Effects of intermittent fasting on periodontal inflammation and subgingival microbiota.

BACKGROUND: Studies on the impact of intermittent fasting on periodontal health are still scarce. Thus, this study evaluated the effects of long-term intermittent fasting on periodontal health and the subgingival microbiota. METHODS: This pilot study was part of a nonrandomized controlled trial. Overweight/obese participants (n = 14) entered an intermittent fasting program, specifically the 5:2 diet, in which they restricted caloric intake to about a quarter of the normal total daily caloric expenditure for two nonconsecutive days/week. Subjects underwent a thorough clinical and laboratory examination, including an assessment of their periodontal condition, at baseline and 6 months after starting the diet. Additionally, subgingival microbiota was assessed by 16S rRNA gene sequencing. RESULTS: After 6 months of intermittent fasting, weight, body mass index, C-reactive protein, hemoglobin A1c (HbA1c), and the cholesterol profile improved significantly (p < 0.05). Moreover, significant reductions were observed in bleeding on probing (p = 0.01) and the presence of shallow periodontal pockets after fasting (p < 0.001), while no significant change was seen in plaque index (p = 0.14). While we did not observe significant changes in α- or ß-diversity of the subgingival microbiota related to dietary intervention (p > 0.05), significant differences were seen in the abundances of several taxa among individuals exhibiting ≥60% reduction (good responders) in probing pocket depth of 4-5 mm compared to those with <60% reduction (bad responders). CONCLUSION: Intermittent fasting decreased systemic and periodontal inflammation. Although the subgingival microbiota was unaltered by this intervention, apparent taxonomic variability was observed between good and bad responders.

Probing the salivary proteome for prognostic biomarkers in response to non-surgical periodontal therapy.

AIM: This prospective study investigated the salivary proteome before and after periodontal therapy. MATERIALS AND METHODS: Ten systemically healthy, non-smoking, stage III, grade C periodontitis patients underwent non-surgical periodontal treatment. Full-mouth periodontal parameters were measured, and saliva (n = 30) collected pre- (T0), and one (T1) and six (T6) months post-treatment. The proteome was investigated by label-free quantitative proteomics. Protein expression changes were modelled over time, with significant protein regulation considered at false discovery rate <0.05. RESULTS: Treatment significantly reduced bleeding scores, percentages of sites with pocket depth ≥5 mm, plaque and gingival indexes. One thousand seven hundred and thirteen proteins were identified and 838 proteins (human = 757, bacterial = 81) quantified (≥2 peptides). At T1, 80 (T1 vs. T0: 60↑:20↓), and at T6, 118 human proteins (T6 vs. T0: 67↑:51↓) were regulated. The salivary proteome at T6 versus T1 remained stable. Highest protein activity post- versus pre-treatment was observed for cellular movement and inflammatory response. The small proline-rich protein 3 (T1 vs. T0: 5.4-fold↑) and lymphocyte-specific protein 1 (T6 vs. T0: 4.6-fold↓) were the top regulated human proteins. Proteins from Neisseria mucosa and Treponema socranskii (T1 vs. T0: 8.0-fold↓, 4.9-fold↓) were down-regulated. CONCLUSIONS: Periodontal treatment reduced clinical disease parameters and these changes were reflected in the salivary proteome. This underscores the potential of utilizing saliva biomarkers as prognostic tools for monitoring treatment outcomes.

Bacterial symbionts in oral niche use type VI secretion nanomachinery for fitness increase against pathobionts.

Microbial ecosystems experience spatial and nutrient restrictions leading to the coevolution of cooperation and competition among cohabiting species. To increase their fitness for survival, bacteria exploit machinery to antagonizing rival species upon close contact. As such, the bacterial type VI secretion system (T6SS) nanomachinery, typically expressed by pathobionts, can transport proteins directly into eukaryotic or prokaryotic cells, consequently killing cohabiting competitors. Here, we demonstrate for the first time that oral symbiont Aggregatibacter aphrophilus possesses a T6SS and can eliminate its close relative oral pathobiont Aggregatibacter actinomycetemcomitans using its T6SS. These findings bring nearer the anti-bacterial prospects of symbionts against cohabiting pathobionts while introducing the presence of an active T6SS in the oral cavity.

