Multi-endpoint in vitro toxicological assessment of snus and tobacco-free nicotine pouch extracts.

'Modern' oral tobacco-free nicotine pouches (NPs) are a nicotine containing product similar in appearance and concept to Swedish snus. A three-step approach was taken to analyse the biological effects of NPs and snus extracts in vitro. ToxTracker was used to screen for biomarkers for oxidative stress, cell stress, protein damage and DNA damage. Cytotoxicity, mutagenicity, and genotoxicity were assessed in the following respective assays: Neutral Red Uptake (NRU), Ames and Mouse Lymphoma Assay (MLA). Targeted analysis of phosphorylation signalling and inflammatory markers under non-toxic conditions was used to investigate any potential signalling pathways or inflammatory response. A reference snus (CRP1.1) and four NPs with various flavours and nicotine strengths were assessed. Test article extracts was generated by incubating one pouch in 20 mL of media (specific to each assay) with the inclusion of the pouch material. NP extracts did not induce any cytotoxicity or mutagenic response, genotoxic response was minimal and limited signalling or inflammatory markers were induced. In contrast, CRP1.1 induced a positive response in four toxicological endpoints in the absence of S9: Srxn1 (oxidative stress), Btg2 (cell stress), Ddit3 (protein damage) and Rtkn (DNA damage), and three endpoints in presence of S9: Srxn1, Ddit3 and Rtkn. CRP1.1 was genotoxic when assessed in MLA and activated signalling pathways involved in proliferation and cellular stress and specifically induced phosphorylation of c-JUN, CREB1, p53, p38 MAPK and to a lesser extent AKT1S1, GSK3α/ß, ERK1/2 and RSK1 in a dose-dependent manner. CRP 1.1 extracts resulted in the release of several inflammatory mediators including cytokines IL-1α, IL5, IL6, IL8, IL-1RA, MIF and TNF-ß, receptor IL-2RA, and growth factors FGF-basic, VEGF and M-CSF. In conclusion these assays contribute to the weight of evidence assessment of the potential comparative health risks of NPs and snus.

TNF-α signals through ITK-Akt-mTOR to drive CD4+ T cell metabolic reprogramming, which is dysregulated in rheumatoid arthritis.

Upon activation, T cells undergo metabolic reprogramming to meet the bioenergetic demands of clonal expansion and effector function. Because dysregulated T cell cytokine production and metabolic phenotypes coexist in chronic inflammatory disease, including rheumatoid arthritis (RA), we investigated whether inflammatory cytokines released by differentiating T cells amplified their metabolic changes. We found that tumor necrosis factor-α (TNF-α) released by human naïve CD4+ T cells upon activation stimulated the expression of a metabolic transcriptome and increased glycolysis, amino acid uptake, mitochondrial oxidation of glutamine, and mitochondrial biogenesis. The effects of TNF-α were mediated by activation of Akt-mTOR signaling by the kinase ITK and did not require the NF-κB pathway. TNF-α stimulated the differentiation of naïve cells into proinflammatory T helper 1 (TH1) and TH17 cells, but not that of regulatory T cells. CD4+ T cells from patients with RA showed increased TNF-α production and consequent Akt phosphorylation upon activation. These cells also exhibited increased mitochondrial mass, particularly within proinflammatory T cell subsets implicated in disease. Together, these findings suggest that T cell-derived TNF-α drives their metabolic reprogramming by promoting signaling through ITK, Akt, and mTOR, which is dysregulated in autoinflammatory disease.

Uptake of long-chain fatty acids from the bone marrow suppresses CD8+ T-cell metabolism and function in multiple myeloma.

