Association between dysbiotic perio-pathogens and inflammatory initiators and mediators in COVID-19 patients with diabetes.

It has been suggested that a corona virus infection is linked to chronic periodontitis (COVID-19). Our objectives were to look at the expression of angiotensin-converting enzyme-2 (ACE2) in periodontal compartments containing periodontal infections to determine if ACE2 is directly or indirectly responsible for the inflammation in periodontal tissues getting worse. In this study, six non-COVID-19 periodontitis patients without diabetes served as controls, and 23 hospitalized periodontitis patients were admitted with PCR-confirmed COVID-19 with diabetes mellitus (Group 1/G1, n = 10), and without diabetes (Group 2/G2, n = 13). We evaluated the mRNA expression of ACE2, IL-6, IL-8, complement C3, and LL-37, as well as the relative proportion of Porphyromonas gingivalis, Fusobacterium nucleatum, and Veillonella parvula to represent the dysbiosis condition in periodontal microenvironment using subgingival plaque and gingival crevicular fluids (GCF) samples and quantitative real time PCR (qPCR). Every analysis was done to ascertain how they related to one another. The area under the curve (AUC) and receiver operating characteristic (ROC) curve were used to determine the sensitivity and specificity of inflammatory indicators. All the grouped patients had ACE2 detected, according to our findings, but only the G1 patients had a positive correlation (p < 0.05) between ACE2 expression and the inflammatory markers. The combination of IL-6 and C3 mRNAs was found to be 0.78 and 0.55 for the G1 group and the G2 group, respectively, based on the ROC and AUC values. According to our research, the relationship between complement C3 and IL-6 may be able to predict the degree of periodontal inflammation in COVID-19 patients who also have diabetes.

Porphyromonas gingivalis association with inflammatory markers and exosomal miRNA-155 in saliva of periodontitis patients with and without diabetes diagnosed with COVID-19.

Background: /Purposes: Studies have indicated that salivary molecules from patients with periodontitis and diabetes are confounded with pathological conditions associated with SARS-CoV-2 infection. The study aimed to address whether the abundance of Porphyromonas gingivalis which causes periodontitis, differed compared with that of Aggregatibacter actinomycetemcomitans (used as control) and to analyze the correlation of periodontitis with the expression levels of severe acute respiratory syndrome coronavirus 2 receptor (ACE2) and periodontitis inflammatory markers (TLR-2/TLR-4, TNFα, and miR-155). Materials and Methods: A saliva sample (5 mL) was obtained from 23 hospitalized patients with COVID-19, categorized into two groups: diabetic (G1, n = 10) and non-diabetic (G2, n = 13). Saliva from patients with periodontitis without diabetes and coronavirus disease 2019 (COVID-19; n = 6) were included as control. The quantitative real-time polymerase chain reaction measured the levels of P. gingivalis and A. actinomycetemcomitans, as well as periodontitis markers in saliva. The obtained data were analyzed using one-way ANOVA and the Spearman correlation test. Results: The abundance of P. gingivalis was observed to be higher (p < 0.05) in saliva of patients with diabetes (G1) than in those without diabetes (G2). A contradictory trend was observed for A. actinomycetemcomitans. The transcription level of ACE2 was comparable in all groups tested, while the expression of periodontitis markers varied. The relationships and sensitivity/specificity among P. gingivalis infection ACE2 expression, and inflammatory markers were also evaluated. Conclusions: This study showed that the association between P. gingivalis infection and ACE2 expression might reflect the characteristics of saliva in COVID-19 patients with and without diabetes. However, the relationships between TLR-4 and miR-155 are more specific in discriminating against COVID-19 patients with and without diabetes.

The utility of salivary CRP and IL-6 as a non-invasive measurement evaluated in patients with COVID-19 with and without diabetes.

Background: The available evidence suggests that inflammatory responses, in both systemic and oral tissue, contribute to the pathology of COVID-19 disease. Hence, studies of inflammation biomarkers in oral fluids, such as saliva, might be useful to better specify COVID-19 features. Methods: In the current study, we performed quantitative real-time PCR to measure salivary levels of C-reactive protein (CRP) and interleukin-6 (IL-6) in saliva obtained from patients diagnosed with mild COVID-19, in a diabetic group (DG; n = 10) and a non-diabetic group (NDG; n = 13). All participants were diagnosed with periodontitis, while six participants with periodontitis but not diagnosed with COVID-19 were included as controls. Results: We found increases in salivary total protein levels in both the DG and NDG compared to control patients. In both groups, salivary CRP and IL-6 levels were comparable. Additionally, the levels of salivary CRP were significantly correlated with total proteins, in which a strong and moderate positive correlation was found between DG and NDG, respectively. A linear positive correlation was also noted in the relationship between salivary IL-6 level and total proteins, but the correlation was not significant. Interestingly, the association between salivary CRP and IL-6 levels was positive. However, a moderately significant correlation was only found in COVID-19 patients with diabetes, through which the association was validated by a receiver operating curve. Conclusions: These finding suggest that salivary CRP and IL-6 are particularly relevant as potential non-invasive biomarker for predicting diabetes risk in mild cases of COVID-19 accompanied with periodontitis.

