Calcium-sensing receptor regulates Kv7 channels via Gi/o protein signalling and modulates excitability of human induced pluripotent stem cell-derived nociceptive-like neurons.

BACKGROUND AND PURPOSE: Neuropathic pain, a debilitating condition with unmet medical needs, can be characterised as hyperexcitability of nociceptive neurons caused by dysfunction of ion channels. Voltage-gated potassium channels type 7 (Kv7), responsible for maintaining neuronal resting membrane potential and thus excitability, reside under tight control of G protein-coupled receptors (GPCRs). Calcium-sensing receptor (CaSR) is a GPCR that regulates the activity of numerous ion channels, but whether CaSR can control Kv7 channel function has been unexplored until now. EXPERIMENTAL APPROACH: Experiments were conducted in recombinant cell models, mouse dorsal root ganglia (DRG) neurons and human induced pluripotent stem cell (hiPSC)-derived nociceptive-like neurons using patch-clamp electrophysiology and molecular biology techniques. KEY RESULTS: Our results demonstrate that CaSR is expressed in recombinant cell models, hiPSC-derived nociceptive-like neurons and mouse DRG neurons, and its activation induced depolarisation via Kv7.2/7.3 channel inhibition. The CaSR-Kv7.2/7.3 channel crosslink was mediated via the Gi/o protein-adenylate cyclase-cyclicAMP-protein kinase A signalling cascade. Suppression of CaSR function demonstrated a potential to rescue hiPSC-derived nociceptive-like neurons from algogenic cocktail-induced hyperexcitability. CONCLUSION AND IMPLICATIONS: This study demonstrates that the CaSR-Kv7.2/7.3 channel crosslink, via a Gi/o protein signalling pathway, effectively regulates neuronal excitability, providing a feasible pharmacological target for neuronal hyperexcitability management in neuropathic pain.

Elevated pro-inflammatory cytokines and chemokines in saliva of cats with feline odontoclastic resorptive lesion.

Feline odontoclastic resorptive lesion (FORL) is an inflammatory oral disease of unknown aetiopathogenesis that affects between 20% to 75% of cats. Twenty immune-associated molecules were measured in saliva of 25 healthy and 40 cats with FORL using a multiplex assay. No statistically significant differences were observed in the levels of these proteins between the healthy group and the diseased group of cats. A two-step cluster analysis of the oral microbiome and salivary cytokine data identified two subgroups of cats with FORL: FORL-1 (subset of cats with a less diverse oral microbiome) and FORL-2 (diseased cats with a microbiome similar to that of healthy animals). The level of some key proinflammatory cytokines (IL-1ß, IL-12p40) and chemokines (IL-8, RANTES, KC) were significantly higher in the FORL-1 subgroup than in the FORL-2 subgroup and the healthy group. In addition, TNF-α levels were greater in the FORL-1 subgroup than in the FORL-2 subgroup. These increases in pro-inflammatory cytokines and chemokines indicate active ongoing inflammation that may promote the osteoclastic/odontoclastic activity associated with FORL.

Limitations of using 16S rRNA microbiome sequencing to predict oral squamous cell carcinoma.

A new era of next-generation sequencing has changed our perception of the oral microbiome in health and disease, and with this there is a growing understanding that the oral microbiome is a contributing factor to oral squamous cell carcinoma (OSCC), a malignancy of the oral cavity. This study aimed to analyse the trends and relevant literature based on the 16S rRNA oral microbiome in head and neck cancer using next-generation sequencing technologies, and to conduct a meta-analysis of the studies with OSCC cases and healthy controls. A literature search using the databases Web of Science and PubMed was conducted in a scoping-like review to collect information based on the study design, and plots were generated using RStudio. We selected case-control studies using 16S rRNA oral microbiome sequencing analysis in OSCC cases versus healthy controls for re-analysis. Statistical analyses were conducted using R. Out of 916 original articles, we filtered and selected 58 studies for review, and 11 studies for meta-analysis. Differences between sampling type, DNA extraction methods, next-generation sequencing technology and region of the 16S rRNA were identified. No significant differences in the α- and ß-diversity between health and oral squamous cell carcinoma were observed (p < 0.05). Random Forest classification marginally improved predictability of four studies (training set) when split 80/20. We found an increase in Selenomonas, Leptotrichia and Prevotella species to be indicative of disease. A number of technological advances have been accomplished to study oral microbial dysbiosis in oral squamous cell carcinoma. There is a clear need for standardization of study design and methodology to ensure 16S rRNA outputs are comparable across the discipline in the hope of identifying 'biomarker' organisms for designing screening or diagnostic tools.

