Glycogen-fuelled metabolism supports rapid mucosal-associated invariant T cell responses.

Mucosal-associated invariant T (MAIT) cells are a subset of unconventional T cells which recognize a limited repertoire of ligands presented by the MHC class-I like molecule MR1. In addition to their key role in host protection against bacterial and viral pathogens, MAIT cells are emerging as potent anti-cancer effectors. With their abundance in human, unrestricted properties, and rapid effector functions MAIT cells are emerging as attractive candidates for immunotherapy. In the current study, we demonstrate that MAIT cells are potent cytotoxic cells, rapidly degranulating and inducing target cell death. Previous work from our group and others has highlighted glucose metabolism as a critical process for MAIT cell cytokine responses at 18 h. However, the metabolic processes supporting rapid MAIT cell cytotoxic responses are currently unknown. Here, we show that glucose metabolism is dispensable for both MAIT cell cytotoxicity and early (<3 h) cytokine production, as is oxidative phosphorylation. We show that MAIT cells have the machinery required to make (GYS-1) and metabolize (PYGB) glycogen and further demonstrate that that MAIT cell cytotoxicity and rapid cytokine responses are dependent on glycogen metabolism. In summary, we show that glycogen-fueled metabolism supports rapid MAIT cell effector functions (cytotoxicity and cytokine production) which may have implications for their use as an immunotherapeutic agent.

Study of Characteristics of COVID-19 Patients with Fatal Outcome during Wave-2 from a Tertiary Care Center in Western India.

BACKGROUND: A large surge of intensive care unit (ICU) admissions leading to mortal outcome was observed in wave-2 of coronavirus disease 2019 (COVID-19) due to the higher virulence of the Delta variant of the COVID-19 virus, which led to the scarcity of resources in hospitals. This study was done to observe the clinical characteristics of COVID-19 patients with fatal outcomeMaterials and methods: We conducted a retrospective cross-sectional study in adults with COVID-19 pneumonia having fatal outcome during wave-2 of COVID-19, and their clinical characteristics were studiedResults: Out of 136 patients included in the study, the most common risk factors leading to adverse outcome were in the male gender, age (middle and elderly), with hypertension and diabetes mellitus (DM) as predominant comorbidities, early onset dyspnea, high C-reactive protein (CRP), high neutrophil to lymphocyte ratio (NLR), high D-dimer, bilateral lower zone involvement of lungs in chest X-ray (CXR), and development of acute kidney injury (AKI)Conclusion: The characteristics of the severely ill COVID-19 patients highlighted in the study could help clinicians in the early identification and management of high-risk patients. This study would help with resource planning and preparation for further COVID-19 waves and future pandemics.

Mucosal-associated invariant T cells are associated with insulin resistance in childhood obesity, and disrupt insulin signalling via IL-17.

AIMS/HYPOTHESIS: Mucosal-associated invariant T cells (MAIT cells) are an abundant population of innate T cells. When activated, MAIT cells rapidly produce a range of cytokines, including IFNγ, TNF-α and IL-17. Several studies have implicated MAIT cells in the development of metabolic dysfunction, but the mechanisms through which this occurs are not fully understood. We hypothesised that MAIT cells are associated with insulin resistance in children with obesity, and affect insulin signalling through their production of IL-17. METHODS: In a cross-sectional observational study, we investigated MAIT cell cytokine profiles in a cohort of 30 children with obesity and 30 healthy control participants, of similar age, using flow cytometry. We then used a cell-based model to determine the direct effect of MAIT cells and IL-17 on insulin signalling and glucose uptake. RESULTS: Children with obesity display increased MAIT cell frequencies (2.2% vs 2.8%, p=0.047), and, once activated, these produced elevated levels of both TNF-α (39% vs 28%, p=0.03) and IL-17 (1.25% vs 0.5%, p=0.008). The IL-17-producing MAIT cells were associated with an elevated HOMA-IR (r=0.65, p=0.001). The MAIT cell secretome from adults with obesity resulted in reduced glucose uptake when compared with the secretome from healthy adult control (1.31 vs 0.96, p=0.0002), a defect that could be blocked by neutralising IL-17. Finally, we demonstrated that recombinant IL-17 blocked insulin-mediated glucose uptake via inhibition of phosphorylated Akt and extracellular signal-regulated kinase. CONCLUSIONS/INTERPRETATIONS: Collectively, these studies provide further support for the role of MAIT cells in the development of metabolic dysfunction, and suggest that an IL-17-mediated effect on intracellular insulin signalling is responsible.

