SARS-CoV-2 Infection of Salivary Glands Compromises Oral Antifungal Innate Immunity and Predisposes to Oral Candidiasis.

Saliva contains antimicrobial peptides considered integral components of host innate immunity, and crucial for protection against colonizing microbial species. Most notable is histatin-5 which is exclusively produced in salivary glands with uniquely potent antifungal activity against the opportunistic pathogen Candida albicans. Recently, SARS-CoV-2 was shown to replicate in salivary gland acinar cells eliciting local immune cell activation. In this study, we performed mechanistic and clinical studies to investigate the implications of SARS-CoV-2 infection on salivary histatin-5 production and Candida colonization. Bulk RNA-sequencing of parotid salivary glands from COVID-19 autopsies demonstrated statistically significant decreased expression of histatin genes. In situ hybridization, coupled with immunofluorescence for co-localization of SARS-CoV-2 spike and histatin in salivary gland cells, showed that histatin was absent or minimally present in acinar cells with replicating viruses. To investigate the clinical implications of these findings, salivary histatin-5 levels and oral Candida burden in saliva samples from three independent cohorts of mild and severe COVID-19 patients and matched healthy controls were evaluated. Results revealed significantly reduced histatin-5 in SARS-CoV-2 infected subjects, concomitant with enhanced prevalence of C. albicans. Analysis of prospectively recovered samples indicated that the decrease in histatin-5 is likely reversible in mild-moderate disease as concentrations tended to increase during the post-acute phase. Importantly, salivary cytokine profiling demonstrated correlations between activation of the Th17 inflammatory pathway, changes in histatin-5 concentrations, and subsequent clearance of C. albicans in a heavily colonized subject. The importance of salivary histatin-5 in controlling the proliferation of C. albicans was demonstrated using an ex vivo assay where C. albicans was able to proliferate in COVID-19 saliva with low histatin-5, but not with high histatin-5. Taken together, the findings from this study provide direct evidence implicating SARS-CoV-2 infection of salivary glands with compromised oral innate immunity, and potential predisposition to oral candidiasis.

Functional Redundancy in Candida auris Cell Surface Adhesins Crucial for Cell-Cell Interaction and Aggregation.

Candida auris is an emerging nosocomial fungal pathogen associated with life-threatening invasive disease due to its persistent colonization, high level of transmissibility and multi-drug resistance. Aggregative and non-aggregative growth phenotypes for C. auris strains with different biofilm forming abilities, drug susceptibilities and virulence characteristics have been described. Using comprehensive transcriptional analysis we identified key cell surface adhesins that were highly upregulated in the aggregative phenotype during in vitro and in vivo grown biofilms using a mouse model of catheter infection. Phenotypic and functional evaluations of generated null mutants demonstrated crucial roles for the adhesins Als5 and Scf1 in mediating cell-cell adherence, coaggregation and biofilm formation. While individual mutants were largely non-aggregative, in combination cells were able to co-adhere and aggregate, as directly demonstrated by measuring cell adhesion forces using single-cell atomic force spectroscopy. This co-adherence indicates their role as complementary adhesins, which despite their limited similarity, may function redundantly to promote cell-cell interaction and biofilm formation. Functional diversity of cell wall proteins may be a form of regulation that provides the aggregative phenotype of C. auris with flexibility and rapid adaptation to the environment, potentially impacting persistence and virulence.

Functional Redundancy in Candida auris Cell Surface Adhesins Crucial for Cell-Cell Interaction and Aggregation.

Candida auris is an emerging nosocomial fungal pathogen associated with life-threatening invasive disease due to its persistent colonization, high level of transmissibility and multi-drug resistance. Aggregative and non-aggregative growth phenotypes for C. auris strains with different biofilm forming abilities, drug susceptibilities and virulence characteristics have been described. Using comprehensive transcriptional analysis we identified key cell surface adhesins that were highly upregulated in the aggregative phenotype during in vitro and in vivo grown biofilms using a mouse model of catheter infection. Phenotypic and functional evaluations of generated null mutants demonstrated crucial roles for the adhesins Als5 and Scf1 in mediating cell-cell adherence, coaggregation and biofilm formation. While individual mutants were largely non-aggregative, in combination cells were able to co-adhere and aggregate, as directly demonstrated by measuring cell adhesion forces using single-cell atomic force spectroscopy. This co-adherence indicates their role as complementary adhesins, which despite their limited similarity, may function redundantly to promote cell-cell interaction and biofilm formation. Functional diversity of cell wall proteins may be a form of regulation that provides the aggregative phenotype of C. auris with flexibility and rapid adaptation to the environment, potentially impacting persistence and virulence.