ACE2 expression in saliva of patients with COVID-19 and its association with Candida albicans and Aggregatibacter actinomycetemcomitans.

Background: A relationship between oral microbiota and susceptibility to SARS-CoV-2 infection has been extensively studied. However, the relationship between oral commensal flora and expression of angiotensin-converting enzyme 2 ( ACE2) remains to be established. In this observational study, we collected saliva from patients with COVID-19 and evaluated the relationship between ACE2 expression and Candida albicans as well as with selected gram-negative bacteria ( Aggregatibacter actin o mycetemcomitans, Fusobacterium nucleatum, and Veillonella parvula). We investigated how this may be directly or indirectly involved in oral dysbiosis in patients with COVID-19. Methods: We included 23 hospitalized patients admitted to Universitas Indonesia Hospital with PCR-confirmed COVID-19, with six healthy participants serving as controls. Saliva and tongue surface swabs were collected from patients with diabetes (DG) and without diabetes (NDG) and subject controls. Using quantitative PCR (qPCR) we assessed the mRNA expression of ACE2, the abundance of C. albicans, and the transcription levels of its biofilm-associated genes, agglutinin-like protein 3 ( ALS3), hyphal wall protein 1 ( HWP1), and yeast-form wall protein 1 ( YWP1). We also counted the relative proportion of the three selected gram-negative oral bacteria in saliva. All analyses were performed to determine the relationship between ACE2 expression and C. albicans and gram-negative bacteria. Results: ACE2 mRNA expression was significantly higher in tongue swab samples than in saliva. However, no significant difference was observed between the patient groups. Conversely, DG patients had a significantly higher abundance of C. albicans in saliva compared to NDG patients and control group patients. The correlation and sensitivity/specificity relationship between ACE2 expression and C. albicans or the selected oral bacteria were also observed. Conclusions: The data show that ACE2 expression can be detected in saliva of patients with COVID-19 and its association with C. albicans and gram-negative oral bacteria might contribute toward developing an oral dysbiosis based predictor for prognosis of COVID-19 severity.

Relationships between Solobacterium moorei and Prevotella intermedia in subgingival microbiota of periodontitis patients with halitosis: A preliminary study using qPCR.

Objective: Solobacterium moorei is suggested to be associated with the production of volatile sulphur compounds (VSCs) and can be found in subgingival plaques of deep periodontal pockets. We examined whether this bacterium's count was reduced in periodontitis patients with halitosis following non-surgical periodontal treatment, while the bacterial count of Prevotella intermedia was measured simultaneously as a control. Material & methods: This clinical study included 20 adults with chronic periodontitis who complained of halitosis. The bacterial relationship in the subgingival plaque sample was measured after 8 weeks post-treatment, including the probing pocket depth (PPD). Quantitative real-time PCR (qPCR) was used to measure the proportion of S. moorei, while the concentrations of H2S and CH3SH were determined using oral ChromaTM. Results: The presence of S. moorei was consistently observed in participants with periodontitis before and after non-surgical periodontal treatment and consistent showed a significantly lower proportion compared with P. intermedia. Solobacterium moorei showed a strong positive correlation with H2S and CH3SH concentrations, but a negative correlation with deep periodontal pocket measurements. Conversely, reduced P. intermedia may be more associated with a deep pocket, independent of the concentration of CH3SH. Conclusion: The study data showed that the proportion of S. moorei in the subgingival biofilm can be related to halitosis in periodontitis patients.

Association between Volatile Sulfur Compounds Prevotella intermedia and Matrix Metalloproteinase-8 Expression

Abstract Objective: To determine the correlation between levels of methyl mercaptan (CH3SH) hydrogen sulfide (H2S), the proportion of Prevotella intermedia (Pi), and matrix metalloproteinase-8 (MMP-8) gene expression levels in periodontitis patients accompanied by halitosis. Material and Methods: Samples were obtained from gingival crevicular fluid (GCF) in the deepest pocket and by swabbing in the tongue coating area in patients with periodontitis presenting with halitosis (n = 23) and healthy subjects as controls (n = 7). The values of CH3SH and H2S were obtained using Oral Chroma. The proportion of Pi and MMP-8 expression levels were evaluated using PCR-RT. All the result was statistically analyzed using SPSS software. Results: The levels of CH3SH and H2S in participants with PD ≥ 6 mm showed a robust negative correlation with the proportion of P. intermedia in GCF and tongue coating. No statistically significant association was detected between CH3SH and H2S levels and MMP-8 expression levels (p>0.05). Conclusion: There is no association between CH3SH and H2S levels, the proportion of P. intermedia, and MMP-8 expression in patients with periodontitis accompanied by halitosis (AU).

Periodontal Ligament Cell Sheets and RGD-Modified Chitosan Improved Regeneration in the Horizontal Periodontal Defect Model.

