Modulation of fast sodium current in airway smooth muscle cells by exchange protein directly activated by cAMP.

Airway smooth muscle (ASM) cells from mouse bronchus express a fast sodium current mediated by NaV1.7. We present evidence that this current is regulated by cAMP. ASM cells were isolated by enzymatic dispersal and studied using the whole cell patch clamp technique at room temperature. A fast sodium current, INa, was observed on holding cells under voltage clamp at -100 mV and stepping to -20 mV. This current was reduced in a concentration-dependent manner by denopamine (10 and 30 µM), a ß-adrenergic agonist. Forskolin (1 µM), an activator of adenylate cyclase, reduced the current by 35%, but 6-MB-cAMP (300 µM), an activator of protein kinase A (PKA), had no effect. In contrast, 8-pCPT-2-O-Me-cAMP-AM (007-AM, 10 µM), an activator of exchange protein directly activated by cAMP (Epac), reduced the current by 48%. The inhibitory effect of 007-AM was still observed in the presence of dantrolene (10 µM), an inhibitor of ryanodine receptors, and when cytosolic [Ca2+] was buffered by inclusion of 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, Sigma (BAPTA) (50 µM) in the pipette solution, suggesting that the inhibition of INa was not due to Ca2+-release from intracellular stores. When 007-AM was tested on the current-voltage relationship, it reduced the current at potentials from -30 to 0 mV, but had no effect on the steady-state activation curve. However, the steady-state inactivation V1/2, the voltage causing inactivation of 50% of the current, was shifted in the negative direction from -76.6 mV to -89.7 mV. These findings suggest that cAMP regulates INa in mouse ASM via Epac, but not PKA.NEW & NOTEWORTHY ß-adrenergic agonists are commonly used in inhalers to treat asthma and chronic obstructive pulmonary disease. These work by causing bronchodilation and reducing inflammation. The present study provides evidence that these drugs have an additional action, namely, to reduce sodium influx into airway smooth muscle cells via fast voltage-dependent channels. This may have the dual effect of promoting bronchodilation and reducing remodeling of the airways, which has a detrimental effect in these diseases.

TRPV2 modulates mechanically Induced ATP Release from Human bronchial epithelial cells.

Repetitive bouts of coughing expose the large airways to significant cycles of shear stress. This leads to the release of alarmins and the tussive agent adenosine triphosphate (ATP) which may be modulated by the activity of ion channels present in the human airway. This study aimed to investigate the role of the transient receptor potential subfamily vanilloid member 2 (TRPV2) channel in mechanically induced ATP release from primary bronchial epithelial cells (PBECs).PBECs were obtained from individuals undergoing bronchoscopy. They were cultured in vitro and exposed to mechanical stress in the form of compressive and fluid shear stress (CFSS) or fluid shear stress (FSS) alone at various intensities. ATP release was measured using a luciferin-luciferase assay. Functional TRPV2 protein expression in human PBECs was investigated by confocal calcium imaging. The role of TRPV2 inhibition on FSS-induced ATP release was investigated using the TRPV2 inhibitor tranilast or siRNA knockdown of TRPV2. TRPV2 protein expression in human lung tissue was also determined by immunohistochemistry.ATP release was significantly increased in PBECs subjected to CFSS compared with control (unstimulated) PBECs (N = 3, ***P < 0.001). PBECs expressed functional TRPV2 channels. TRPV2 protein was also detected in fixed human lung tissue. ATP release from FFS stimulated PBECs was decreased by the TRPV2 inhibitor tranilast (N = 3, **P < 0.01) (vehicle: 159 ± 17.49 nM, tranilast: 25.08 ± 5.1 nM) or by TRPV2 siRNA knockdown (N = 3, *P < 0.05) (vehicle: 197 ± 24.52 nM, siRNA: 119 ± 26.85 nM).In conclusion, TRPV2 is expressed in the human airway and modulates ATP release from mechanically stimulated PBECs.

Immunological isolation and characterization of neuronal progenitors from human dental pulp: A laboratory-based investigation.