A Longitudinal Metagenomic Comparative Analysis of Oral Microbiome Shifts in Patients Receiving Proton Radiation versus Photon Radiation for Head and Neck Cancer.

Introduction: Due to the radiation-sparing effects on salivary gland acini, changes in the composition of the oral microbiome may be a driver for improved outcomes in patients receiving proton radiation, with potentially worse outcomes in patients exposed to photon radiation therapy. To date, a head-to-head comparison of oral microbiome changes at a metagenomic level with longitudinal sampling has yet to be performed in these patient cohorts. Methods and Materials: To comparatively analyze oral microbiome shifts during head and neck radiation therapy, a prospective pilot cohort study was performed at the Maryland Proton Treatment Center and the University of Maryland Marlene and Stewart Greenebaum Comprehensive Cancer Center. A longitudinal metagenomic comparative analysis of oral microbiome shifts was performed at three time points (pre-radiation, during radiation, and immediately post-radiation). Head and neck cancer patients receiving proton radiation (n = 4) were compared to photon radiation (n = 4). Additional control groups included healthy age- and sex-matched controls (n = 5), head and neck cancer patients who never received radiation therapy (n = 8), and patients with oral inflammatory disease (n = 3). Results: Photon therapy patients presented with lower microbial alpha diversity at all timepoints, and there was a trend towards reduced species richness as compared with proton therapy. Healthy controls and proton patients exhibited overall higher and similar diversity. A more dysbiotic state was observed in patients receiving photon therapy as compared to proton therapy, in which oral microbial homeostasis was maintained. Mucositis was observed in 3/4 photon patients and was not observed in any proton patients during radiation therapy. The bacterial de novo pyrimidine biosynthesis pathway and the nitrate reduction V pathway were comparatively higher following photon exposure. These functional changes in bacterial metabolism may suggest that photon exposure produces a more permissive environment for the proliferation of pathogenic bacteria. Conclusion: Oral microbiome dysbiosis in patients receiving photon radiation may be associated with increased mucositis occurrence. Proton radiation therapy for head and neck cancer demonstrates a safer side effect profile in terms of oral complications, oral microbiome dysbiosis, and functional metabolic status.

Novel Remineralizing and Antibiofilm Low-Shrinkage-Stress Nanocomposites to Inhibit Salivary Biofilms and Protect Tooth Structures.

Recurrent caries remain a persistent concern, often linked to microleakage and a lack of bioactivity in contemporary dental composites. Our study aims to address this issue by developing a low-shrinkage-stress nanocomposite with antibiofilm and remineralization capabilities, thus countering the progression of recurrent caries. In the present study, we formulated low-shrinkage-stress nanocomposites by combining triethylene glycol divinylbenzyl ether and urethane dimethacrylate, incorporating dimethylaminododecyl methacrylate (DMADDM), along with nanoparticles of calcium fluoride (nCaF2) and nanoparticles of amorphous calcium phosphate (NACP). The biofilm viability, biofilm metabolic activity, lactic acid production, and ion release were evaluated. The novel formulations containing 3% DMADDM exhibited a potent antibiofilm activity, exhibiting a 4-log reduction in the human salivary biofilm CFUs compared to controls (p < 0.001). Additionally, significant reductions were observed in biofilm biomass and lactic acid (p < 0.05). By integrating both 10% NACP and 10% nCaF2 into one formulation, efficient ion release was achieved, yielding concentrations of 3.02 ± 0.21 mmol/L for Ca, 0.5 ± 0.05 mmol/L for P, and 0.37 ± 0.01 mmol/L for F ions. The innovative mixture of DMADDM, NACP, and nCaF2 displayed strong antibiofilm effects on salivary biofilm while concomitantly releasing a significant amount of remineralizing ions. This nanocomposite is a promising dental material with antibiofilm and remineralization capacities, with the potential to reduce polymerization-related microleakage and recurrent caries.

Pharmacist-Mediated Thyroid-Stimulating Hormone (TSH) Test Guideline Monitoring Program- Outcomes of a Retrospective Study In Patients On Levothyroxine.