T cells demonstrate impaired function in multiple myeloma (MM) but suppressive mechanisms in the bone marrow microenvironment remain poorly defined. We observe that bone marrow CD8+ T-cell function is decreased in MM compared with controls, and is also consistently lower within bone marrow samples than in matched peripheral blood samples. These changes are accompanied by decreased mitochondrial mass and markedly elevated long-chain fatty acid uptake. In vitro modeling confirmed that uptake of bone marrow lipids suppresses CD8+ T function, which is impaired in autologous bone marrow plasma but rescued by lipid removal. Analysis of single-cell RNA-sequencing data identified expression of fatty acid transport protein 1 (FATP1) in bone marrow CD8+ T cells in MM, and FATP1 blockade also rescued CD8+ T-cell function, thereby identifying this as a novel target to augment T-cell activity in MM. Finally, analysis of samples from cohorts of patients who had received treatment identified that CD8+ T-cell metabolic dysfunction resolves in patients with MM who are responsive to treatment but not in patients with relapsed MM, and is associated with substantial T-cell functional restoration.

Canagliflozin impairs T cell effector function via metabolic suppression in autoimmunity.

Augmented T cell function leading to host damage in autoimmunity is supported by metabolic dysregulation, making targeting immunometabolism an attractive therapeutic avenue. Canagliflozin, a type 2 diabetes drug, is a sodium glucose co-transporter 2 (SGLT2) inhibitor with known off-target effects on glutamate dehydrogenase and complex I. However, the effects of SGLT2 inhibitors on human T cell function have not been extensively explored. Here, we show that canagliflozin-treated T cells are compromised in their ability to activate, proliferate, and initiate effector functions. Canagliflozin inhibits T cell receptor signaling, impacting on ERK and mTORC1 activity, concomitantly associated with reduced c-Myc. Compromised c-Myc levels were encapsulated by a failure to engage translational machinery resulting in impaired metabolic protein and solute carrier production among others. Importantly, canagliflozin-treated T cells derived from patients with autoimmune disorders impaired their effector function. Taken together, our work highlights a potential therapeutic avenue for repurposing canagliflozin as an intervention for T cell-mediated autoimmunity.

Advances in whole aerosol approaches for in vitro e-cigarette testing.

Electronic-cigarette regulation and risk assessment is a prominent and developing field, as the popularity and prevalence of this product category increases. Over the last 10 years since their emergence, there have been many advances and adaptations to current in vitro testing techniques to better assess and predict absolute consumer risk. However, there are still requirements to create a cross-field harmonised approach to appropriate exposure and experimental design. With many assessments still being carried out using methods developed and optimised for cigarette smoke, there must first be an acknowledgement regarding the differences between cigarette smoke and tobacco-free e-cigarette aerosols before we can accurately assess these distinct products. Here, we discuss five published studies from within our own research to demonstrate how in vitro testing techniques have evolved to improve determination of risk by considering appropriate dosimetry and exposure for both e-cigarette and cigarette aerosols and how we can contextualise the data through human consumption and dose extrapolation, ultimately giving more relevance to in vitro data. Furthermore, we have demonstrated the evolution of techniques, which has allowed us to bridge between platforms, simplify exposure set-up, experimental design and demonstrate technology evolution within our products, thus fulfilling a responsible duty of care to consumers via an appropriate and robust in vitro product assessment.

Design and Usability of an Avatar-Based Learning Program to Support Diabetes Education: Quality Improvement Study in Colombia.

BACKGROUND: This quality improvement study, entitled Avatar-Based LEarning for Diabetes Optimal Control (ABLEDOC), explored the feasibility of delivering an educational program to people with diabetes in Colombia. The aim was to discover how this approach could be used to improve awareness and understanding of the condition, the effects of treatment, and strategies for effective management of blood-glucose control. METHODS: Individuals with diabetes were recruited by Colombian endocrinologists to a human-centered study to codesign the educational program, using the Double Diamond model. Participants contributed to two phases. The first phase focused on gathering unmet educational needs and choice of curriculum. Three prototypes were developed as a result. During phase 2, a different group of participants engaged with the program for several weeks, before reporting back. RESULTS: Thirty-six participants completed a Web survey during phase 1, and five were also interviewed by telephone. The majority (33 of 36; 91%) were receptive to the prospect of educational interventions and ranked the chosen topic of hypoglycemia highly. In phase 2, the three prototypes were tested by 17 participants, 10 of whom also gave feedback in focus groups. The response was overwhelmingly positive, with 16 of 17 (94%) stating they would use a program like this again. The 3D version was the most highly rated. CONCLUSIONS: Immersive, avatar-based programs, delivered through smartphone, have the potential to deliver educational information that is trusted, engaging, and useful. Future work includes expansion of the curriculum, evaluation with a larger group, and exploration of the prospective role of artificial intelligence in personalizing this form of educational intervention.