AIMS: Dental pulp stem cells (DPSCs) contain a population of stem cells with a broad range of differentiation potentials, as well as more lineage-committed progenitors. Such heterogeneity is a significant obstacle to experimental and clinical applications. The aim of this study is to isolate and characterize a homogenous neuronal progenitor cell population from human DPSCs. METHODOLOGY: Polysialylated-neural cell adhesion molecule (PSA-NCAM+) neural progenitors were isolated from the dental pulp of three independent donors using magnetic-activated cell sorting (MACS) technology. Immunofluorescent staining with a panel of neural and non-neural markers was used to characterize the magnetically isolated PSA-NCAM+ fraction. PSA-NCAM+ cells were then cultured in Neurobasal A supplemented with neurotrophic factors: dibutyryl cyclic-AMP, neurotrophin-3, B27 and N2 supplements to induce neuronal differentiation. Both PSA-NCAM+ and differentiated PSA-NCAM+ cells were used in Ca2+ imaging studies to assess the functionality of P2X3 receptors as well as membrane depolarization. RESULTS: PSA-NCAM+ neural progenitors were isolated from a heterogeneous population of hDPSCs using magnetic-activated cell sorting and anti-PSA-NCAM MicroBeads. Flow cytometry analysis demonstrated that immunomagnetic sorting significantly increased the purity of PSA-NCAM+ cells. Immunofluorescent staining revealed expression of pan-neuronal and mature neuronal markers, PGP9.5 and MAP2, respectively, as well as weak expression of the mature sensory markers, peripherin and islet1. ATP-induced response was mediated predominately by P2X3 receptors in both undifferentiated and differentiated cells, with a greater magnitude observed in the latter. In addition, membrane depolarizations were also detected in cells before and after differentiation when loaded with fast-voltage-responding fluorescent molecule, FluoVolt™ in response to potassium chloride. Interestingly, only differentiated PSA-NCAM+ cells were capable of spontaneous membrane oscillations. CONCLUSIONS: In summary, DPSCs contain a population of neuronal progenitors with enhanced neural differentiation and functional neural-like properties that can be effectively isolated with magnetic-activated cell sorting (MACS).

An international consensus study to identify "what" outcomes should be included in a core outcome set for endodontic treatments (COSET) for utilization in clinical practice and research.

BACKGROUND: Development of a standardized set of topic-specific outcomes known as a Core Outcome Set (COS) is important to address issues of heterogeneity in reporting research findings in order to streamline evidence synthesis and clinical decision making. AIM: The aim of the current international consensus study is to identify "what" outcomes to include in the Core Outcome Set for Endodontic Treatments (COSET). Outcomes of various endodontic treatments (non-surgical root canal treatment, surgical endodontics, vital pulp treatment and revitalization procedures) performed on permanent teeth were considered. METHODS: A standard validated methodology for COS development and reporting was adopted. The process involved identification of existing outcomes through four published scoping reviews. This enabled creation of a list of outcomes to be prioritized via semi-structured patient interviews, e-Delphi process and a consensus meeting with a range of relevant global stakeholders. Outcomes were prioritized using a 1-9 Likert scale, with outcomes rated 7-9 considered critical, 4-6 are important and 1-3 are less important. Outcomes rated 7-9 by ≥70% and 1-3 by <15% of participants were considered to achieve consensus for inclusion in the COS. The outcomes that did not achieve consensus in the first round were considered for further prioritization in the second Delphi round and consensus meeting. Final decisions about the outcomes to include in COSET were made by voting during the consensus panel meeting using the Zoom Poll function. RESULTS: A total of 95 participants including patients contributed to the COS development process. The consensus panel recommended, with strong consensus, eight outcomes shared across all treatment modalities for inclusion in COSET: pain; signs of infection (swelling, sinus tract); further intervention/exacerbation; tenderness to percussion/palpation; radiographic evidence of disease progression/healing; function; tooth survival; and patient satisfaction. Additional treatment specific outcomes were also recommended. DISCUSSION: Many of the outcomes included in COSET are patient reported. All should be included in future outcomes studies. CONCLUSION: COSET identified outcomes that are important for patients and clinicians and validated these using a rigorous methodology. Further work is ongoing to determine "how" and "when" these outcomes should be measured.

Novel Antibacterial Properties of the Human Dental Pulp Multipotent Mesenchymal Stromal Cell Secretome.