Acetylcholine Receptors in Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) are well known for their regenerative potential. Even though the ability of MSCs to proliferate and differentiate has been studied extensively, there remains much to learn about the signaling mechanisms and pathways that control proliferation and influence the differentiation phenotype. In recent years, there has been growing evidence for the utility of non-neuronal cholinergic signaling systems and that acetylcholine (ACh) plays an important ubiquitous role in cell-to-cell communication. Indeed, cholinergic signaling is hypothesized to occur in stem cells and ACh synthesis, as well as in ACh receptor (AChR) expression, has been identified in several stem cell populations, including MSCs. Furthermore, AChRs have been found to influence MSC regenerative potential. In humans, there are two major classes of AChRs, muscarinic AChRs and nicotinic AChRs, with each class possessing several subtypes or subunits. In this review, the expression and function of AChRs in different types of MSC are summarized with the aim of highlighting how AChRs play a pivotal role in regulating MSC regenerative function.

Expression of Toll-like receptor and cytokine mRNAs in feline odontoclastic resorptive lesion (FORL) and feline oral health.

Feline odontoclastic resorptive lesion (FORL) is a common chronic inflammatory condition whose aetiopathogenesis remains unclear. FORL affects 20-75% of cats and causes excruciating pain and tooth loss. The purpose of this study was to evaluate chronic inflammation in FORL by assessing differences in Toll-like receptor (TLR) and cytokine transcripts in gingival tissues between diseased and healthy cats. Gingival tissue samples were collected from 14 healthy cats with no known clinical signs of oral disease and 41 cats with FORL. Levels of mRNA encoding TLR2, TLR3, TLR4, TLR7, TLR9 and the cytokines interleukin-1ß (IL-1ß), IL-4, IL-6, IL-10, IL-12, interferon-γ (IFN-γ) and tumour necrosis factor-α (TNF-α) was evaluated using quantitative real-time PCR. Statistical significance of the results was assessed using non-parametric tests. Levels of TLR and cytokine transcripts were upregulated in gingival tissue from cats with FORL as compared with healthy gingival tissue: TLR2, TLR3 and TLR9, p ≤ 0.001; TLR4 and TLR7, p ≤ 0.01; IFN-γ, IL-4, IL-6, IL-10, IL-12, IL-1ß and TNF-α, p ≤ 0.001). In conclusion, expression of TLR and both pro- and anti-inflammatory cytokines were significantly increased, confirming an ongoing chronic inflammatory response to the microbiome in FORL. It is likely that dysbiosis of the oral microbiota in cats with FORL activates the innate immune response, leading to active inflammation that results in tooth resorption.

Screening the Tocriscreen™ bioactive compound library in search for inhibitors of Candida biofilm formation.

Biofilms formed by Candida species present a significant clinical problem due to the ineffectiveness of many conventional antifungal agents, in particular the azole class. We urgently require new and clinically approved antifungal agents quickly for treatment of critically ill patients. To improve efficiency in antifungal drug development, we utilized a library of 1280 biologically active molecules within the Tocriscreen 2.0 Micro library. Candida auris NCPF 8973 and Candida albicans SC5314 were initially screened for biofilm inhibitory activity using metabolic and biomass quantitative assessment methods, followed up by targeted evaluation of five selected hits. The initial screening (80% metabolic inhibition rate) revealed that there was 90 and 87 hits (approx. 7%) for C. albicans and C. auris, respectively. Additionally, all five compounds selected from the initial hits exhibited a biofilm inhibition effect against several key Candida species tested, including C. glabrata and C. krusei. Toyocamycin displayed the most potent activity at concentrations as low as 0.5 µg/mL, though was limited to inhibition. Darapladib demonstrated an efficacy for biofilm inhibition and treatment at a concentration range from 8 to 32 µg/mL and from 16 to 256 µg/mL, respectively. Combinational testing with conventional antifungals against C. albicans strains demonstrated a range of synergies for planktonic cells, and notably an anti-biofilm synergy for darapladib and caspofungin. Together, these data provide new insights into antifungal management possibilities for Candida biofilms.