Bacteriome and mycobiome dysbiosis in oral mucosal dysplasia and oral cancer.

It has long been considered that the oral microbiome is tightly connected to oral health and that dysbiotic changes can be detrimental to the occurrence and progression of dysplastic oral mucosal lesions or oral cancer. Improved understanding of the concepts of microbial dysbiosis together with advances in high-throughput molecular sequencing of these pathologies have charted in greater microbiological detail the nature of their clinical state. This review discusses the bacteriome and mycobiome associated with oral mucosal lesions, oral candidiasis, and oral squamous cell carcinoma, aiming to delineate the information available to date in pursuit of advancing diagnostic and prognostic utilities for oral medicine.

A Mapping Review of the Pathogenesis of Peri-Implantitis: The Biofilm-Mediated Inflammation and Bone Dysregulation (BIND) Hypothesis.

This mapping review highlights the need for a new paradigm in the understanding of peri-implantitis pathogenesis. The biofilm-mediated inflammation and bone dysregulation (BIND) hypothesis is proposed, focusing on the relationship between biofilm, inflammation, and bone biology. The close interactions between immune and bone cells are discussed, with multiple stable states likely existing between clinically observable definitions of peri-implant health and peri-implantitis. The framework presented aims to explain the transition from health to disease as a staged and incremental process, where multiple factors contribute to distinct steps towards a tipping point where disease is manifested clinically. These steps might be reached in different ways in different patients and may constitute highly individualised paths. Notably, factors affecting the underlying biology are identified in the pathogenesis of peri-implantitis, highlighting that disruptions to the host-microbe homeostasis at the implant-mucosa interface may not be the sole factor. An improved understanding of disease pathogenesis will allow for intervention on multiple levels and a personalised treatment approach. Further research areas are identified, such as the use of novel biomarkers to detect changes in macrophage polarisation and activation status, and bone turnover.

Clinical, oral immunological and microbiological shifts during and after pregnancy.

OBJECTIVES: Physiological changes and shifts in the oral microbiota composition during pregnancy may affect the maternal immune system. Uncomplicated pregnancy is associated with a T-helper (Th) 2 predominant cytokine regulation (anti-inflammatory), while oral health deterioration during pregnancy is reflected by severe gingival inflammation, a primarily Th1 cytokine phenotype (pro-inflammatory), and oral microbiome alterations. This prospective observational study aimed to evaluate Th cytokine shifts and changes in the oral microbiota composition in saliva of women before and after birth. MATERIAL AND METHODS: Saliva (n = 96) was collected before and 6 months after birth, and medical, oral health, and periodontal status were assessed. In a multiplex immunoassay, 10 cytokines were simultaneously analyzed and cumulative Th1 and Th2 cytokine levels and Th1/Th2 ratio were calculated for all groups. Putative periodontal pathogens (n = 6) were evaluated by quantitative real-time polymerase chain reaction. RESULTS: Th2 cytokine levels were significantly lower (p = 0.014) while pro-inflammatory cytokine levels were significantly higher (p < 0.01) during pregnancy than postpartum. Similar Th1 levels were found between the groups (p = 0.143). Th1 and Th2 cytokines positively correlated with periodontal parameters (p < 0.001) and levels of studied bacteria during pregnancy (p < 0.05). CONCLUSIONS: This study identified a significantly increased Th1/Th2 cytokine ratio during pregnancy and a positive association with putative periodontal pathogens. This immunological and microbiological deregulation in the oral milieu during pregnancy is suggestive of a destructive inflammatory periodontal profile. STUDY REGISTRATION: Clinical Trials.gov (Record BAP-2015). CLINICAL RELEVANCE: Understanding altered oral immunological and microbiological regulation patterns during pregnancy may help improve the inflammatory periodontal profile in pregnant women.

Salivary inflammatory burden in pre- and postmenopausal women: Associations with body mass index, patient-reported health, serum cytokines, and periodontal parameters.

