vampirovibrio sp. having a positive correlation with non-RA group, and seven ASVs belonging to Sphingomonas insulae, Sphingobium sp., Novosphingobium aromaticivorans, Delftia acidovorans, Aquabacterium spp. and Sphingomonas echinoides with a positive correlation with RA group. Among the detected inflammatory mediators in saliva samples, TWEAK/TNFSF12, IL-35, IFN-α2, pentraxin-3, gp130/sIL6Rb, sIL-6Ra, IL-19 and sTNF-R1 were found to be significantly increased in patients with periodontitis and RA compared to non-RA group with periodontitis. Moreover, correlations between ASVs and inflammatory mediators using sPLS-DA analysis revealed that TWEAK/TNFSF12, pentraxin-3 and IL-19 were positively correlated with the ASVs Sphingobium sp., Acidovorax delafieldii, Novosphingobium sp., and Aquabacterium sp. Conclusion: Our results suggest that the combination of microbes and host inflammatory mediators could be more efficient to be used as a predictable biomarker associated with periodontitis and RA, as compared to microbes and inflammatory mediators alone.

Intra-individual cytokine profile in peri-implantitis and periodontitis: A cross-sectional study.

AIM: To study cytokine profiles and intra-individual correlations in crevicular fluid samples at periodontitis, peri-implantitis, and healthy sites. MATERIALS AND METHODS: Samples from gingival crevicular fluid (GCF) and peri-implant crevicular fluid (PICF) were collected from healthy and diseased sites in patients who had had dental implants for a minimum of 10 years. Cytokine levels were analyzed using the Bio-Plex Pro Human inflammation kit, which included biomarkers for the tumor necrosis factor-α (TNF-α) superfamily, regulatory T Cell (Treg) cytokines, and interferon (IFN) proteins. RESULTS: Gingival crevicular fluid/PICF cytokine levels, determined in samples from 163 patients, were frequently lower for healthy tooth and implant sites compared to sites with periodontitis or peri-implantitis. In contrast, there were no significant differences in cytokine levels between peri-implant sites and periodontitis sites. Intra-individual correlations between cytokines at peri-implant sites were frequently significant. In addition, the cytokines IFN-λ1 and TNFSF12 were significantly correlated with the presence of peri-implantitis. CONCLUSION: Within the limits of this study, the intra-individual cytokine profile did not differ between sites diagnosed with periodontitis and those diagnosed with peri-implantitis, but did differ between healthy tooth and healthy implant sites. Studying intra-individual cytokine profiles is a method to elucidate possible differences between the etiopathogeneses of periodontitis and peri-implantitis, since it is well known that immune responses to dysbiosis vary between individuals according to host factors. Thus, the findings of the present study are potentially relevant to the advancement of knowledge in this field.

Pleckstrin Levels Are Increased in Patients with Chronic Periodontitis and Regulated via the MAP Kinase-p38α Signaling Pathway in Gingival Fibroblasts.

Chronic periodontitis (CP) is a bacteria-driven inflammatory disease characterized by the breakdown of gingival tissue, the periodontal ligament, and alveolar bone, leading ultimately to tooth loss. We previously reported the pleckstrin gene (PLEK) to be highly upregulated in gingival tissue of patients with CP and the only gene concurrently upregulated in other inflammatory diseases including rheumatoid arthritis and cardiovascular diseases. Using saliva from 169 individuals diagnosed with CP and healthy controls, we investigated whether pleckstrin could serve as a novel biomarker of periodontitis. Additionally, we explored signal pathways involved in the regulation of PLEK using human gingival fibroblasts (HGFs). Pleckstrin levels were significantly higher (p < 0.001) in the saliva samples of patients with CP compared to controls and closely associated with CP severity. Immunohistochemical analysis revealed the expression of pleckstrin in inflammatory cells and gingival fibroblasts of CP patients. To explore the signal pathways involved in pleckstrin regulation, we stimulated HGFs with either interleukin-1ß (IL-1ß) or lipopolysaccharides (LPS) alone, or in combination with inhibitors targeting c-Jun N-terminal kinase, tyrosine kinase, protein kinase C, or p38 MAP kinase. Results showed that IL-1ß and LPS significantly increased PLEK mRNA and pleckstrin protein levels. VX-745, the p38 MAP kinase inhibitor significantly decreased IL-1ß- and LPS-induced pleckstrin levels at both the mRNA and the protein level. Together, these findings show that pleckstrin could serve as a salivary biomarker for the chronic inflammatory disease periodontitis and a regulator of inflammation via the p38 MAP kinase pathway.