Assessing the inflammatory response to in vitro polymicrobial wound biofilms in a skin epidermis model.

Wounds can commonly become infected with polymicrobial biofilms containing bacterial and fungal microorganisms. Microbial colonization of the wound can interfere with sufficient healing and repair, leading to high rates of chronicity in certain individuals, which can have a huge socioeconomic burden worldwide. One route for alleviating biofilm formation in chronic wounds is sufficient treatment of the infected area with topical wound washes and ointments. Thus, the primary aim here was to create a complex in vitro biofilm model containing a range of microorganisms commonly isolated from the infected wound milieu. These polymicrobial biofilms were treated with three conventional anti-biofilm wound washes, chlorhexidine (CHX), povidone-iodine (PVP-I), and hydrogen peroxide (H2O2), and efficacy against the microorganisms assessed using live/dead qPCR. All treatments reduced the viability of the biofilms, although H2O2 was found to be the most effective treatment modality. These biofilms were then co-cultured with 3D skin epidermis to assess the inflammatory profile within the tissue. A detailed transcriptional and proteomic profile of the epidermis was gathered following biofilm stimulation. At the transcriptional level, all treatments reduced the expression of inflammatory markers back to baseline (untreated tissue controls). Olink technology revealed a unique proteomic response in the tissue following stimulation with untreated and CHX-treated biofilms. This highlights treatment choice for clinicians could be dictated by how the tissue responds to such biofilm treatment, and not merely how effective the treatment is in killing the biofilm.

Recurrent Vulvovaginal Candidiasis: a Dynamic Interkingdom Biofilm Disease of Candida and Lactobacillus.

Despite the strikingly high worldwide prevalence of vulvovaginal candidiasis (VVC), treatment options for recurrent VVC (RVVC) remain limited, with many women experiencing failed clinical treatment with frontline azoles. Further, the cause of onset and recurrence of disease is largely unknown, with few studies identifying potential mechanisms of treatment failure. This study aimed to assess a panel of clinical samples from healthy women and those with RVVC to investigate the influence of Candida, the vaginal microbiome, and how their interaction influences disease pathology. 16S rRNA sequencing characterized disease by a reduction in specific health-associated Lactobacillus species, such as Lactobacillus crispatus, coupled with an increase in Lactobacillus iners. In vitro analysis showed that Candida albicans clinical isolates are capable of heterogeneous biofilm formation, and we found the presence of hyphae and C. albicans aggregates in vaginal lavage fluid. Additionally, the ability of Lactobacillus to inhibit C. albicans biofilm formation and biofilm-related gene expression was demonstrated. Using RNA sequencing technology, we were able to identify a possible mechanism by which L. crispatus may contribute to re-establishing a healthy vaginal environment through amino acid acquisition from C. albicans. This study highlights the potential formation and impact of Candida biofilms in RVVC. Additionally, it suggests that RVVC is not entirely due to an arbitrary switch in C. albicans from commensal to pathogen and that understanding interactions between this yeast and vaginal Lactobacillus species may be crucial to elucidating the cause of RVVC and developing appropriate therapies. IMPORTANCE RVVC is a significant burden, both economically and for women's health, but its prevalence is poorly documented globally due to the levels of self-treatment. Identifying triggers for development and recurrence of VVC and the pathogenesis of the microbes involved could considerably improve prevention and treatment options for women with recurrent, azole-resistant cases. This study therefore aimed to examine the interkingdom dynamics from healthy women and those with RVVC using next-generation sequencing techniques and to further investigate the molecular interactions between C. albicans and L. crispatus in a relevant biofilm coculture system.