Differential association of plasma angiopoietin-like proteins 3 and 4 with lipid and metabolic traits.

OBJECTIVE: Angiopoietin-like protein 3 (ANGPTL3) and 4 (ANGPTL4) are secreted proteins that inhibit lipoprotein lipase in vitro. Genetic variants at the ANGPTL3 and ANGPTL4 gene loci are significantly associated with plasma lipid traits. The aim of this study was to evaluate the association of plasma ANGPTL3 and ANGPTL4 concentrations with lipid and metabolic traits in a large community-based sample. APPROACH AND RESULTS: Plasma ANGPTL3 and ANGPTL4 levels were measured in 1770 subjects using a validated ELISA assay. A Pearson unadjusted correlation analysis and a linear regression analysis adjusting for age, sex, and race were performed. ANGPTL3 levels were significantly positively associated with low-density lipoprotein cholesterol and high-density lipoprotein cholesterol levels (both P<2×10(-5)) but not triglycerides. In contrast, ANGPTL4 levels were significantly negatively associated with low-density lipoprotein cholesterol and high-density lipoprotein cholesterol (both P<2×10(-5)) and positively associated with triglycerides (P=0.003). In addition, ANGPTL4, but not ANGPTL3, levels were significantly positively associated with fasting blood glucose and metabolic syndrome. CONCLUSIONS: Despite having similar biochemical effects in vitro, plasma ANGPTL3 and ANGPTL4 concentrations have nearly opposite relationships with plasma lipids. ANGPTL4 is strongly negatively associated with low-density lipoprotein cholesterol and high-density lipoprotein cholesterol and positively with multiple features of the metabolic syndrome including triglycerides, whereas ANGPTL3 is positively associated with low-density lipoprotein cholesterol and high-density lipoprotein cholesterol and not with metabolic syndrome traits including triglycerides. Although ANGPTL3 and ANGPTL4 both inhibit lipoprotein lipase in vitro and influence lipoprotein metabolism in vivo, the physiology of these related proteins and their effects on lipoproteins is clearly divergent and complex.

One size does not fit all: on how Markov model order dictates performance of genomic sequence analyses.

The structural simplicity and ability to capture serial correlations make Markov models a popular modeling choice in several genomic analyses, such as identification of motifs, genes and regulatory elements. A critical, yet relatively unexplored, issue is the determination of the order of the Markov model. Most biological applications use a predetermined order for all data sets indiscriminately. Here, we show the vast variation in the performance of such applications with the order. To identify the 'optimal' order, we investigated two model selection criteria: Akaike information criterion and Bayesian information criterion (BIC). The BIC optimal order delivers the best performance for mammalian phylogeny reconstruction and motif discovery. Importantly, this order is different from orders typically used by many tools, suggesting that a simple additional step determining this order can significantly improve results. Further, we describe a novel classification approach based on BIC optimal Markov models to predict functionality of tissue-specific promoters. Our classifier discriminates between promoters active across 12 different tissues with remarkable accuracy, yielding 3 times the precision expected by chance. Application to the metagenomics problem of identifying the taxum from a short DNA fragment yields accuracies at least as high as the more complex mainstream methodologies, while retaining conceptual and computational simplicity.

An Expanded Genetic Toolbox to Accelerate the Creation of Acholeplasma laidlawii Driven by Synthetic Genomes.