Improved methodology to determine the fate and transport of microplastics in a secondary wastewater treatment plant.

Wastewater treatment plants are a significant pathway of microplastics (MPs) to aquatic environments. To develop suitable management options and reduce microplastic emissions in treated effluent and sludge, we must first develop a reliable method to understand their transport and fate throughout the treatment process. An improved methodology was applied to determine the size, shape, polymer type and partitioning behaviour of MPs using a combination of oxidation treatment, fluorescent staining and Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy (ATR-FTIR) to detect small MPs ≤20 µm in wastewater. The mean number of MPs (<5 mm) detected using this methodology was 11.80 ± 1.10 MP/L in raw sewage, 5.23 ± 0.25 MP/L in degritted wastewater, 7.91 ± 0.44 MP/L in waste activated sludge and 2.76 ± 0.11 MP/L in the final treated effluent. An increase in MPs following primary screening suggests that WAS is a sink for several stealth microplastics, including glitter, which is returned to the effluent during the treatment process. The estimated MP removal efficiency for the studied WWTP is relatively poor compared with global data for secondary WWTPs, which is likely due to the release of partially treated effluent into the Pacific Ocean via a second discharge point. The information generated through this study can be useful to; firstly, inform on-site wastewater management practices, and secondly, reduce MP concentrations in final treated effluents discharged to the marine environment.

Identification of Salivary Microbiota and Its Association With Host Inflammatory Mediators in Periodontitis.

Periodontitis is a microbial-induced chronic inflammatory disease, which may not only result in tooth loss, but can also contribute to the development of various systemic diseases. The transition from healthy to diseased periodontium depends on microbial dysbiosis and impaired host immune response. Although periodontitis is a common disease as well as associated with various systemic inflammatory conditions, the taxonomic profiling of the salivary microbiota in periodontitis and its association with host immune and inflammatory mediators has not been reported. Therefore, the aim of this study was to identify key pathogens and their potential interaction with the host's inflammatory mediators in saliva samples for periodontitis risk assessment. The microbial 16S rRNA gene sequencing and the levels of inflammatory mediators were performed in saliva samples from patients with chronic periodontitis and periodontally healthy control subjects. The salivary microbial community composition differed significantly between patients with chronic periodontitis and healthy controls. Our analyses identified a number of microbes, including bacteria assigned to Eubacterium saphenum, Tannerella forsythia, Filifactor alocis, Streptococcus mitis/parasanguinis, Parvimonas micra, Prevotella sp., Phocaeicola sp., and Fretibacterium sp. as more abundant in periodontitis, compared to healthy controls. In samples from healthy individuals, we identified Campylobacter concisus, and Veillonella sp. as more abundant. Integrative analysis of the microbiota and inflammatory mediators/cytokines revealed associations that included positive correlations between the pathogens Treponema sp. and Selenomas sp. and the cytokines chitinase 3-like 1, sIL-6Rα, sTNF-R1, and gp130/sIL-6Rß. In addition, a negative correlation was identified between IL-10 and Filifactor alocis. Our results reveal distinct and disease-specific patterns of salivary microbial composition between patients with periodontitis and healthy controls, as well as significant correlations between microbiota and host-mediated inflammatory cytokines. The positive correlations between the pathogens Treponema sp. and Selenomas sp. and the cytokines chitinase 3-like 1, sIL-6Rα, sTNF-R1, and gp130/sIL-6Rß might have the future potential to serve as a combined bacteria-host salivary biomarker panel for diagnosis of the chronic infectious disease periodontitis. However, further studies are required to determine the capacity of these microbes and inflammatory mediators as a salivary biomarker panel for periodontitis.

Periodontal Health and Oral Microbiota in Patients with Rheumatoid Arthritis.