It is well recognized that clearance of bacterial infection within the dental pulp precedes pulpal regeneration. However, although the regenerative potential of the human dental pulp has been investigated extensively, its antimicrobial potential remains to be examined in detail. In the current study bactericidal assays were used to demonstrate that the secretome of dental pulp multipotent mesenchymal stromal cells (MSCs) has direct antibacterial activity against the archetypal Gram-positive and Gram-negative bacteria, Staphylococcus aureus and Escherichia coli, respectively, as well as the oral pathogens Streptococcus mutans, Lactobacillus acidophilus, and Fusobacterium nucleatum. Furthermore, a cytokine/growth factor array, enzyme-linked immunosorbent assays, and antibody blocking were used to show that cytokines and growth factors present in the dental pulp MSC secretome, including hepatocyte growth factor, angiopoietin-1, IL-6, and IL-8, contribute to this novel antibacterial activity. This study elucidated a novel and diverse antimicrobial secretome from human dental pulp MSCs, suggesting that these cells contribute to the antibacterial properties of the dental pulp. With this improved understanding of the secretome of dental pulp MSCs and its novel antibacterial activity, new evidence for the ability of the dental pulp to fight infection and restore functional competence is emerging, providing further support for the biological basis of pulpal repair and regeneration.

Subgingival microbial diversity and respiratory decline: A cross-sectional study.

AIM: To investigate whether there is an association between subgingival microbial diversity and reduced respiratory function. MATERIALS AND METHODS: A group of dentate 58-72-year-old men in Northern Ireland had a comprehensive periodontal examination including subgingival plaque sampling. DNA was extracted from plaque samples and the V1-V3 regions of the 16S rRNA gene were analysed by high-throughput sequencing and a microbial diversity index (MDI) was derived. Spirometry measurements were made using a wedge bellows spirometer. The primary outcome variable of interest was the percentage of predicted forced expiratory volume in 1 s (% predicted FEV1 ). Analysis included multiple linear regression with adjustment for various confounders. RESULTS: Five-hundred and seven men were included in the analysis. The mean age was 63.6 years (SD = 3.1). Of these, 304 (60.0%) men had no or mild periodontitis, 105 (20.7%) had moderate periodontitis and 98 (19.3%) had severe periodontitis. Multiple linear regression analysis showed that a one unit increase in MDI was associated with a 0.71% loss (95% confidence interval: 0.06%-1.35%; p = .03) in % predicted FEV1 after adjustment for all confounders. CONCLUSIONS: In this group of dentate men from Northern Ireland, subgingival microbial diversity was associated with reduced respiratory function.

Regulation of caries-induced pulp inflammation by NLRP3 inflammasome: A laboratory-based investigation.

AIM: To evaluate the expression and function of the nod-like receptor pyrin domain containing 3 (NLRP3) inflammasome in caries induced pulpitis. METHODOLOGY: NLRP3 expression was determined with immunohistochemistry in the dental pulp and qPCR in dental pulp cells (DPCs). THP-1 macrophages expressing the apoptosis-related speck-like protein (ASC) and green fluorescent protein (GFP) fusion protein were used to assess NLRP3 inflammasome activation by live cell imaging, following treatment with lipopolysaccharide (LPS) and lipoteichoic acid (LTA). Caspase I inhibitor was used to confirm inflammasome activation. An ex-vivo pulpitis model in which the DPCs were co-cultured with THP-1 macrophages was used to study the effect of the NLRP3 inflammasome inhibitor (MCC950), and cytokines were measured using ELISA and multiplex array. Data were analysed using the t-test or anova followed by a Bonferroni post hoc test with the level of significance set at p ≤ .05. RESULTS: NLRP3 inflammasome was differentially expressed in dental pulp of sound and carious teeth. Treatment of DPCs with LTA significantly upregulates NLRP3 and IL-1 ß-expression (p < .05) and in induces more ASC specks formation compared to LPS. IL-ß release in response to LTA treatment is significantly reduced with Caspase I inhibitor suggesting inflammasome dependent mechanism (p < .01). NLRP3-specific inhibitor, MCC950, significantly reduced IL-1ß and IL-6 in an ex-vivo pulpitis model (p < .01) but had no effect on IL-8 or matrix metalloproteinase-9 (MMP-9). CONCLUSIONS: Expression and upregulation of NLRP3 inflammasome with caries and LTA treatment suggest a role in caries-induced pulpitis. NLRP3 inhibitor attenuated the release of selective inflammatory cytokines and could be a potential treatment target that merit further investigation.