OBJECTIVE: The aim of this study was to examine the potential of periodontal ligament (PDL) cells sheet and arginine-glycyl-aspartic acid (RGD)-modified chitosan scaffold for periodontal tissue regeneration in horizontal periodontal defect model. MATERIALS AND METHODS: PDL cell cytotoxicity was tested with 3-[4,5- dimethylthiazol-2yl]-2,5-diphenyl-2H-tetrazolium bromide assay. Cell migration toward the chitosan-based materials was analyzed with trans-well migration assay. Horizontal periodontal defect model was created in four maxillary and mandibular lateral incisors of Macaque nemestrina. Following periodontal therapy, the sites were transplanted with various regenerative materials: (1) chitosan, (2) RGD-modified chitosan, (3) PDL cell sheet with chitosan, (4) PDL cell sheet with RGD-modified chitosan. The periodontal tissue regeneration was evaluated clinically and radiographically. Gingival crevicular fluids were collected each week to evaluate cementum protein-1 (CEMP-1) expression with enzyme-linked immunosorbent assay, while the biopsies were retrieved after 4 weeks for histological and microcomputed tomography evaluation. STATISTICAL ANALYSIS: Data was statistically analyzed using GraphPad Prism 6 for MacOS X. Normality was tested using the Shapiro-Wilk normality test. The Kruskal-Wallis test was used to compare the groups. Significance was accepted when p < 0.05. RESULTS: Clinical examination revealed more epithelial attachment was formed in the group with PDL cell sheet with RGD-modified chitosan. Similarly, digital subtraction radiography analysis showed higher gray scale, an indication of higher alveolar bone density surrounded the transplanted area, as well as higher CEMP-1 protein expression in this group. The incorporation of RGD peptide to chitosan scaffold in the group with or without PDL cells sheet reduced the distance of cement-enamel junction to the alveolar bone crest; hence, more periodontal tissue formed. CONCLUSIONS: Horizontal periodontal defect model could be successfully created in M. nemestrina model. Combination of PDL cell sheet and RGD-modified chitosan resulted in the higher potential for periodontal tissue regeneration. The results of this study highlight the PDL cell sheet and RGD-modified chitosan as a promising approach for future clinical use in periodontal regeneration.

Expression of Toll-Like Receptor 4 and Matrix Metalloproteinase 8 in Gingival Crevicular Fluid in Patients with Periodontitis

Abstract Objective: To determine the expression of TLR4 and MMP8 in gingival crevicular fluid [GCF] in patients with periodontitis. Material and Methods: Clinical samples were collected from 23 gingival crevicular fluid of periodontal disease subjects (n = 14) and healthy periodontal subjects (n=9). Measurement of Clinical parameters of probing pocket depth (PPD), bleeding on probing (BOP), and clinical attachment loss (CAL) were included as diagnostic criteria. Pocket Depth (PD) and CAL were defined as present if the PPD was ≥ 4 mm and the CAL ≥ 1 mm. Expression of TLR4 and MMP8 in the gingival crevicular fluid of deep pockets (PD≥ 6mm), shallow pockets (PD 4-5 mm) and healthy periodontal sulcus (0-3 mm) were evaluated by quantitative real-time polymerase chain reaction (qRT-PCR). Statistical analysis to compare the pocket was using Independent t-test and Mann-Whitney test. Correlation between mRNA expression and clinical parameters was analyzed using Spearman's correlation test Results: Expression of TLR4 was higher in shallow pockets compared to the control group, but the difference was not statistically significant (p>0.05). The expression of MMP8 was higher in shallow pockets compared to the control group, but the difference was not statistically significant (p>0.05) either. There is no significant correlation between TLR4 and MMP8 with clinical periodontal parameters Conclusion: TLR4 and MMP8 mRNA expression levels should not be used as a clinical biomarker in periodontitis diagnostic tools.

AI-2 of Aggregatibacter actinomycetemcomitans inhibits Candida albicans biofilm formation.

Aggregatibacter actinomycetemcomitans, a Gram-negative bacterium, and Candida albicans, a polymorphic fungus, are both commensals of the oral cavity but both are opportunistic pathogens that can cause oral diseases. A. actinomycetemcomitans produces a quorum-sensing molecule called autoinducer-2 (AI-2), synthesized by LuxS, that plays an important role in expression of virulence factors, in intra- but also in interspecies communication. The aim of this study was to investigate the role of AI-2 based signaling in the interactions between C. albicans and A. actinomycetemcomitans. A. actinomycetemcomitans adhered to C. albicans and inhibited biofilm formation by means of a molecule that was secreted during growth. C. albicans biofilm formation increased significantly when co-cultured with A. actinomycetemcomitans luxS, lacking AI-2 production. Addition of wild-type-derived spent medium or synthetic AI-2 to spent medium of the luxS strain, restored inhibition of C. albicans biofilm formation to wild-type levels. Addition of synthetic AI-2 significantly inhibited hypha formation of C. albicans possibly explaining the inhibition of biofilm formation. AI-2 of A. actinomycetemcomitans is synthesized by LuxS, accumulates during growth and inhibits C. albicans hypha- and biofilm formation. Identifying the molecular mechanisms underlying the interaction between bacteria and fungi may provide important insight into the balance within complex oral microbial communities.