Exploring the roles of neuropeptides in trigeminal neuropathic pain: A systematic review and narrative synthesis of animal studies.

OBJECTIVE: This systematic review aims to explore the changes in expression of neuropeptides and/or their receptors following experimental trigeminal neuropathic pain in animals. DESIGN: MEDLINE, Embase, and Scopus were searched for publications up to 31st March 2021. Study selection and data extraction were completed by two independent reviewers based on the eligibility criteria. The quality of articles was judged based on the Systematic Review Centre for Laboratory Animal Experimentation (SYRCLE) risk-of-bias tool. RESULTS: A total of 19 studies satisfied the eligibility criteria and were included for narrative synthesis. Methods of trigeminal neuropathic pain induction were nerve ligation, nerve compression/crush, nerve transection and dental pulp injury. Animal behaviours used for pain verification were evoked responses to mechanical and thermal stimuli. Non-evoked behaviours, including vertical exploration, grooming and food consumption, were also employed in some studies. Calcitonin gene-related peptide (CGRP) and substance P were the most frequently reported neuropeptides. Overall, unclear to high risk of bias was identified in the included studies. CONCLUSIONS: Limited evidence has suggested the pro-nociceptive role of CGRP in trigeminal neuropathic pain. In order to further translational pain research, animal models of trigeminal neuropathic pain and pain validation methods need to be optimised. Complete reporting of future studies based on available guidelines to improve confidence in research is encouraged.

Microbiome analysis of feline odontoclastic resorptive lesion (FORL) and feline oral health.

Introduction. Feline odontoclastic resorptive lesion (FORL) is one of the most common and painful oral diseases of the cat. It is characterised by tooth resorption due to destructive activity of odontoclasts. FORL can result in tooth loss. While the aetiology of FORL is not clearly understood, it is thought to be multifactorial and bacteria are likely to play a major role.Hypothesis. Dysbiosis of the normal feline oral microbiota leads to an alteration in commensal bacteria populations, which results in the development of FORL.Aim. The purpose of the current study was to determine the composition of the microbiomes associated with feline oral health and FORL.Methodology. Supragingival plaque was collected from 25 cats with a healthy oral cavity and 40 cats with FORL. DNA was extracted from each sample, the V4 region of the 16S rRNA gene amplified by polymerase chain reaction and amplicons sequenced. Diversity and species richness analyses were performed, principal component analysis was used to explore differences between the oral microbiomes of healthy cats and those with FORL, and linear discriminant analysis effect size was used to assess differences between the groups.Results. The six most abundant bacterial genera identified were Bergeyella, Capnocytophaga, Lampropedia, Morexella, Porphyromonas and Treponema. Two-step cluster analysis of the data identified two FORL sub-groups (FORL-1, FORL-2). The FORL-2 sub-group was very similar to the healthy group, whilst the FORL-1 sub-group was clearly different from both the FORL-2 sub-group and the healthy groups. In this analysis, Capnocytophaga (P <0.001) and Lampropedia (P <0.01) were found at significantly lower levels and Porphyromonas at a slightly higher level in the FORL-1 sub-group compared to the healthy and FORL-2 sub-groups. Microbial diversity was found to be less in the FORL-1 sub-group than in the healthy group. Lampropedia sp., a phosphate-accumulating oral commensal species, was significantly lower in the FORL-1 sub-group.Conclusion. The oral microbiota associated with the FORL-1 sub-group is distinct from that found in the healthy group and FORL-2 sub-group. Lampropedia species may influence the local calcium-phosphate ratio, which could be a factor in tooth and bone resorption observed in FORL.

Evaluating aerosol and splatter following dental procedures: Addressing new challenges for oral health care and rehabilitation.