Molecular biomarkers for objective assessment of symptomatic pulpitis: A systematic review and meta-analysis.

BACKGROUND: Inflammatory biomarkers are potentially useful targets for pulpal diagnostic tests that can identify pulp status and predict vital pulp treatment (VPT) outcome, however, their accuracy is unknown. OBJECTIVES: (1) Calculate sensitivity, specificity and diagnostic odds ratio (DOR) of previously investigated pulpitic biomarkers; (2) Determine if biomarker levels discriminate between clinical diagnoses of pulpitis based on the presence or absence of spontaneous pain (3) Evaluate if biomarker level can predict VPT outcome. METHODS: Searches: PubMed/MEDLINE, Ovid SP, Cochrane Central Register of Controlled Trials (CENTRAL), International Clinical Trials Registry Platform (ICTRP), ClinicalTrials.gov, Embase, Web of Science and Scopus in May 2023. INCLUSION: prospective and retrospective observational studies and randomized trials. Participants were humans with vital permanent teeth and a well-defined pulpal diagnosis. EXCLUSION: deciduous teeth, in vitro and animal studies. Risk of bias was assessed with modified-Downs and Black quality assessment checklist. Meta-analysis was performed using bivariate random effect model in Meta-DiSc 2.0 and RevMan and the quality of the evidence was assessed using Grading of Recommendations Assessment, Development and Evaluation. RESULTS: Fifty-six studies were selected, reporting >70 individual biomolecules investigating pulpal health and disease at the gene and protein level. Most studies were of low and fair quality. Among the biomolecules investigated, IL-8 and IL-6 demonstrated a level of diagnostic accuracy with high sensitivity, specificity and DOR to discriminate between healthy pulps and those exhibiting spontaneous pain suggestive of IRP (low-certainty evidence). However, none was shown to have high DOR and the ability to discriminate between pulpitic states (very low certainty evidence). Limited data suggests high levels of matrix metalloproteinase 9 correlate with poorer outcomes of full pulpotomy. DISCUSSION: The inability of identified molecular inflammatory markers to discriminate between dental pulps with spontaneous and non-spontaneous pain should shift the focus to improved study quality or the pursuit of other molecules potentially associated with healing and repair. CONCLUSIONS: Low-quality evidence suggests IL-8 and IL-6 demonstrated level of diagnostic accuracy to discriminate between healthy pulps and those exhibiting spontaneous pain. There is a need for standardized biomarker diagnostic and prognostic studies focusing on solutions that can accurately determine the degree of pulp inflammation. REGISTRATION: PROSPERO CRD42021259305.

The Role of Extracellular Matrix (ECM) Adhesion Motifs in Functionalised Hydrogels.

To create functional tissue engineering scaffolds, biomaterials should mimic the native extracellular matrix of the tissue to be regenerated. Simultaneously, the survival and functionality of stem cells should also be enhanced to promote tissue organisation and repair. Hydrogels, but in particular, peptide hydrogels, are an emerging class of biocompatible scaffolds which act as promising self-assembling biomaterials for tissue engineering and regenerative therapies, ranging from articular cartilage regeneration at joint defects, to regenerative spinal cord injury following trauma. To enhance hydrogel biocompatibility, it has become imperative to consider the native microenvironment of the site for regeneration, where the use of functionalised hydrogels with extracellular matrix adhesion motifs has become a novel, emerging theme. In this review, we will introduce hydrogels in the context of tissue engineering, provide insight into the complexity of the extracellular matrix, investigate specific adhesion motifs that have been used to generate functionalised hydrogels and outline their potential applications in a regenerative medicine setting. It is anticipated that by conducting this review, we will provide greater insight into functionalised hydrogels, which may help translate their use towards therapeutic roles.

Quantitative oral health assessments in mechanically ventilated patients: A scoping review.