Management of Clostridioides difficile infection in adults and challenges in clinical practice: review and comparison of current IDSA/SHEA, ESCMID and ASID guidelines.

Clostridioides difficile infection (CDI) remains a significant clinical challenge both in the management of severe and severe-complicated disease and the prevention of recurrence. Guidelines released by the Infectious Diseases Society of America and Society for Healthcare Epidemiology of America (IDSA/SHEA) and ESCMID had some consensus as well as some discrepancies in disease severity classification and treatment recommendations. We review and compare the key clinical strategies from updated IDSA/SHEA, ESCMID and current Australasian guidelines for CDI management in adults and discuss relevant issues for clinicians, particularly in the management of severe-complicated infection. Updated IDSA/SHEA and ESCMID guidelines now reflect the increased efficacy of fidaxomicin in preventing recurrence and have both promoted fidaxomicin to first-line therapy with an initial CDI episode in both non-severe and severe disease and endorsed the role of bezlotoxumab in the prevention of recurrent infection. Vancomycin remains acceptable therapy and metronidazole is not preferred. For severe-complicated infection the IDSA/SHEA recommends high-dose oral ±â€Šrectal vancomycin and IV metronidazole, whilst in an important development, ESCMID has endorsed fidaxomicin and tigecycline as part of combination anti-CDI therapy, for the first time. The role of faecal microbiota transplantation (FMT) in second CDI recurrence is now clearer, but timing and mode of FMT in severe-complicated refractory disease still requires further study.

Succinate uptake by T cells suppresses their effector function via inhibition of mitochondrial glucose oxidation.

Succinate dehydrogenase (SDH) loss-of-function mutations drive succinate accumulation in tumor microenvironments, for example in the neuroendocrine tumors pheochromocytoma (PC) and paraganglioma (PG). Control of innate immune cell activity by succinate is described, but effects on T cells have not been interrogated. Here we report that exposure of human CD4+ and CD8+ T cells to tumor-associated succinate concentrations suppresses degranulation and cytokine secretion, including of the key anti-tumor cytokine interferon-γ (IFN-γ). Mechanistically, this is associated with succinate uptake-partly via the monocarboxylate transporter 1 (MCT1)-inhibition of succinyl coenzyme A synthetase activity and impaired glucose flux through the tricarboxylic acid cycle. Consistently, pharmacological and genetic interventions restoring glucose oxidation rescue T cell function. Tumor RNA-sequencing data from patients with PC and PG reveal profound suppression of IFN-γ-induced genes in SDH-deficient tumors compared with those with other mutations, supporting a role for succinate in modulating the anti-tumor immune response in vivo.

Ciliary Beat Frequency: Proceedings and Recommendations from a Multi-laboratory Ring Trial Using 3-D Reconstituted Human Airway Epithelium to Model Mucociliary Clearance.