BACKGROUND: Dental procedures often produce aerosol and splatter which have the potential to transmit pathogens such as SARS-CoV-2. The existing literature is limited. OBJECTIVE(S): To develop a robust, reliable and valid methodology to evaluate distribution and persistence of dental aerosol and splatter, including the evaluation of clinical procedures. METHODS: Fluorescein was introduced into the irrigation reservoirs of a high-speed air-turbine, ultrasonic scaler and 3-in-1 spray, and procedures were performed on a mannequin in triplicate. Filter papers were placed in the immediate environment. The impact of dental suction and assistant presence were also evaluated. Samples were analysed using photographic image analysis and spectrofluorometric analysis. Descriptive statistics were calculated and Pearson's correlation for comparison of analytic methods. RESULTS: All procedures were aerosol and splatter generating. Contamination was highest closest to the source, remaining high to 1-1.5 m. Contamination was detectable at the maximum distance measured (4 m) for high-speed air-turbine with maximum relative fluorescence units (RFU) being: 46,091 at 0.5 m, 3,541 at 1.0 m and 1,695 at 4 m. There was uneven spatial distribution with highest levels of contamination opposite the operator. Very low levels of contamination (≤0.1% of original) were detected at 30 and 60 minutes post-procedure. Suction reduced contamination by 67-75% at 0.5-1.5 m. Mannequin and operator were heavily contaminated. The two analytic methods showed good correlation (r = 0.930, n = 244, P < .001). CONCLUSION: Dental procedures have potential to deposit aerosol and splatter at some distance from the source, being effectively cleared by 30 minutes in our setting.

Evaluating contaminated dental aerosol and splatter in an open plan clinic environment: Implications for the COVID-19 pandemic.

OBJECTIVES: Identify splatter/aerosol distribution from dental procedures in an open plan clinic and explore aerosol settling time after dental procedures. METHODS: In two experimental designs using simulated dental procedures on a mannequin, fluorescein dye was introduced: (1) into the irrigation system of an air-turbine handpiece; (2) into the mannequin's mouth. Filter papers were placed in an open plan clinic to collect fluorescein. An 8-metre diameter rig was used to investigate aerosol settling time. Analysis was by fluorescence photography and spectrofluorometry. RESULTS: Contamination distribution varied across the clinic depending on conditions. Unmitigated procedures have the potential to deposit contamination at large distances. Medium volume dental suction (159 L/min air) reduced contamination in the procedural bay by 53%, and in other areas by 81-83%. Low volume suction (40 L/min air) was similar. Cross-ventilation reduced contamination in adjacent and distant areas by 80-89%. In the most realistic model (fluorescein in mouth, medium volume suction), samples in distant bays (≥5 m head-to-head chair distance) gave very low or zero readings (< 0.0016% of the fluorescein used during the procedure). Almost all (99.99%) of the splatter detected was retained within the procedural bay/walkway. After 10 min, very little additional aerosol settled. CONCLUSIONS: Cross-infection risk from dental procedures in an open plan clinic appears small when bays are ≥ 5 m apart. Dilution effects from instrument water spray were observed, and dental suction is of benefit. Most settled aerosol is detected within 10 min indicating environmental cleaning may be appropriate after this. CLINICAL SIGNIFICANCE: Aerosols produced by dental procedures have the potential to contaminate distant sites and the majority of settled aerosol is detectable after 10 min. Dental suction and ventilation have a substantial beneficial effect. Contamination is likely to be minimal in open plan clinics at distances of 5 m or more.

Detection, treatment and prevention of endodontic biofilm infections: what's new in 2020?

Endodontic disease, a biofilm infection of the root canal space, is a significant cause of dental morbidity worldwide. Endodontic treatment, or root canal treatment, as it is commonly known is founded on the ability to eradicate microbial biofilm infection and prevent re-infection of the highly complex root canal space. Despite many "advances" in clinical endodontics we have seen little improvement in outcomes. The aim of this critical review paper is to provide a contemporary view of endodontic microbiology and biofilm polymicrobiality, provide an understanding of the host response, and how together these impact upon clinical treatment. Ultimately, it is intended to provide insight into novel opportunities and strategies for the future diagnostics, treatment, and prevention of endodontic disease.

Effects of smoking on non-surgical periodontal therapy in patients with periodontitis Stage III or IV, and Grade C.