BACKGROUND: Oral health is a key contributor to a person's overall health. Previous studies indicate that oral health deteriorates throughout ventilation and may contribute to the development of ventilator-associated pneumonia (VAP). Oral health at the time of initial ventilation may impact on this deterioration. AIMS: To determine the quantitative clinical assessment methods used to measure oral health and what is currently known regarding the oral health of patients at the time of initial ventilation. STUDY DESIGN: A systematic literature search using electronic bibliographic databases MEDLINE/PubMed, Embase, CINAHL, and the Cochrane Library was undertaken for this scoping review. Studies were included if patients were >18 years old and mechanically ventilated for <48 h at the time of the first oral assessment. RESULTS: In total, 12 studies were included. The review demonstrates a limited understanding of clinical oral health at the time of initial ventilation. Significant variation in both assessment methods and reporting of oral health makes comparison of results difficult resulting in a poor overall understanding of oral health at the time of intubation. CONCLUSION: Standardized assessment and reporting methods may improve clinical application of findings and help direct future research. We suggest developing a core outcome set to ensure consistent use of assessment tools as well as standardized reporting of results. RELEVANCE TO CLINICAL PRACTICE: It is essential that a good understanding of oral health at the time of initial ventilation is gained so that patients receive more targeted oral hygiene intervention in ICU, potentially leading to improved patient outcomes.

Oral health care in adult intensive care units: A national point prevalence study.

BACKGROUND: The importance of good oral hygiene for patients in Intensive Care Units (ICUs) is well recognized, however, the most effective way to achieve good oral care in the ICU is unclear. AIM: This study aimed to provide a national picture of oral care practices in adult ICUs in the United Kingdom (UK) to identify areas for improvement. STUDY DESIGN: A national one-day point prevalence study was undertaken in adult ICUs in the UK in the period from 30th September to 14th October 2021. Data were collected on all patients in the ICU on the date of data collection. Using a validated electronic data collection form, anonymised data were collected on methods and frequency of oral care provided, and the use of oral care protocols within the ICU. Data were analysed using descriptive analysis. RESULTS: Data from 195 patients in 15 ICUs in England, Wales and Northern Ireland were collected. Written oral care protocols were available for use in the care of 65% (n = 127) of patients. 73% (n = 142) of patients received oral care within the 24-h period. Oral care methods included toothbrushing 41% (n = 79), foam sticks 3% (n = 5), moisturizing the oral cavity 10% (n = 19) and mouth rinse with chlorhexidine 3% (n = 5) and other oral care methods not specified 12% (n = 23). 44% (n = 85) of patients had an oral assessment within the 24-h period and variable assessment methods were used. CONCLUSION: There is large variability in oral care provision and methods for intubated ICU patients and a lack of consensus was revealed in the study. Oral assessment is conducted less frequently using multiple tools. Optimal oral care standards and further research into oral care provision is pivotal to address this important patient-relevant practice. RELEVANCE TO CLINICAL PRACTICE: Oral care is a fundamental part of care for ICU patients, however, there is a large degree of variability, and oral care is often not based upon oral assessment. The use of an oral care protocol and oral assessments would help to improve patient care, ease of use for staff and provide a tailored oral care plan for patients, improving efficiency and preventing wasted resources.

Functional expression of NaV1.7 channels in freshly dispersed mouse bronchial smooth muscle cells.

Isolated smooth muscle cells (SMCs) from mouse bronchus were studied using the whole cell patch-clamp technique at ∼21°C. Stepping from -100 mV to -20 mV evoked inward currents of mean amplitude -275 pA. These inactivated (tau = 1.1 ms) and were abolished when external Na+ was substituted with N-Methyl-d-glucamine. In current-voltage protocols, current peaked at -10 mV and reversed between +20 and +30 mV. The V1/2s of activation and inactivation were -25 and -86 mV, respectively. The current was highly sensitive to tetrodotoxin (IC50 = 1.5 nM) and the NaV1.7 subtype-selective blocker, PF-05089771 (IC50 = 8.6 nM), consistent with NaV1.7 as the underlying pore-forming α subunit. Two NaV1.7-selective antibodies caused membrane-delineated staining of isolated SMC, as did a nonselective pan-NaV antibody. RT-PCR, performed on groups of ∼15 isolated SMCs, revealed transcripts for NaV1.7 in 7/8 samples. Veratridine (30 µM), a nonselective NaV channel activator, reduced peak current evoked by depolarization but induced a sustained current of 40 pA. Both effects were reversed by tetrodotoxin (100 nM). In tension experiments, veratridine (10 µM) induced contractions that were entirely blocked by atropine (1 µM). However, in the presence of atropine, veratridine was able to modulate the pattern of activity induced by a combination of U-46619 (a thromboxane A2 mimetic) and PGE2 (prostaglandin E2), by eliminating bursts in favor of sustained phasic contractions. These effects were readily reversed to control-like activity by tetrodotoxin (100 nM). In conclusion, mouse bronchial SMCs functionally express NaV1.7 channels that are capable of modulating contractile activity, at least under experimental conditions.