BACKGROUND: The decline of estrogen levels during menopause impacts weight, mood, and overall health, both orally and systemically. This study assessed salivary levels of tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), IL-10, and IL-7 in postmenopausal (PMW) and regularly menstruating premenopausal (RMPW) women, while considering serum cytokine levels, body mass index (BMI), periodontal health, and self-reported physical and emotional well-being. METHODS: In this study, 75 PMW and 71 RMPW were included. Clinical and periodontal parameters were evaluated, and perceived health was assessed with the Women's Health Questionnaire (WHQ). Cytokine levels in both saliva and serum were quantified by enzyme-linked immunosorbent assay (ELISA). Covariate evaluations of salivary cytokines were conducted using hierarchical linear regression modeling. RESULTS: Cytokines were detectable in saliva from 71 PMW and 67 RMPW. In the initial unadjusted model, IL-7, IL-10, and TNF-α exibited significant differences between RMPW and PMW. However, these differences became non-significant (p > 0.05) in the final model after adjusting for age, which implies a negligible effect of the investigated covariates on salivary cytokine levels when age was considered. Lower levels of IL-6 in PMW, which initially showed no significant difference, became borderline (p = 0.054) in the final model after adjusting for age. CONCLUSIONS: After adjusting for multiple factors, no significant difference was found in the salivary levels of the investigated cytokines between RMPW and PMW. Factors such as BMI, perceived health, serum cytokine levels, and periodontal parameters seem to minimally influence these levels in PMW. However, age may be a stronger confounding factor.

Value of gingival crevicular fluid TREM-1, PGLYRP1, and IL-1ß levels during menopause.

OBJECTIVE: This study aimed to investigate the association of GCF TREM-1, PGLYRP1, and IL-1ß levels with periodontal health in pre- and postmenopausal women. BACKGROUND: Triggering receptor expressed on myeloid cells 1 (TREM-1), activated through its ligand peptidoglycan recognition protein 1 (PGLYRP1), stimulates proinflammatory cytokine production, such as interleukin (IL)-1ß, during periodontal inflammation. Postmenopausal changes may modulate these immune-inflammatory functions. No clinical study has yet investigated the effect of menopause on TREM-1, PGLYRP1, and IL-1ß levels in gingival crevicular fluid (GCF). METHODS: This cross-sectional study included 148 women (age range = 35-65 years), divided into postmenopausal women (PMW) (n = 76, mean age = 54 ± 5 years) and regularly menstruating premenopausal women (RMPW) (n = 72, mean age = 40 ± 4 years). Clinical periodontal parameters were recorded. TREM-1, PGLYRP1, and IL-1ß levels were quantified with enzyme-linked immunosorbent assays. Pearson's Chi-squared test and Mann-Whitney-U test were used to compare categorical and numerical variables, respectively. Spearman's Rho correlation analysis was used to test the linear relationship between variables. Analyte level data were categorized based on the periodontal diagnosis and menopause status (2 × 2 nonparametric factorial ANOVA). RESULTS: No significant differences in TREM-1, PGLYRP1, and IL-1ß levels between PMW and RMPW were observed (p > .05). Mean values of periodontal indexes including probing depth did not differ significantly between PMW and RMPW groups (p = .474). TREM-1 levels were significantly higher in both PMW and RMPW with periodontitis, compared to gingivitis or health (p = .0021). CONCLUSION: Menopause-related changes have no observable effect on GCF levels of TREM-1, PGLYRP1, and IL-1ß. Higher GCF TREM-1 levels in women with periodontitis regardless of their menopausal status indicate that TREM-1 may be an indicator for periodontitis both in premenopausal and postmenopausal women.

Association of salivary TREM-1 and PGLYRP1 inflammatory markers with non-communicable diseases.