BACKGROUND: To evaluate possible effects of smoking on clinical, biochemical, and microbiological outcomes of non-surgical periodontal treatment in patients with periodontitis Stage III or IV and Grade C. METHODS: Conventional quadrant-wise non-surgical periodontal treatment was performed and whole-mouth periodontal measurements were recorded at baseline, 1, 3, and 6 months after completion of treatment. Saliva, gingival crevicular fluid, subgingival plaque, and blood samples were obtained at the same time points. Inflammatory cytokine levels, presence, and quantities of 11 different bacterial species were determined. Smoking status was validated by cotinine assay. RESULTS: Fourteen smoker and 13 non-smoker patients completed the study protocol and revealed similar clinical findings except for the higher plaque scores in the non-smokers at 6 months (P <0.01). Significant differences were found between the study groups in biofluid cytokine levels at 1 and 3 months (P <0.01). Gram-negative bacteria were more abundant in the smokers at baseline and so were Gram-positive bacteria in the non-smokers (P <0.01). Gram-negative bacteria repopulated in the smokers faster than in the non-smokers (P <0.01). CONCLUSIONS: The present findings suggest that smoker patients with periodontitis Stage III and IV, Grade C respond well to the non-surgical periodontal treatment during the 6-month follow-up. However, smokers exhibit faster repopulation of Gram-negative bacteria.

Candida albicans Biofilm Heterogeneity and Tolerance of Clinical Isolates: Implications for Secondary Endodontic Infections.

AIM: Endodontic infections are caused by the invasion of various microorganisms into the root canal system. Candida albicans is a biofilm forming yeast and the most prevalent eukaryotic microorganism in endodontic infections. In this study we investigated the ability of C. albicans to tolerate treatment with standard endodontic irrigants NaOCl (sodium hypochlorite), ethylenediaminetetraacetic acid (EDTA) and a combination thereof. We hypothesized that biofilm formed from a panel of clinical isolates differentially tolerate disinfectant regimens, and this may have implications for secondary endodontic infections. METHODOLOGY: Mature C. albicans biofilms were formed from 30 laboratory and oral clinical isolates and treated with either 3% NaOCl, 17% EDTA or a sequential treatment of 3% NaOCl followed by 17% EDTA for 5 min. Biofilms were then washed, media replenished and cells reincubated for an additional 24, 48 and 72 h at 37 °C. Regrowth was quantified using metabolic reduction, electrical impedance, biofilm biomass and microscopy at 0, 24, 48 and 72 h. RESULTS: Microscopic analysis and viability readings revealed a significant initial killing effect by NaOCl, followed by a time dependent significant regrowth of C. albicans, but with inter-strain variability. In contrast to NaOCl, there was a continuous reduction in viability after EDTA treatment. Moreover, EDTA significantly inhibited regrowth after NaOCl treatment, though viable cells were still observed. CONCLUSIONS: Our results indicate that different C. albicans biofilm phenotypes grown in a non-complex surface topography have the potential to differentially tolerate standard endodontic irrigation protocols. This is the first study to report a strain dependent impact on efficacy of endodontic irrigants. Its suggested that within the complex topography of the root canal, a more difficult antimicrobial challenge, that existing endodontic irrigant regimens permit cells to regrow and drive secondary infections.

Cholinergic signalling mechanisms and early implant healing phases in healthy versus generalized aggressive periodontitis patients: A prospective, case-control study.

AIMS: Periodontal diseases negatively affect implant osseointegration. Perturbations in non-neuronal cholinergic signalling mechanisms are associated with periodontitis; however, their role in generalized aggressive periodontitis (GAgP) is unknown. The aim of this prospective case-control study was to determine the relationship between non-neuronal cholinergic signalling mechanisms, secreted Ly-6/uPAR-related protein-1 (SLURP-1), interleukin-17 (IL-17) family cytokines and healing of dental implants in health and GAgP. MATERIAL AND METHODS: Thirteen GAgP patients and seven periodontally healthy individuals (PH) were recruited. Peri-implant crevicular fluid (PICF) was obtained at baseline and 1 month post-placement. Acetylcholine (ACh) levels and cholinesterase activity were determined biochemically. SLURP-1, IL-17A and IL-17E levels were determined by ELISA. Marginal bone loss (MBL) at 1 and 6 months post-placement was determined radiographically. RESULTS: The concentration of ACh, cholinesterase activity and IL-17A levels was elevated in PICF of patients with GAgP compared to PH individuals at baseline and 1 month post-placement. The concentration of ACh and cholinesterase activity levels in PICF correlated with levels of IL-17A and MBL around implants 1 month post-placement in patients with GAgP. CONCLUSIONS: Non-neuronal cholinergic mechanisms may play a role in the aetiopathogenesis of GAgP and may directly or indirectly, through modulation of IL-17A, influence early implant osseointegration and potential long-term implant survival.