Endogenous Mas-related G-protein-coupled receptor X1 activates and sensitizes TRPA1 in a human model of peripheral nerves.

Mas-related G-protein-coupled receptor X1 (MrgprX1) is a human-specific Mrgpr and its expression is restricted to primary sensory neurons. However, its role in nociception and pain signaling pathways is largely unknown. This study aims to investigate a role for MrgprX1 in nociception via interaction with the pain receptor, Transient Receptor Potential Ankyrin 1 (TRPA1), using in-vitro and in-vivo human neuronal models. MrgprX1 protein expression in human trigeminal nociceptors was investigated by the immunolabeling of the dental pulp and cultured peripheral neuronal equivalent (PNE) cells. MrgprX1 receptor signaling was monitored by Fura-2-based Ca2+ imaging using PNEs and membrane potential responses were measured using FluoVoltTM . Immunofluorescent staining revealed MrgprX1 expression in-vivo in dental afferents, which was more intense in inflamed compared to healthy dental pulps. Endogenous MrgprX1 protein expression was confirmed in the in-vitro human PNE model. MrgprX1 receptor signaling and the mechanisms through which it couples to TRPA1 were studied by Ca2+ imaging. Results showed that MrgprX1 activates TRPA1 and induces membrane depolarization in a TRPA1 dependent manner. In addition, MrgprX1 sensitizes TRPA1 to agonist stimulation via Protein Kinase C (PKC). The activation and sensitization of TRPA1 by MrgprX1 in a model of human nerves suggests an important role for this receptor in the modulation of nociception.

Differential regulation of NPY and SP receptor expression in STRO-1+ve PDLSCs by inflammatory cytokines.

OBJECTIVES: The aims of this study were to investigate neuropeptide receptor expression regulation on STRO-1 +ve periodontal ligament stem cells (PDLSCs) in response to inflammatory cytokines and to investigate a potential osteogenic effect of neuropeptides. BACKGROUND: Nerve fibres innervating the periodontal tissues in humans contain several neuropeptides including neuropeptide Y and substance P. The role of neuropeptide receptors on PDLSCs, including their response to the local inflammatory environment of periodontitis, is currently unknown. METHODS: A homogenous population of STRO-1 +ve PDLSCs was prepared by immunomagnetic separation of cells obtained by the tissue out-growth method from healthy premolar teeth from a single donor. Regulation of gene expression of the neuropeptide Y Y1 receptor and substance P receptor tachykinin receptor 1 was investigated. A potential osteogenic effect of neuropeptide Y and substance P was also investigated by measuring alkaline phosphatase (ALP) activity, Alizarin red staining and quantifying osteogenic gene expression. RESULTS: Treatment of STRO-1 +ve PDLSCs with tumour necrosis factor-alpha or interleukin 1-beta up-regulated the expression of the neuropeptide Y's Y1 receptor, but down-regulated substance P's receptor. Significantly increased ALP activity was observed in STRO-1 +ve PDLSCs treated with neuropeptide Y but not substance P. Further studies showed that neuropeptide Y had a modest osteogenic effect on cells at both a functional level and a gene level. CONCLUSIONS: Expression of the neuropeptide Y Y1 receptor gene on STRO-1 +ve PDLSCs was sensitive to local inflammatory cytokines. Treatment of cells with neuropeptide Y was found to produce a modest enhanced osteogenic effect.

Outcomes reporting in systematic reviews on vital pulp treatment: A scoping review for the development of a core outcome set.