AIM: Triggering receptor expressed on myeloid cells 1 (TREM-1) and peptidoglycan recognition protein 1 (PGLYRP1) are elevated in biofluids in the presence of various inflammatory conditions. This cross-sectional study aimed to evaluate the effect of age, sex, smoking and different oral and systemic non-communicable diseases on the levels of TREM-1 and PGLYRP1 in saliva. MATERIALS AND METHODS: In total, 445 individuals (mean age 48.7 ± 16.9 years, female:male 51%:49%) were included. All provided self-reported information on smoking and systemic diseases and whole stimulated saliva. Periodontal and cariological parameters were recorded. Salivary levels of TREM-1, PGLYRP1 and total protein were measured using commercially available assays. RESULTS: Salivary TREM-1 levels were significantly higher in stages III-IV periodontitis compared to other periodontal diagnoses (p < .05). Smoking, bleeding on probing (BOP), percentage of pockets ≥4 mm and the number of manifest caries were associated with TREM-1 (p < .05), while sex, BOP, number of manifest caries and muscle and joint diseases were associated with PGLYRP1 (p < .05). CONCLUSIONS: Salivary TREM-1 is associated with periodontitis and caries, while PGLYRP1 is associated with gingival inflammation and caries. Additionally, TREM-1 levels are modified by smoking, while PGLYRP1 is modified by sex and muscle and joint diseases. TREM-1 and PGLYRP1 in saliva could serve as potential biomarkers for detecting and monitoring non-communicable diseases.

Precision periodontal care: from omics discoveries to chairside diagnostics.

The interface of molecular science and technology is guiding the transformation of personalized to precision healthcare. The application of proteomics, genomics, transcriptomics, and metabolomics is shaping the suitability of biomarkers for disease. Prior validation of such biomarkers in large and diverse patient cohorts helps verify their clinical usability. Incorporation of molecular discoveries into routine clinical practice relies on the development of customized assays and devices that enable the rapid delivery of analytical data to the clinician, while the patient is still in session. The present perspective review addresses this topic under the prism of precision periodontal care. Selected promising research attempts to innovate technological platforms for oral diagnostics are brought forward. Focus is placed on (a) the suitability of saliva as a conveniently sampled biological specimen for assessing periodontal health, (b) proteomics as a high-throughput approach for periodontal disease biomarker identification, and (c) chairside molecular diagnostic assays as a technological funnel for transitioning from the laboratory benchtop to the clinical point-of-care.

Community dynamics of subgingival microbiome in periodontitis and targets for microbiome modulation therapy.

The microbial aetiology for periodontitis has been widely studied and deciphered for more than a century. The evolving and changing concepts about periodontal microbiology can be attributed to continuously developing laboratory techniques. The current sequencing platforms have not only expanded the catalog of periodontal pathogens but have also facilitated the understanding of functional interactions of the ecological framework. However, the translation of this new knowledge to advance periodontal therapeutics is minimal. We contend that novel clinical interventions directed beyond conventional therapies need to be emphasized. A clear understanding of the structural and functional dynamics of subgingival microbiota is a pre-requisite for developing any microbiome-based interventions for applications in periodontal health care. In this review, we discuss the 16 s-rRNA gene sequencing-based knowledge of the subgingival microbial community structure, its interactions and functions, and our perspective on the potential to engineer it for periodontal therapeutics. Harnessing this next-generation sequencing-based knowledge, microbiome modulation therapies are poised to change microbiome therapeutics' face.

Proteome Analysis of Oral Biofluids in Periodontal Health and Disease Using Mass Spectrometry.

Mass spectrometry-based proteomic approaches permit the high-throughput assessment of proteins from oral biofluids, therefore, allowing a deeper insight into the mechanistic study of periodontal disease. Here we describe an entire experimental design of proteomic workflow for oral biofluids, exemplified by saliva and gingival crevicular fluid collected from periodontal health or disease subjects and using a label-free quantification strategy for mass spectrometric data acquisition.

Salivary secretory leukocyte protease inhibitor levels in patients with stage 3 grade C periodontitis: a comparative cross-sectional study.

Secretory leukocyte protease inhibitor (SLPI) is an anti-protease that protects mucosal tissue integrity owing to its anti-microbial and immunomodulatory properties. This study aimed to investigate SLPI levels in periodontal diseases, and analyze the potential correlation with clinical periodontal parameters. Whole saliva samples were obtained from healthy (n = 24), gingivitis (n = 24) and patients with stage 3 grade C periodontitis (n = 24). SLPI was measured by ELISA and normalized by total protein. Receiver operating characteristics (ROC) curve was used for estimating the area under the curve (AUC). The normalized SLPI levels were significantly reduced in periodontitis compared with gingivitis (4.84-fold) or health (1.83-fold) and negatively correlated with periodontal parameters. The ROC curves showed a good predictor value of the SLPI for differentiation of periodontitis versus health or gingivitis (AUC ≥ 0.80). This study demonstrates that the levels of SLPI are high in periodontal health, further elevated in gingivitis, but eventually decreased in severe periodontitis beyond the former two states. This observation may have broader implications in the context of inflammatory diseases affecting the oral mucosa, as it shows that the bacterial burden is disturbing the homeostatic balances of anti-microbial and anti-protease factors in the oral cavity.