Repurposing Pilocarpine Hydrochloride for Treatment of Candida albicans Infections.

Acetylcholine modulates the virulence of Candidaalbicans and regulates an appropriate immune response to infection in a Galleria mellonella infection model. Indeed, the evidence suggests that C. albicans possesses a functional cholinergic receptor that can regulate filamentous growth and biofilm formation. Furthermore, G. mellonella immune cell subsets possess repertories of cholinergic receptors which regulate an effective and appropriate cellular immune response to C. albicans infection. This study aimed to investigate the cholinergic receptor subtype involved in regulation of filamentous growth and biofilm formation by C. albicans and determine the roles of cholinergic receptors in modulation of G. mellonella immune cell subsets. The general muscarinic receptor agonist, pilocarpine hydrochloride, inhibited C. albicans biofilm formation and pathogenicity, a phenomenon that could be reversed using the general muscarinic receptor antagonist, scopolamine. Pilocarpine hydrochloride protected G. mellonella larvae from C. albicans infection via inhibition of C. albicans filamentation and appropriate regulation of cellular immunity. However, scopolamine abrogated the capacity of pilocarpine hydrochloride to protect G. mellonella larvae from C. albicans infection. Furthermore, acetylcholine and pilocarpine hydrochloride exhibited differential modulatory capabilities on Galleria mellonella hemocyte responses to C. albicans The data in this article demonstrate that a muscarinic receptor modulates C. albicans filamentation and biofilm formation. Furthermore, the results suggest that G. mellonella hemocyte subsets possess unique repertoires of cholinergic receptors that regulate their differentiation, activation, and function in contrasting manners. Therefore, targeting cholinergic receptors by repurposing currently licensed cholinergic drugs may offer novel therapeutic solutions for the prevention or treatment of fungal infections.IMPORTANCECandida albicans is the most common human fungal pathogen with an estimated crude mortality rate of 40%. The ability of the organism to switch from the yeast to hyphal form and produce biofilms are important virulence factors. C. albicans infections are combatted by the host immune system. However, Candida triggers a strong inflammatory response that, if not appropriately regulated, can damage host tissues. Therefore, it is important that the host immune response eliminates the fungus but limits tissue damage. This study provides evidence that targeting cholinergic receptors cannot only curb the virulence of C. albicans by inhibiting filamentous growth and biofilm formation but can also appropriately regulate the host immune response to induce rapid clearance with limited damage to vital tissues. This article provides evidence that repurposing licensed drugs that target cholinergic receptors may offer novel therapeutic solutions for the prevention or treatment of fungal infections.

Interkingdom interactions on the denture surface: Implications for oral hygiene.

BACKGROUND: Evidence to support the role of Candida species in oral disease is limited. Often considered a commensal, this opportunistic yeast has been shown to play a role in denture related disease, though whether it is an active participant or innocent bystander remains to be determined. This study sought to understand the role of Candida species alongside the bacterial microbiome in a denture patient cohort, exploring how the microbiology of the denture was affected by oral hygiene practices. MATERIALS AND METHODS: In vitro denture cleansing studies were performed on a complex 9-species interkingdom denture biofilm model, with quantitative assessment of retained bacterial and fungal viable bioburdens. Patient hygiene measures were also collected from 131 patients, including OHIP, frequency of denture cleansing, oral hygiene measure and patient demographics. The bacterial microbiome was analysed from each patient, alongside quantitative PCR assessment of ITS (fungal) and 16S (bacterial) bioburden from denture, mucosa and intact dentition. RESULTS: It was shown that following in vitro denture cleansing C. albicans were unresponsive to treatment, whereas bacterial biofilms could repopulate 100-fold, but were susceptible to subsequent treatment. Within the patient cohort, oral hygiene did not impact candidal or bacterial composition, nor diversity. The levels of Candida did not significantly influence the bacterial microbiome, though an observed gradient was suggestive of a microbial composition change in response to Candida load, indicating interkingdom interaction rather than an oral hygiene effect. Indeed, correlation analysis was able to show significant correlations between Candida species and key genera (Lactobacillus, Scardovia, Fusobacterium). CONCLUSIONS: Overall, this study has shown that the denture microbiome/mycobiome is relatively resilient to oral hygiene challenges, but that Candida species have potential interactions with key oral genera. These interactions may have a bearing on shaping community structure and a shift from health to disease when the opportunity arises.