BACKGROUND: A large number of research reports on vital pulp treatment (VPT) has been published over the last two decades. However, heterogeneity in reporting outcomes of VPT is a significant challenge for evidence synthesis and clinical decision-making. OBJECTIVES: To identify outcomes assessed in VPT studies and to evaluate how and when outcomes are measured. A subsidiary aim was to assess evidence for selective reporting bias in the included studies. The results of this review will be used to inform the development of a core outcome set (COS) for endodontic treatments. METHODS: Multiple healthcare bibliographic databases, including PubMed/MEDLINE, Ovid EMBASE, Scopus, Cochrane Database of Systematic Reviews and Web of Science were searched for systematic reviews published between 1990 and 2020, reporting on VPT. Screening, data extraction and risk of bias assessment were completed independently by two reviewers. Outcomes' information was extracted and aligned with a healthcare taxonomy into five core areas: survival, clinical/physiological changes, life impact, resource use and adverse events. RESULTS: Thirty-six systematic reviews were included, 10 reporting on indirect pulp capping or selective caries removal, nine on direct pulp capping, eight on pulpotomy and nine on combined VPTs. There was considerable variation in the outcomes reported in these reviews and their included studies. Clinician-reported outcomes were used considerably more often than patient-reported outcomes. A range of instruments and time points were used for measuring outcomes. Several of the reviews were assessed as having low risk of selective reporting bias, but many did not specifically report this domain, whilst others did not provide risk of bias assessment at all. DISCUSSION: Considerable variation in selection of outcomes and how and when they are measured and reported was evident, and this heterogeneity has implications for evidence synthesis and clinical decision-making. CONCLUSIONS: Whilst there is a lack of consistency, several potentially important outcomes for VPT, including pulp survival, incidence of post-operative pain and need for further intervention, have been identified which could inform the development of a COS for endodontic treatment. REGISTRATION: Core Outcome Measures in Effectiveness Trials (COMET) (No. 1879).

Non-typeable Haemophilus influenzae chronic colonization in chronic obstructive pulmonary disease (COPD).

Haemophilus influenzae is the most common cause of bacterial infection in the lungs of chronic obstructive pulmonary disease (COPD) patients and contributes to episodes of acute exacerbation which are associated with increased hospitalization and mortality. Due to the ability of H. influenzae to adhere to host epithelial cells, initial colonization of the lower airways can progress to a persistent infection and biofilm formation. This is characterized by changes in bacterial behaviour such as reduced cellular metabolism and the production of an obstructive extracellular matrix (ECM). Herein we discuss the multiple mechanisms by which H. influenzae contributes to the pathogenesis of COPD. In particular, mechanisms that facilitate bacterial adherence to host airway epithelial cells, biofilm formation, and microbial persistence through immune system evasion and antibiotic tolerance will be discussed.

Fusobacterium nucleatum and oral cancer: a critical review.

There is a growing level of interest in the potential role inflammation has on the initiation and progression of malignancy. Notable examples include Helicobacter pylori-mediated inflammation in gastric cancer and more recently Fusobacterium nucleatum-mediated inflammation in colorectal cancer. Fusobacterium nucleatum is a Gram-negative anaerobic bacterium that was first isolated from the oral cavity and identified as a periodontal pathogen. Biofilms on oral squamous cell carcinomas are enriched with anaerobic periodontal pathogens, including F. nucleatum, which has prompted hypotheses that this bacterium could contribute to oral cancer development. Recent studies have demonstrated that F. nucleatum can promote cancer by several mechanisms; activation of cell proliferation, promotion of cellular invasion, induction of chronic inflammation and immune evasion. This review provides an update on the association between F. nucleatum and oral carcinogenesis, and provides insights into the possible mechanisms underlying it.

Oral health care for the critically ill: a narrative review.