We have developed genetic tools for the atypical bacterium Acholeplasma laidlawii. A. laidlawii is a member of the class Mollicutes, which lacks cell walls, has small genomes, and has limited metabolic capabilities, requiring many metabolites from their hosts. Several of these traits have facilitated the development of genome transplantation for some Mollicutes, consequently enabling the generation of synthetic cells. Here, we propose the development of genome transplantation for A. laidlawii. We first investigated a donor-recipient relationship between two strains, PG-8A and PG-8195, through whole-genome sequencing. We then created multihost shuttle plasmids and used them to optimize an electroporation protocol. We also evolved a superior strain for DNA uptake via electroporation. We created a PG-8A donor strain with a Tn5 transposon carrying a tetracycline resistance gene. These tools will enhance Acholeplasma research and accelerate the effort toward creating A. laidlawii strains with synthetic genomes.

Pharmacologic interactions of multidrug therapy for dyslipidemia.

Despite the best available medical therapy inclusive of statins, substantial residual risk remains for atherothrombotic cardiovascular disease. Non-statin lipid-lowering therapy may help address this critical unmet need through reduction of the levels of low-density lipoprotein and other atherogenic lipoproteins. In the past few years, several landmark trials have provided important information regarding the efficacy and safety of non-statin therapy for dyslipidemia and cardiovascular risk reduction.

Shear bond strength of acrylic teeth to acrylic denture base after different surface conditioning methods.

BACKGROUND AND OBJECTIVES: Acrylic resin ruled the dental profession for 60 years, and this success is attributed to its aesthetics, handling properties, physical and biological compatibility, its stability in oral environment and its cost effectiveness. The objective of this study is to evaluate and compare the bond strength of acrylic resin teeth treated with various conditioning materials like monomer and silane coupling agent. METHDOLOGY: A study was carried out in which 96 samples were grouped into 3 groups with a sample size of 32 each (16 premolars, 16 molars). They were conditioned with different conditioning materials i,e monomer and silane coupling agent. Monomer, Silane coupling agent are coated on the ridge lap area before thermocycling and cured according to the manufacturer recommendations. The samples are retained from the fask; trimmed and polished. The samples are then subjected to shear bond strength using the Insteron Universal Testing Machine. RESULTS: In the present study it was found that application of monomer increased the bond strength between acrylic teeth and denture base, when compared to the conventionally processed samples. However it was found that application of silane coupling agent further increased the shear bond strength between acrylic teeth and denture base. Interprations and CONCLUSIONS: Within the confnes of this study it is found that there was a signifcant improvement in the bond strength between the acrylic teeth and denture base when silane coupling agent and monomer were used as surface conditioning material. The order of shear strength of samples is control > monomer > silane coupling agent.

Assessment of utilisation of government programmes and services by pregnant women in India.

BACKGROUND: Since the implementation of various maternal health programs, Maternal Mortality Ratio (MMR) has significantly declined in India through improvements in maternal health services. However, inequality persists at the regional and socio-economic levels. In light of this, the present study aims to assess the existing regional disparities in utilising various government initiatives for safe motherhood in India. METHODS: National-level datasets such as National Family and Health Surveys (NFHS-3 (2005-06); NFHS-4 (2015-16) and NFHS-5(2019-21); Health Management Information System (HMIS), 2019-20; Sample Registrar System (SRS), 2001-2018) were used in the study. In addition, composite Index and inequality measures (Range, Ratio, and Gini) were calculated to examine inequality. At the same time, the Pearson correlation was used to investigate the correlation between various components of maternal health services and Maternal Mortality Rate (MMR). RESULTS: The composite index score (0.65) reflects that India is still far behind the targets of the utilisation of maternal health care services. Within the utilisation of services, the Gini coefficient reveals that the least inequality was recorded in skilled birth assistance deliveries (0.03) and institutional deliveries (0.04). In contrast, the highest inequality was recorded in receiving Iron and Folic Acid (IFA) Tablets for 100 days (0.19) and four Antenatal Care (ANC) visits (0.13) among selected states. Based on the composite score for maternal health utilisation, Kerala, Tamil Nadu, Andhra Pradesh, Odisha, and Delhi were amongst the best performers, whereas Bihar, Jharkhand, Uttar Pradesh, and Assam were amongst the worst performers. CONCLUSION: This indicates that the government's single-minded focus on enhancing institutional deliveries and skilled health-assisted deliveries has detracted from other essential interventions related to maternal health. Therefore, the states with the utilisation of maternal services need to initiate immediate action to increase the ANC and Post-natal Care (PNC utilisation with more attention towards better implementation of existing ANC programmes by the government.