Implementing of aMMP-8 point-of-care test with a modified new disease classification in Finnish adolescent cohorts.

OBJECTIVES: Periodontitis is a multifactorial biofilm-induced inflammatory disease; however, clinical and radiographic information reflects events that have already occurred. aMMP-8 lateral-flow chairside or point-of-care-test (POC-test) results have also come to complement the overall status of the patient's current situation. The current study aimed to clarify the usefulness of aMMP-8 chairside-test (aMMP-8 POCT) results to alert the initial or early periodontitis in adolescents, a potential periodontitis risk group with cut off 20 ng/ml in Stage I periodontitis, according to the current periodontitis classification system. MATERIAL AND METHODS: A total of 117 adolescents were aMMP-8 POC tested for aMMP-8 levels and examined for the full mouth and were interviewed for questions concerning health behavior. RESULTS: Of all 117 participants, N = 38 (32.5%) was aMMP-8 POCT positive, and N = 34 (29.1%) had at least one 4 mm periodontal deepened pocket; however, only N = 16 (13.7%) had both characteristics. The subclinical stage (N = 47) could not be classified either as gingivitis or Stage I. Of the participants, who did not have any deepened periodontal pockets, 18  tested negative. CONCLUSIONS: Stage I is preceded by antecedent stages that should be tackled by oral healthcare prevention and personalized treatment modalities by professionals. Elevated (>20 ng/ml), positive aMMP-8 POCT results can be regarded as initial alarmer such as emerging risk. This should be utilized in the preventive personalized interventions by oral health professionals.

ImmunoDisk-A Fully Automated Bead-Based Immunoassay Cartridge with All Reagents Pre-Stored.

In this paper, we present the ImmunoDisk, a fully automated sample-to-answer centrifugal microfluidic cartridge, integrating a heterogeneous, wash-free, magnetic- and fluorescent bead-based immunoassay (bound-free phase detection immunoassay/BFPD-IA). The BFPD-IA allows the implementation of a simple fluidic structure, where the assay incubation, bead separation and detection are performed in the same chamber. The system was characterized using a C-reactive protein (CRP) competitive immunoassay. A parametric investigation on air drying of protein-coupled beads for pre-storage at room temperature is presented. The key parameters were buffer composition, drying temperature and duration. A protocol for drying two different types of protein-coupled beads with the same temperature and duration using different drying buffers is presented. The sample-to-answer workflow was demonstrated measuring CRP in 5 µL of human serum, without prior dilution, utilizing only one incubation step, in 20 min turnaround time, in the clinically relevant concentration range of 15-115 mg/L. A reproducibility assessment over three disk batches revealed an average signal coefficient of variation (CV) of 5.8 ± 1.3%. A CRP certified reference material was used for method verification with a concentration CV of 8.6%. Our results encourage future testing of the CRP-ImmunoDisk in clinical studies and its point-of-care implementation in many diagnostic applications.

aMMP-8 Oral Fluid PoC Test in Relation to Oral and Systemic Diseases.