Thirty to sixty percent of individuals taking levothyroxine were either under or overtreated, which leads to organ damage and excess mortality. This study aims to assess the gaps in the "thyroid-stimulating hormone (TSH) test guideline compliance rate" and validate the scope of ambulatory care pharmacist-mediated practice in patients on levothyroxine. At the study site, pharmacists offered patient-centered telephonic counseling to patients on levothyroxine who had been non-compliant with TSH tests for more than a year. A two-month quantitative retrospective analysis of this practice was conducted to assess its impact on TSH lab test adherence and dose modification outcomes. 415 patients met the study's inclusion criteria who received pharmacist counseling with documented intervention. Pharmacists bridged the significant gap in practice by creating new TSH lab requests with counseling in 81.2% (n = 337) of the study population who did not have TSH lab requests prior to the program. The non-compliance rate population dropped from 79.27% (n = 329) to 17.59% (n = 73) in the study population who had been non-compliant with the TSH test for 13 and 24 months. 74.5% (n = 309) were found to have performed their TSH test after the pharmacist's intervention. Among 100, 66% (n = 66) patients with abnormal TSH values consulted their physician for advice, of which 60.6% (n=40) had their levothyroxine dose adjusted (χ2=82.702, P < 0.01. The study suggests that pharmacists can significantly mediate between patients and physicians to enhance TSH test compliance and essential dose adjustment in patients prescribed levothyroxine.

The oral mycobiome: Oral epithelial dysplasia and oral squamous cell carcinoma.

Fungi, a diverse group of eukaryotic organisms, play distinct roles in health and disease. Recent advances in the field of mycobiology have enabled the characterization of the "human mycobiome." The human mycobiome has extensively been studied in various disease models. However, to date, the role of the oral mycobiome in oral carcinogenesis has yet to be elucidated. Candida albicans, the most common oral colonizer, has been speculated to display tumorigenic effects; however, the literature lacks consistent documentation from mechanistic studies on whether oral mycobiota act as drivers, facilitators, or passive colonizers of oral premalignancy and cancer. This review article provides an overview of existing hypothesis-driven mechanistic models that outline the complex interplay between the oral mycobiome and oral epithelial dysplasia as well as their potential clinical implications.

Long-Term Post-COVID-19 Associated Oral Inflammatory Sequelae.

The oral cavity remains an underappreciated site for SARS-CoV-2 infection despite the myriad oral conditions observed in COVID-19 patients. Recently, replicating SARS-CoV-2 was found inside salivary epithelial cells resulting in inflammation and atrophy of salivary glands. Saliva possesses healing properties crucial for maintaining the health of the oral mucosa. Specifically, salivary antimicrobial peptides, most notable, histatin-5 exclusively produced in salivary glands, plays a vital role in innate immunity against colonizing microbial species. The demonstration of SARS-CoV-2 destruction of gland tissue where histatin-5 is produced strongly indicate that histatin-5 production is compromised due to COVID-19. Here we present a case of a patient presenting with unexplained chronic oral dysesthesia and dysgeusia post-recovery from COVID-19. To explore potential physiological mechanisms behind the symptoms, we comparatively analyzed saliva samples from the patient and matched healthy subject for histatin-5 and key cytokines. Findings demonstrated significantly reduced histatin-5 levels in patient's saliva and activation of the Th17 inflammatory pathway. As histatin-5 exhibits potent activity against the opportunistic oral pathogen Candida albicans, we evaluated saliva potency against C. albicans ex vivo. Compared to control, patient saliva exhibited significantly reduced anti-candidal efficacy. Although speculative, based on history and salivary analysis we hypothesize that salivary histatin-5 production may be compromised due to SARS-CoV-2 mediated salivary gland destruction. With the current lack of emphasis on implications of COVID-19 on oral health, this report may provide lacking mechanistic insights that may lead to reassessment of risks for oral opportunistic infections and mucosal inflammatory processes in acutely-ill and recovered COVID-19 patients.

In vitro demineralization prevention by fluoride and silver nanoparticles when applied to sound enamel and enamel caries-like lesions of varying severities.