The use of reconstituted human airway (RHuA) epithelial tissues to assess functional endpoints is highly relevant in respiratory toxicology, but standardised methods are lacking. In June 2015, the Institute for In Vitro Sciences (IIVS) held a technical workshop to evaluate the potential for standardisation of methods, including ciliary beat frequency (CBF). The applicability of a protocol suggested in the workshop was assessed in a multi-laboratory ring study. This report summarises the findings, and uses the similarities and differences identified between the laboratories to make recommendations for researchers in the absence of a validated method. Two software platforms for the assessment of CBF were used - Sisson-Ammons Video Analysis (SAVA; Ammons Engineering, Clio, MI, USA) and ciliaFA (National Institutes of Health, Bethesda, MD, USA). Both were utilised for multiple read temperatures, one objective strength (10×) and up to four video captures per tissue, to assess their utility. Two commercial RHuA tissue cultures were used: MucilAir™ (Epithelix, Geneva, Switzerland) and EpiAirway™ (MatTek, Ashland, MA, USA). IL-13 and procaterol were used to induce CBF-specific responses as positive controls. Further testing addressed the impact of tissue acclimation duration, the number of capture fields and objective strengths on baseline CBF readings. Both SAVA and ciliaFA reliably collected CBF data. However, ciliaFA failed to generate accurate CBF measurements above ∼10 Hz. The positive controls were effective, but were subject to inter-laboratory variability. CBF endpoints were generally uniform across replicate tissues, objective strengths and laboratories. Longer tissue acclimation increased the percentage active area, but had minimal impact on CBF. Taken together, these findings support the development and validation of a standardised CBF measurement protocol.

1,25-Dihydroxyvitamin D3 suppresses CD4+ T-cell effector functionality by inhibition of glycolysis.

In CD4+ T helper cells, the active form of vitamin D3 , 1,25-dihydroxyvitamin D3 (1,25D) suppresses production of inflammatory cytokines, including interferon-gamma (IFN-γ), but the mechanisms for this are not yet fully defined. In innate immune cells, response to 1,25D has been linked to metabolic reprogramming. It is unclear whether 1,25D has similar effects on CD4+ T cells, although it is known that antigen stimulation of these cells promotes an anabolic metabolic phenotype, characterized by high rates of aerobic glycolysis to support clonal expansion and effector cytokine expression. Here, we performed in-depth analysis of metabolic capacity and pathway usage, employing extracellular flux and stable isotope-based tracing approaches, in CD4+ T cells treated with 1,25D. We report that 1,25D significantly decreases rates of aerobic glycolysis in activated CD4+ T cells, whilst exerting a lesser effect on mitochondrial glucose oxidation. This is associated with transcriptional repression of Myc, but not repression of mTOR activity under these conditions. Consistent with the modest effect of 1,25D on mitochondrial activity, it also did not impact CD4+ T-cell mitochondrial mass or membrane potential. Finally, we demonstrate that inhibition of aerobic glycolysis by 1,25D substantially contributes to its immune-regulatory capacity in CD4+ T cells, since the suppression of IFN-γ expression was significantly blunted in the absence of aerobic glycolysis. 1,25-Dihydroxyvitamin D3 (1,25D) suppresses the production of inflammatory cytokines such as interferon-gamma (IFN-γ) by CD4+ T cells, but the underpinning mechanisms are not yet fully defined. Here, we identify that 1,25D inhibits aerobic glycolysis in activated CD4+ T cells, associated with decreased c-Myc expression. This mechanism appears to substantially contribute to the suppression of IFN-γ by 1,25D, since this is significantly blunted in the absence of aerobic glycolysis.

How staying in a single room affects the experiences of haematology inpatients in an Australian cancer hospital.