Plaque Accumulation and Inflammation Adjacent to Restorations of Amorphous Calcium Phosphate-containing Composite in Early Childhood Caries.

PURPOSE: To evaluate the clinical, biochemical, and microbiological reactions to nanocomposite containing amorphous calcium phosphate (ACP) in comparison to a traditional composite restorative material in early childhood caries. MATERIALS AND METHODS: Eighteen teeth were restored with the test material (ACP-containing resin) and 18 teeth were restored with the control material (traditional composite, TC) in fourteen paediatric patients using a split-mouth design. One caries- and restoration-free intact tooth in each patient was selected as the healthy control. Gingival crevicular fluid (GCF) and supragingival plaque samples were collected at baseline before the treatment and also on days 1, 7, 14 and 30 after treatment. Unstimulated whole saliva samples were obtained from each patient at baseline, and 1 and 6 months after restoration. GCF and saliva samples were assayed for IL-17A, IL-17F IL-17A/F, IL-17E, OPG and RANKL levels by ELISA, and plaque composition was assessed using RT-PCR. RESULTS: Clinical evaluation indicated no statistically significant differences between the two restorative materials according to the FDI criteria surface lustre, material retention and marginal adaptation properties. Pro-inflammatory IL-17 levels decreased statistically significantly at 6 months compared to baseline and 1-month values (p < 0.05). The baseline pro-inflammatory IL-17 cytokine levels in GCF samples around the carious teeth were higher than those obtained around the healthy teeth (p < 0.05), but similar in GCF from the ACP-test and TC teeth. Microbiological findings were similar in the ACP and T groups. CONCLUSION: It may be suggested that both ACP-containing and traditional resin composites show similar antimicrobial and biochemical effects in early childhood caries.

Clinical associations between acetylcholine levels and cholinesterase activity in saliva and gingival crevicular fluid and periodontal diseases.

AIM: The oral mucosa possesses a non-neuronal cholinergic system. This study aimed to determine clinical evidence for a role of cholinergic mechanisms in the pathogenesis of periodontal diseases. MATERIALS AND METHODS: Fifty healthy participants, 52 patients with gingivitis and 49 with periodontitis were recruited. Full periodontal parameters were recorded and saliva and gingival crevicular fluid (GCF) collected. Levels of acetylcholine and inflammatory mediators were quantified using commercially available assay kits. Acetylcholinesterase and butyrylcholinesterase activities were measured using a published biochemical assay. RESULTS: Acetylcholine levels are significantly elevated in saliva and GCF, whereas GCF levels of butyrylcholinesterase activity are significantly decreased, in patients with periodontal diseases. Acetylcholine levels in saliva and GCF correlated positively with clinical markers of disease severity and with increased levels of IL-17A and IL-17F. In contrast, butyrylcholinesterase activity levels in GCF showed significant negative correlations with clinical markers of disease severity and IL-17A and IL-17F levels. None of the findings were due to smoking. CONCLUSIONS: Elevated acetylcholine levels and reduced butyrylcholinesterase activity are clinically associated with periodontal diseases and elevated levels of IL-17A and IL-17F. Therefore, non-neuronal cholinergic mechanisms may influence IL-17 biology and the aetiopathogenesis of periodontal diseases and therefore are possible therapeutic targets.