BACKGROUND: The link between oral bacteria and respiratory infections is well documented. Dental plaque has the potential to be colonized by respiratory pathogens and this, together with microaspiration of oral bacteria, can lead to pneumonia particularly in the elderly and critically ill. The provision of adequate oral care is therefore essential for the maintenance of good oral health and the prevention of respiratory complications. MAIN BODY: Numerous oral care practices are utilised for intubated patients, with a clear lack of consensus on the best approach for oral care. This narrative review aims to explore the oral-lung connection and discuss in detail current oral care practices to identify shortcomings and offer suggestions for future research. The importance of adequate oral care has been recognised in guideline interventions for the prevention of pneumonia, but practices differ and controversy exists particularly regarding the use of chlorhexidine. The oral health assessment is also an important but often overlooked element of oral care that needs to be considered. Oral care plans should ideally be implemented on the basis of an individual oral health assessment. An oral health assessment prior to provision of oral care should identify patient needs and facilitate targeted oral care interventions. CONCLUSION: Oral health is an important consideration in the management of the critically ill. Studies have suggested benefit in the reduction of respiratory complication such as Ventilator Associated Pneumonia associated with effective oral health care practices. However, at present there is no consensus as to the best way of providing optimal oral health care in the critically ill. Further research is needed to standardise oral health assessment and care practices to enable development of evidenced based personalised oral care for the critically ill.

Identification and validation of novel biomarkers and therapeutics for pulpitis using connectivity mapping.

AIM: To create an irreversible pulpitis gene signature from microarray data of healthy and inflamed dental pulps, followed by a bioinformatics approach using connectivity mapping to identify therapeutic compounds that could potentially treat pulpitis. METHODOLOGY: The Gene Expression Omnibus (GEO) database, an international public repository of genomics data sets, was searched for human microarray datasets assessing pulpitis. An irreversible pulpitis gene expression signature was generated by differential expression analysis. The statistically significant connectivity map (ssCMap) method was used to identify compounds with a highly correlating gene expression pattern. qPCR was used to validate novel pulpitis genes. An ex vivo pulpitis model was used to test the effects of the compounds identified, and the level of inflammatory cytokines was measured with qPCR, ELISA and multiplex array. Means were compared using the t-test or ANOVA with the level of significance set at p ≤ .05. RESULTS: Pulpitis gene signatures were created using differential gene expression analysis at cutoff points p = .0001 and .000018. Top upregulated genes were selected as potential pulpitis biomarkers. Among these, IL8, IL6 and MMP9 were previously identified as pulpitis biomarkers. Novel upregulated genes, chemokine (C-C motif) ligand 21 (CCL21), metallothionein 1H (MT1H) and aquaporin 9 (AQP9) were validated in the pulp tissue of teeth clinically diagnosed with irreversible pulpitis using qPCR. ssCMap analysis identified fluvastatin (Statin) and dequalinium chloride (Quaternary ammonium) as compounds with the strongest correlation to the gene signatures (p = .0001). Fluvastatin reduced IL8, IL6, CCL21, AQP9 (p < .001) and MMP9 (p < .05) in the ex vivo pulpitis model, while dequalinium chloride reduced AQP9 (p < .001) but had no significant effect on the other biomarkers. CONCLUSIONS: AQP9, MT1H and CCL21 were identified and validated as novel biomarkers for pulpitis. Fluvastatin and dequalinium chloride identified by the ssCMap as potential therapeutics for pulpitis reduced selected pulpitis biomarkers in an ex vivo pulpitis model. In vivo testing of these licenced drugs is warranted.

Mammalian Neuropeptides as Modulators of Microbial Infections: Their Dual Role in Defense versus Virulence and Pathogenesis.

The regulation of infection and inflammation by a variety of host peptides may represent an evolutionary failsafe in terms of functional degeneracy and it emphasizes the significance of host defense in survival. Neuropeptides have been demonstrated to have similar antimicrobial activities to conventional antimicrobial peptides with broad-spectrum action against a variety of microorganisms. Neuropeptides display indirect anti-infective capacity via enhancement of the host's innate and adaptive immune defense mechanisms. However, more recently concerns have been raised that some neuropeptides may have the potential to augment microbial virulence. In this review we discuss the dual role of neuropeptides, perceived as a double-edged sword, with antimicrobial activity against bacteria, fungi, and protozoa but also capable of enhancing virulence and pathogenicity. We review the different ways by which neuropeptides modulate crucial stages of microbial pathogenesis such as adhesion, biofilm formation, invasion, intracellular lifestyle, dissemination, etc., including their anti-infective properties but also detrimental effects. Finally, we provide an overview of the efficacy and therapeutic potential of neuropeptides in murine models of infectious diseases and outline the intrinsic host factors as well as factors related to pathogen adaptation that may influence efficacy.