BACKGROUND: It has been suggested that single rooms for patients improve patient dignity and privacy and reduce infection transmission, but they can be socially isolating. It is not well understood how single rooms affect long-stay patients. AIMS: To understand the experience of being an inpatient in a ward with single-room design. METHODS: A qualitative, phenomenological study was conducted using semi-structured interviews with patients (n=10) in a newly built cancer hospital with a 100% single-room haematology ward. Interviews were analysed using Colaizzi's (1978) seven-step analysis. FINDINGS: Patients described their experiences of their acute stay using the concepts of privacy, isolation and independence, as well as enabling sleep. Privacy enabled patients to have their own toilet, was perceived to aid infection control and provided silence. Privacy came at a cost of isolation, but patients re-framed this as expected and necessary for self-preservation. Furthermore, they were unsure as to whether other patients would reciprocate social contact and instead relied on the healthcare team. Patients sought independence during their acute stay as it enabled them to control the environment and create a space for healing. The ability to sleep and be rested was also a critical feature of patients' stay. CONCLUSION: The research highlighted that haematology patients prefer single rooms. However, because they experienced isolation, it also highlighted the importance of facilitating and enabling peer support within the haematology setting.

Autoimmune disease and interconnections with vitamin D.

Vitamin D has well-documented effects on calcium homeostasis and bone metabolism but recent studies suggest a much broader role for this secosteroid in human health. Key components of the vitamin D system, notably the vitamin D receptor (VDR) and the vitamin D-activating enzyme (1α-hydroxylase), are present in a wide array of tissues, notably macrophages, dendritic cells and T lymphocytes (T cells) from the immune system. Thus, serum 25-hydroxyvitamin D (25D) can be converted to hormonal 1,25-dihydroxyvitamin D (1,25D) within immune cells, and then interact with VDR and promote transcriptional and epigenomic responses in the same or neighbouring cells. These intracrine and paracrine effects of 1,25D have been shown to drive antibacterial or antiviral innate responses, as well as to attenuate inflammatory T cell adaptive immunity. Beyond these mechanistic observations, association studies have reported the correlation between low serum 25D levels and the risk and severity of human immune disorders including autoimmune diseases such as inflammatory bowel disease, multiple sclerosis, type 1 diabetes and rheumatoid arthritis. The proposed explanation for this is that decreased availability of 25D compromises immune cell synthesis of 1,25D leading to impaired innate immunity and over-exuberant inflammatory adaptive immunity. The aim of the current review is to explore the mechanistic basis for immunomodulatory effects of 25D and 1,25D in greater detail with specific emphasis on how vitamin D-deficiency (low serum levels of 25D) may lead to dysregulation of macrophage, dendritic cell and T cell function and increase the risk of inflammatory autoimmune disease.

Low rates of venous thromboembolism in hospitalised COVID-19 patients: an Australian experience.

BACKGROUND: Venous thromboembolic (VTE) complications appear common in hospitalised COVID-19 patients, particularly among critically ill patients in intensive care units. However, there is significant heterogeneity in the reported use of thromboprophylaxis. AIMS: The primary objective was to determine rates of symptomatic VTE in hospitalised COVID-19 patients. Secondary objectives were to assess adherence to an institutional risk-adapted thromboprophylaxis guideline, and rates of bleeding complications. METHODS: A retrospective, single-centre, cohort study was performed in consecutive hospitalised COVID-19 patients over a 6-month period (March to August 2020). Enoxaparin was used as thromboprophylaxis in all patients without a contraindication, with dose adjusted according to disease severity, weight and renal function. RESULTS: Among 86 hospitalised COVID-19 patients, no VTE were identified. Eighty-one (94%) patients received anticoagulation, with 90% adherence to institutional thromboprophylaxis guidelines. Four bleeding events occurred, with one clinically relevant non-major bleeding event and three minor bleeding events. CONCLUSION: Low rates of VTE were identified in hospitalised COVID-19 patients using a risk-adapted thromboprophylaxis protocol.

Survival Analysis of Training Methodologies and Other Risk Factors for Musculoskeletal Injury in 2-Year-Old Thoroughbred Racehorses in Queensland, Australia.