The manuscript uses the previously published literature and highlights the benefits of active-matrix metalloproteinase (aMMP)-8 chairside/point-of-care (PoC) diagnostic tools as adjunctive measures in oral and systemic diseases. Previous studies suggest that as a biomarker, aMMP-8 is more precise than total MMP-8, MMP-9, MMP-2, MMP-3, MMP-13, MMP-7, MMP-1, calprotectin, myeloperoxidase (MPO), human neutrophil elastase (HNE), tissue inhibitor of matrix metalloproteinase (TIMP)-1, and bleeding of probing (BOP). Therefore, aMMP-8 could be implemented as the needed key biomarker for the new disease classification for both periodontitis and peri-implantitis. With a sensitivity to the tune of 75-85% and specificity in the range of 80-90%, lateral flow aMMP-8 PoC testing is comparable to catalytic protease activity assays for aMMP-8. The test can be further applied to estimate the glycemic status of an individual, to ascertain whether a person is at risk for COVID-19, in managing the oral side effects of radiotherapy carried in head and neck cancers, and in selected cases pertaining to reproductive health. In the future, aMMP-8 could find application as a potential systemic biomarker in diseases affecting the cardiovascular system, cancers, bacteremia, sepsis, diabetes, obesity, meningitis, as well as pancreatitis. The aMMP-8 PoCT is the first practical test in the emerging new dental clinical field, that is, oral clinical chemistry representing oral medicine, clinical chemistry, peri-implantology, and periodontology.

Association of peptidoglycan recognition protein 1 to post-myocardial infarction and periodontal inflammation: A subgroup report from the PAROKRANK (Periodontal Disease and the Relation to Myocardial Infarction) study.

BACKGROUND: Peptidoglycan recognition protein 1 (PGLYRP1) is an antimicrobial and proinflammatory innate immunity protein activated during infections. We aimed to investigate whether PGYLRP1 and associated molecules of the immune response in saliva is a cumulative outcome result of both MI and periodontal inflammation. METHODS AND RESULTS: Two hundred patients with MI and another 200 matched non-MI controls were included. A full-mouthexamination was conducted to assess periodontal inflammation and collection of stimulated saliva was performed 6 to 10 weeks after the first MI. PGLYRP1, triggering receptor expressed on myeloid cells 1 (TREM-1), interleukin-1 beta (IL-1ß) were analyzed by ELISA. Matrix metalloproteinase (MMP)-8 levels were determined by IFMA. Compared to controls, MI patients showed higher salivary PGLYRP1, but not TREM-1 levels. The difference in PGLYRP1 levels remained after adjustment for covariates. In MI patients, the PGLYRP1 levels positively correlated with BOP and PPD 4 to 5 mm. Among non-MI subjects, the levels of PGLYRP1 correlated positively and significantly with BOP and total PPD. Salivary PGLYRP1 concentrations also showed strong positive correlations with levels of TREM-1, IL-1ß and MMP-8. In multivariate linear regression analysis, in MI patients, BOP and former smokingstatus displayed an association with salivary PGLYRP1 concentration. CONCLUSION: MI patients showed higher salivary PGLYRP1 levels than healthy controls, also after adjusting for smoking, sex, age and periodontal health status. Salivary levels of PGLYRP1 may reflect the overall inflammatory burden to chronic bacterial exposure, possibly underpinning the observed associations between periodontitis and exposure with MI.

Cytokine profiles and the dynamic of gingivitis development in humans.

AIM: To investigate the relationship between cytokine profiles and "fast" and "slow" patterns of gingival inflammation development. MATERIALS AND METHODS: Forty-two adults participated in an experimental gingivitis study, comprising a 2-week hygiene phase (clinical examination and professional cleaning); a 3-week induction phase (absence of oral hygiene); and a 2-week resolution phase (re-establishment of oral hygiene). Plaque and gingival inflammation scores were assessed. Interferon-gamma (IFN-γ), interleukin (IL)-1ß, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, and tumour necrosis factor-alpha (TNF-α) from gingival crevicular fluid were collected and measured by multiplex ELISA. Group-based-trajectory-modelling (GBTM) was used to model cytokine profiles over the induction phase. The effect of gingival inflammation on cytokine levels over time was estimated with mixed-effects modelling. RESULTS: GBTM analysis revealed two cytokine profiles, "non-organized response" (IL-4, IL-6, IL-8, IL-12, and IL-13) and "organized response" (IL-2, IL-10, and TNF-α). Among the "slow" responders, neither cytokine profile was associated with gingivitis. In contrast, a "fast" response was associated with a higher "non-organized response" factor (coef. 0.14) and a lower "organized response" factor (coef. -0.03). CONCLUSION: A "fast" gingivitis development was associated with a higher "non-organized response" and a lower "organized response", which may elucidate the role of individual variability in gingivitis susceptibility.