OBJECTIVES: To investigate the effect of fluoride and silver nanoparticles on the prevention of in vitro demineralization of sound enamel and enamel caries-like lesions of varying severities. METHODS: Caries-like lesions of different severities (1/6/15 days) were created in bovine enamel specimens. One group remained sound. All specimens were demineralized again using a partially saturated acetic acid solution. Mimicking the intra-oral retention of fluoride and silver in vitro, this solution was supplemented with fluoride (0/1/10 ppm) and/or silver nanoparticles (0/10 ppm) in a factorial design. Changes in lesion depth (ΔL) and integrated mineral loss (ΔΔZ) were evaluated by digital transverse microradiography. Data was analyzed using three-way ANOVA. RESULTS: Lesion severity significantly affected ΔΔZ and ΔL, after no treatment and after the treatment of fluoride and silver independently (p = 0.012 and p = 0.037, respectively). Fluoride and the fluoride × lesion severity interaction were shown to be significant (p < 0.001) on ΔΔZ and ΔL. Silver nanoparticles significantly affected ΔΔZ (p = 0.041), but not ΔL (p = 0.15). The silver nanoparticles × lesion severity interaction was significant for ΔΔZ and ΔL (p = 0.032 and p = 0.024, respectively). No interaction was observed for ΔΔZ and ΔL between fluoride and silver (p = 0.962 and p = 0.971, respectively) as well as lesion severity and the use of fluoride and silver combined (p = 0.722 and p = 0.158, respectively). CONCLUSION: Fluoride and silver nanoparticles had a significant effect on the prevention of in vitro demineralization of sound enamel and enamel caries-like lesions of varying severities. CLINICAL SIGNIFICANCE: Fluoride and silver nanoparticles may potentially allow for more tailored caries prevention.

Impact of caffeine on metabolic activity and biofilm formation of Candida albicans on acrylic denture resin in the presence of nicotine.

STATEMENT OF PROBLEM: Candida albicans has been implicated in denture stomatitis, and this effect is exacerbated by nicotine exposure. However, studies have also suggested that caffeine exposure inhibits the growth of C. albicans. The interaction effects of nicotine and caffeine are not yet clear on the growth of C. albicans. PURPOSE: The purpose of this in vitro study was to determine the effect of caffeine on metabolic activity and biofilm formation of C. albicans growing on acrylic denture resin while simultaneously exposed to nicotine and, if an effect were to be identified, whether this effect would vary depending on the caffeine concentration. MATERIAL AND METHODS: A total of 240 acrylic resin specimens were divided into 2 equal groups (120 each). Specimens in one group were processed to measure C. albicans metabolic activity, and those in the other group were processed to measure C. albicans biofilm attachment. Ten subgroups (n=12) were established within each group with different concentration combinations of nicotine and caffeine to test the interaction effect. The first subgroup was designed as a negative control, containing 0 mg/mL of nicotine and caffeine. The following subgroups all contained 8.00 mg/mL of nicotine, and the caffeine concentrations were prepared at the following 9 levels: 0, 0.25, 0.50, 1.00, 2.00, 4.00, 8.00, 16.00, and 32.00 mg/mL. Metabolic activity was measured by using a 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-carboxanilide (XTT) assay. Biofilm attachment was measured by using spiral plating and calculated in terms of the number of colony-forming units (CFUs)/mL. Descriptive statistics and a 2-way ANOVA were conducted to determine whether the concentrations of nicotine and caffeine used affected the biofilm attachment and metabolic activity of C. albicans (α=.05). RESULTS: The presence of 8 mg/mL of nicotine increased the metabolic activity and biofilm formation of C. albicans. When compared with the 0 mg/mL of caffeine and 8.00 mg/mL of nicotine group, caffeine from 1.00 to 4.00 mg/mL significantly increased C. albicans biofilm metabolic activity. Caffeine at 16.00 and 32.00 mg/mL significantly decreased C. albicans biofilm metabolic activity in the presence of 8 mg/mL of nicotine. Caffeine from 1.00 to 32.00 mg/mL significantly decreased the biofilm formation of C. albicans in the presence of 8 mg/mL of nicotine. CONCLUSIONS: The presence of 8 mg/mL of nicotine alone increased the metabolic activity and biofilm formation of C. albicans. In the presence of 8 mg/mL of nicotine with different caffeine concentrations, the results suggest that, overall, caffeine at higher concentrations (16 and 32 mg/mL) inhibited the metabolic activity and biofilm formation of C. albicans on acrylic denture resin most.