Musculoskeletal injuries remain a global problem for the Thoroughbred racing industry and there is conflicting evidence regarding the effect of age on the incidence of injuries. The ideal time to commence race training is strongly debated, with limited supporting literature. There is also conflicting evidence regarding the effect of high-speed exercise on musculoskeletal injuries. There is a strong interest in developing training and management strategies to reduce the frequency of injuries. The types of musculoskeletal injuries vary between 2-year-old and older horses, with dorsal metacarpal disease the most common injury in 2-year-old horses. It is likely that risk factors for injury in 2-year-old horses are different than those for older horses. It is also likely that the risk factors may vary between types of injury. This study aimed to determine the risk factors for musculoskeletal injuries and dorsal metacarpal disease. We report the findings of a large scale, prospective observational study of 2-year-old horses in Queensland, Australia. Data were collected weekly for 56-weeks, from 26 trainers, involving 535 2-year-old Thoroughbred racehorses, 1, 258 training preparations and 7, 512-weeks of exercise data. A causal approach was used to develop our statistical models, to build on the existing literature surrounding injury risk, by incorporating the previously established causal links into our analyses. Where previous data were not available, industry experts were consulted. Survival analyses were performed using Cox proportional hazards or Weibull regression models. Analysis of musculoskeletal injuries overall revealed the hazard was reduced with increased exposure to high-speed exercise [Hazard ratio (HR) 0.89, 95% Confidence Interval (CI) 0.84, 0.94, p < 0.001], increased number of training preparations (HR 0.58, 95% CI 0.50, 0.67, p < 0.001), increased rest before the training preparation (HR 0.89, 95% CI 0.83, 0.96, p = 0.003) and increased dam parity (HR 0.86, 95% CI 0.77, 0.97, p = 0.01). The hazard of injury was increased with increasing age that training commenced (HR 1.13, 95% CI 1.06, 1.19, p < 0.001). Analyses were then repeated with the outcome of interest dorsal metacarpal disease. Factors that were protective against dorsal metacarpal disease and musculoskeletal injuries overall included: increased total cumulative distance (HR 0.89, 95% CI 0.82, 0.97, p = 0.001) and total cumulative days exercised as a gallop (HR 0.96, 95% CI 0.92, 0.99, p = 0.03), the number of the training preparations (HR 0.43, 95% CI 0.30, 0.61, p < 0.001). The age that training commenced was harmful for both dorsal metacarpal disease (HR 1.17, 95% CI 1.07, 1.28, p < 0.001 and overall musculoskeletal injuries.). The use of non-ridden training modalities was protective for dorsal metacarpal disease (HR 0.89, 95% CI 0.81, 0.97, p = 0.008), but not musculoskeletal injuries overall. The male sex increased the hazard of DMD compared to females (HR 2.58, 95% CI 1.20, 5.56, p = 0.02), but not MSI overall. In summary, the hazard of musculoskeletal injury is greatest for 2-year-old horses that are born from uniparous mares, commence training at a later age, are in their first training preparation, have undertaken little high-speed exercise or had limited rest before their training preparation. The hazard of dorsal metacarpal disease is greatest for 2-year-old horses that are males, commence training at a later age, are in their first training preparation, have undertaken little high-speed exercise or had limited use of non-ridden training modalities. Close monitoring of these high-risk horses during their training program could substantially reduce the impact of MSI. Furthermore, an understanding of how training methodologies affect the hazard of MSI facilitates modification of training programs to mitigate the risk impact of injury. The strengths of this study include a large sample size, a well-defined study protocol and direct trainer interviews. The main limitation is the inherent susceptibility to survival bias.

A Prospective Study of Training Methods for Two-Year-Old Thoroughbred Racehorses in Queensland, Australia, and Analysis of the Differences in Training Methods between Trainers of Varying Stable Sizes.