This study aimed to investigate the periodontal health of patients with established rheumatoid arthritis (RA) in relation to oral microbiota, systemic and oral inflammatory mediators, and RA disease activity. Forty patients underwent full-mouth dental/periodontal and rheumatological examination, including collection of blood, saliva, gingival crevicular fluid (GCF) and subgingival plaque. Composition of plaque and saliva microbiota were analysed using 16S rRNA sequencing and levels of inflammatory mediators by multiplex-immunoassay. The majority of the patients (75%) had moderate or severe periodontitis and the rest had no/mild periodontitis. Anti-citrullinated protein antibody (ACPA) positivity was significantly more frequent in the moderate/severe periodontitis (86%) compared to the no/mild group (50%). No significance between groups was observed for RA disease duration or activity, or type of medication. Levels of sCD30/TNFRSF8, IFN-α2, IL-19, IL-26, MMP-1, gp130/sIL-6Rß, and sTNF-R1 were significantly higher in serum or GCF, and April/TNFSF13 was significantly higher in serum and saliva samples in moderate/severe periodontitis. The microbial composition in plaque also differed significantly between the two groups. In conclusion, the majority of RA patients had moderate/severe periodontitis and that this severe form of the disease was significantly associated with ACPA positivity, an altered subgingival microbial profile, and increased levels of systemic and oral inflammatory mediators.

Gene expression profiling of periodontitis-affected gingival tissue by spatial transcriptomics.

Periodontitis is a highly prevalent chronic inflammatory disease of the periodontium, leading ultimately to tooth loss. In order to characterize the gene expression of periodontitis-affected gingival tissue, we have here simultaneously quantified and localized gene expression in periodontal tissue using spatial transcriptomics, combining RNA sequencing with histological analysis. Our analyses revealed distinct clusters of gene expression, which were identified to correspond to epithelium, inflamed areas of connective tissue, and non-inflamed areas of connective tissue. Moreover, 92 genes were identified as significantly up-regulated in inflamed areas of the gingival connective tissue compared to non-inflamed tissue. Among these, immunoglobulin lambda-like polypeptide 5 (IGLL5), signal sequence receptor subunit 4 (SSR4), marginal zone B and B1 cell specific protein (MZB1), and X-box binding protein 1 (XBP1) were the four most highly up-regulated genes. These genes were also verified as significantly higher expressed in gingival tissue of patients with periodontitis compared to healthy controls, using reverse transcription quantitative polymerase chain reaction. Moreover, the protein expressions of up-regulated genes were verified in gingival biopsies by immunohistochemistry. In summary, in this study, we report distinct gene expression signatures within periodontitis-affected gingival tissue, as well as specific genes that are up-regulated in inflamed areas compared to non-inflamed areas of gingival tissue. The results obtained from this study may add novel information on the genes and cell types contributing to pathogenesis of the chronic inflammatory disease periodontitis.

Mucin 4 and matrix metalloproteinase 7 as novel salivary biomarkers for periodontitis.

AIM: Periodontitis is a chronic inflammatory disease, characterized by irreversible destruction of tooth-supporting tissue including alveolar bone. We recently reported mucin 4 (MUC4) and matrix metalloproteinase 7 (MMP7) as highly associated with periodontitis in gingival tissue biopsies. The aim of this study was to further investigate the levels of MUC4 and MMP7 in saliva and gingival crevicular fluid (GCF) samples of patients with periodontitis. MATERIALS AND METHODS: Saliva and GCF samples were collected from periodontitis patients and healthy controls. The levels of MUC4, MMP7, and total protein concentrations were analysed using ELISA or Bradford assay. RESULTS: MUC4 levels were significantly lower in saliva and GCF from periodontitis patients relative to healthy controls. MMP7 levels were significantly higher in saliva and GCF from periodontitis patients. Multivariate analysis revealed that MUC4 was significantly associated with periodontitis after adjusting for age and smoking habits and, moreover, that the combination of MUC4 and MMP7 accurately discriminated periodontitis from healthy controls. CONCLUSIONS: MUC4 and MMP7 may be utilized as possible novel biomarkers for periodontitis.

An automated approach to prepare tissue-derived spatially barcoded RNA-sequencing libraries.

Sequencing the nucleic acid content of individual cells or specific biological samples is becoming increasingly common. This drives the need for robust, scalable and automated library preparation protocols. Furthermore, an increased understanding of tissue heterogeneity has lead to the development of several unique sequencing protocols that aim to retain or infer spatial context. In this study, a protocol for retaining spatial information of transcripts has been adapted to run on a robotic workstation. The method spatial transcriptomics is evaluated in terms of robustness and variability through the preparation of reference RNA, as well as through preparation and sequencing of six replicate sections of a gingival tissue biopsy from a patient with periodontitis. The results are reduced technical variability between replicates and a higher throughput, processing four times more samples with less than a third of the hands on time, compared to the standard protocol.

Effects by periodontitis on pristane-induced arthritis in rats.

BACKGROUND: An infection-immune association of periodontal disease with rheumatoid arthritis has been suggested. This study aimed to investigate the effect of pre-existing periodontitis on the development and the immune/inflammatory response of pristane-induced arthritis. METHODS: We investigated the effect of periodontitis induced by ligature placement and Porphyromonas gingivalis (P. gingivalis) infection, in combination with Fusobacterium nucleatum to promote its colonization, on the development of pristane-induced arthritis (PIA) in rats (Dark Agouti). Disease progression and severity of periodontitis and arthritis was monitored using clinical assessment, micro-computed tomography (micro-CT)/intraoral radiographs, antibody response, the inflammatory markers such as α-1-acid glycoprotein (α-1-AGP) and c-reactive protein (CRP) as well as cytokine multiplex profiling at different time intervals after induction. RESULTS: Experimentally induced periodontitis manifested clinically (P < 0.05) prior to pristane injection and progressed steadily until the end of experiments (15 weeks), as compared to the non-ligated arthritis group. Injection of pristane 8 weeks after periodontitis-induction led to severe arthritis in all rats demonstrating that the severity of arthritis was not affected by the pre-existence of periodontitis. Endpoint analysis showed that 89% of the periodontitis-affected animals were positive for antibodies against arginine gingipain B and furthermore, the plasma antibody levels to a citrullinated P. gingivalis peptidylarginine deiminase (PPAD) peptide (denoted CPP3) were significantly (P < 0.05) higher in periodontitis rats with PIA. Additionally, there was a trend towards increased pro-inflammatory and anti-inflammatory cytokine levels, and increased α-1-AGP levels in plasma from periodontitis-challenged PIA rats. CONCLUSIONS: Pre-existence of periodontitis induced antibodies against citrullinated peptide derived from PPAD in rats with PIA. However, there were no differences in the development or severity of PIA between periodontitis challenged and periodontitis free rats.

Visualization and analysis of gene expression in tissue sections by spatial transcriptomics.

Analysis of the pattern of proteins or messengerRNAs (mRNAs) in histological tissue sections is a cornerstone in biomedical research and diagnostics. This typically involves the visualization of a few proteins or expressed genes at a time. We have devised a strategy, which we call "spatial transcriptomics," that allows visualization and quantitative analysis of the transcriptome with spatial resolution in individual tissue sections. By positioning histological sections on arrayed reverse transcription primers with unique positional barcodes, we demonstrate high-quality RNA-sequencing data with maintained two-dimensional positional information from the mouse brain and human breast cancer. Spatial transcriptomics provides quantitative gene expression data and visualization of the distribution of mRNAs within tissue sections and enables novel types of bioinformatics analyses, valuable in research and diagnostics.

Transcriptome analysis reveals mucin 4 to be highly associated with periodontitis and identifies pleckstrin as a link to systemic diseases.

The multifactorial chronic inflammatory disease periodontitis, which is characterized by destruction of tooth-supporting tissues, has also been implicated as a risk factor for various systemic diseases. Although periodontitis has been studied extensively, neither disease-specific biomarkers nor therapeutic targets have been identified, nor its link with systemic diseases. Here, we analyzed the global transcriptome of periodontitis and compared its gene expression profile with those of other inflammatory conditions, including cardiovascular disease (CVD), rheumatoid arthritis (RA), and ulcerative colitis (UC). Gingival biopsies from 62 patients with periodontitis and 62 healthy subjects were subjected to RNA sequencing. The up-regulated genes in periodontitis were related to inflammation, wounding and defense response, and apoptosis, whereas down-regulated genes were related to extracellular matrix organization and structural support. The most highly up-regulated gene was mucin 4 (MUC4), and its protein product was confirmed to be over-expressed in periodontitis. When comparing the expression profile of periodontitis with other inflammatory diseases, several gene ontology categories, including inflammatory response, cell death, cell motion, and homeostatic processes, were identified as common to all diseases. Only one gene, pleckstrin (PLEK), was significantly overexpressed in periodontitis, CVD, RA, and UC, implicating this gene as an important networking link between these chronic inflammatory diseases.