Worldwide, musculoskeletal injuries remain a major problem for the Thoroughbred racing industry. There is a strong interest in developing training and management strategies to reduce the impact of musculoskeletal injuries, however, progress has been limited by studies reporting conflicting findings, and a limited understanding of the role of different training methods in preventing injury. There is little data on patterns of rest periods and exercise data and how these vary between trainers. This prospective study of two-year-old racehorses was conducted in Queensland, Australia and involved weekly personal structured interviews with 26 trainers over 56 weeks. Detailed daily exercise data for 535 horses providing 1258 training preparations and 7512 weeks at risk were collected. Trainers were categorised into three groups by the mean number of two-year-old horses that they had in work each week over the study duration: (1) Small stables with five or less, (2) Medium stables with 6 to 15 and (3) Large stables with greater than 15 horses in training. Differences between trainers with small, medium and large stable sizes were evaluated using linear regression, Kruskal-Wallis equality-of-populations rank test if linear models were mis-specified or Chi-squared tests for categorical variables. Significant differences were observed between trainers, with horses from larger stables accumulating a greater high-speed exercise volume (p < 0.001), attaining training milestones more frequently (p = 0.01) and taking less time to reach their training milestones (p = 0.001). This study provides detailed data to which training practices from other locations can be compared. Presenting actual training data rather than trainers' estimation of a typical program provides a more accurate assessment of training practices. Understanding how training practices vary between regions improves comparability of studies investigating risk factors and is an important step towards reducing the impact of musculoskeletal injuries.

Control of T Cell Metabolism by Cytokines and Hormones.

Dynamic, coordinated changes in metabolic pathway activity underpin the protective and inflammatory activity of T cells, through provision of energy and biosynthetic precursors for effector functions, as well as direct effects of metabolic enzymes, intermediates and end-products on signaling pathways and transcriptional mechanisms. Consequently, it has become increasingly clear that the metabolic status of the tissue microenvironment directly influences T cell activity, with changes in nutrient and/or metabolite abundance leading to dysfunctional T cell metabolism and interlinked immune function. Emerging evidence now indicates that additional signals are integrated by T cells to determine their overall metabolic phenotype, including those arising from interaction with cytokines and hormones in their environment. The impact of these on T cell metabolism, the mechanisms involved and the pathological implications are discussed in this review article.

Tolerogenic effects of 1,25-dihydroxyvitamin D on dendritic cells involve induction of fatty acid synthesis.

The active form of vitamin D, 1,25-dihydroxyvitamin D (1,25D) is a potent regulator of immune function, promoting anti-inflammatory, tolerogenic T cell responses by modulating antigen presentation by dendritic cells (DC). Transcriptomic analyses indicate that DC responses to 1,25D involve changes in glycolysis, oxidative phosphorylation, electron transport and the TCA cycle. To determine the functional impact of 1,25D-mediated metabolic remodelling, human monocyte-derived DC were differentiated to immature (+vehicle, iDC), mature (+LPS, mDC), and immature tolerogenic DC (+1,25D, itolDC) and characterised for metabolic function. In contrast to mDC which showed no change in respiration, itolDC showed increased basal and ATP-linked respiration relative to iDC. Tracer metabolite analyses using 13C -labeled glucose showed increased lactate and TCA cycle metabolites. Analysis of lipophilic metabolites of 13C-glucose revealed significant incorporation of label in palmitate and palmitoleate, indicating that 1,25D promotes metabolic fatty acid synthesis in itolDC. Inhibition of fatty acid synthesis in itolDC altered itolDC morphology and suppressed expression of CD14 and IL-10 by these cells. These data indicate that the ability of 1,25D to induce tolerogenic DC involves metabolic remodelling leading to synthesis of fatty acids.

Vitamin D and Immune Regulation: Antibacterial, Antiviral, Anti-Inflammatory.

Regulation of immune function continues to be one of the most well-recognized extraskeletal actions of vitamin D. This stemmed initially from the discovery that antigen presenting cells such as macrophages could actively metabolize precursor 25-hydroxyvitamin D (25D) to active 1,25-dihydroxyvitamin D (1,25D). Parallel observation that activated cells from the immune system expressed the intracellular vitamin D receptor (VDR) for 1,25D suggested a potential role for vitamin D as a localized endogenous modulator of immune function. Subsequent studies have expanded our understanding of how vitamin D exerts effects on both the innate and adaptive arms of the immune system. At an innate level, intracrine synthesis of 1,25D by macrophages and dendritic cells stimulates expression of antimicrobial proteins such as cathelicidin, as well as lowering intracellular iron concentrations via suppression of hepcidin. By potently enhancing autophagy, 1,25D may also play an important role in combatting intracellular pathogens such as M. tuberculosis and viral infections. Local synthesis of 1,25D by macrophages and dendritic cells also appears to play a pivotal role in mediating T-cell responses to vitamin D, leading to suppression of inflammatory T helper (Th)1 and Th17 cells, and concomitant induction of immunotolerogenic T-regulatory responses. The aim of this review is to provide an update on our current understanding of these prominent immune actions of vitamin D, as well as highlighting new, less well-recognized immune effects of vitamin D. The review also aims to place this mechanistic basis for the link between vitamin D and immunity with studies in vivo that have explored a role for vitamin D supplementation as a strategy for improved immune health. This has gained prominence in recent months with the global coronavirus disease 2019 health crisis and highlights important new objectives for future studies of vitamin D and immune function. © 2020 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Optimization of aqueous aerosol extract (AqE) generation from e-cigarettes and tobacco heating products for in vitro cytotoxicity testing.

Electronic cigarettes (e-cigarettes) and tobacco heating products (THPs) have reduced yields of toxicants and have recently emerged as a potentially safer alternative to combustible cigarettes. To understand if reduced toxicant exposure is associated with reductions in biological responses, there is a need for high-quality pre-clinical in vitro studies. Here, we investigated the cytotoxic response of human umbilical vein endothelial cells to conventional cigarette aqueous aerosol extracts (AqE) and highly concentrated AqEs from e-cigarettes (two generations of atomisers) and THPs (two variants). All AqE samples were generated by a standardized methodology and characterized for nicotine, propylene glycol and vegetable glycerol. The cigarette AqE caused a maximum 100 ± 0.00 % reduction in cell viability at 35 % dose (2.80 puffs) as opposed to 96.63 ± 2.73 % at 50 % (20 puffs) and 99.85 ± 0.23 % at 75 % (30 puffs) for the two THP variants (glo Bright Tobacco, glo Rich Tobacco), and 99.07 ± 1.61 % at the neat ePen2.0 e-cigarette (200 puffs). The AqE of the remaining e-cigarettes either resulted in an incomplete dose-response or did not elicit any response. The methods utilized were suitably sensitive to not only differentiate between cigarette, THP and e-cigarette aerosols but also to distinguish between products within each product category.

Evaluation of a high-throughput in vitro endothelial cell migration assay for the assessment of nicotine and tobacco delivery products.

Endothelial cell migration is a critical process in the maintenance of healthy blood vessels. Impaired endothelial migration is reportedly associated with the development of cardiovascular diseases. Here, we report on the development of a 96-well in vitro endothelial migration assay for the purpose of comparative toxicological assessment of a novel THP relative to cigarette smoke, to be able to rapidly inform regulatory decision making. Uniform scratches were induced in confluent human umbilical vein endothelial cells using the 96-pin wound maker and exposed to 3R4F cigarette or THP aqueous extracts (AqE). Endothelial migration was recorded over 24 h, and the rate of wound closure calculated using mean relative wound density rather than migration rate as previously reported. This self-normalising parameter accounts for starting wound size, by comparing the density of the scratch to the outer region at each time-point. Furthermore, wound width acceptance criteria was defined to further increase the sensitivity of the assay. 3R4F and THP AqE samples were tested at comparable nicotine concentrations. 3R4F showed significant cytotoxicity and inhibition of wound healing whereas THP AqE did not show any response in either endpoint. This 96-well endothelial migration assay was suitably sensitive to distinguish combustible cigarette and THP test articles.