The proliferation of human mucosal-associated invariant T cells requires a MYC-SLC7A5-glycolysis metabolic axis.

Mucosal-associated invariant T (MAIT) cells are an abundant population of innate T cells that recognize bacterial ligands and play a key role in host protection against bacterial and viral pathogens. Upon activation, MAIT cells undergo proliferative expansion and increase their production of effector molecules such as cytokines. In this study, we found that both mRNA and protein abundance of the key metabolism regulator and transcription factor MYC was increased in stimulated MAIT cells. Using quantitative mass spectrometry, we identified the activation of two MYC-controlled metabolic pathways, amino acid transport and glycolysis, both of which were necessary for MAIT cell proliferation. Last, we showed that MAIT cells isolated from people with obesity showed decreased MYC mRNA abundance upon activation, which was associated with defective MAIT cell proliferation and functional responses. Collectively, our data uncover the importance of MYC-regulated metabolism for MAIT cell proliferation and provide additional insight into the molecular basis for the functional defects of MAIT cells in obesity.

MAITabolism2 - the emerging understanding of MAIT cell metabolism and their role in metabolic disease.

Mucosal associated invariant T (MAIT) cells are a population of unconventional innate T cells due to their non-MHC restriction and rapid effector responses. MAIT cells can recognise bacterial derived antigens presented on the MHC-like protein via their semi-restricted T cell receptor (TCR). Upon TCR triggering MAIT cells rapidly produce a range of effector molecules including cytokines, lytic granules and chemokines. This rapid and robust effector response makes MAIT cells critical in host responses against many bacterial pathogens. MAIT cells can also respond independent of their TCR via innate cytokines such as interleukin (IL)-18, triggering cytokine production, and are important in anti-viral responses. In addition to their protective role, MAIT cells have been implicated in numerous inflammatory diseases, including metabolic diseases often contributing to the pathogenesis via their robust cytokine production. Effector cells such as MAIT cells require significant amounts of energy to support their potent responses, and the type of nutrients available can dictate the functionality of the cell. Although data on MAIT cell metabolism is just emerging, several recent studies are starting to define the intrinsic metabolic requirements and regulators of MAIT cells. In this review we will outline our current understanding of MAIT cell metabolism, and outline their role in metabolic disease, and how disease-related changes in extrinsic metabolism can alter MAIT cell responses.

Cytokine-induced natural killer cell training is dependent on cellular metabolism and is defective in obesity.

Natural killer (NK) cells are a population of innate immune cells that can rapidly kill cancer cells and produce cytokines such as interferon-γ. A key feature of NK cells is their ability to respond without prior sensitization; however, it is now well established that NK cells can possess memory-like features. After activation with cytokines, NK cells demonstrate enhanced effector functions upon restimulation days or weeks later. This demonstrates that NK cells may be trained to be more effective killers and harnessed as more potent cancer immunotherapy agents. We have previously demonstrated that cellular metabolism is essential for NK cell responses, with NK cells upregulating both glycolysis and oxidative phosphorylation upon cytokine stimulation. Limiting NK cell metabolism results in reduced cytotoxicity and cytokine production. We have also demonstrated that defective NK cell responses in obesity are linked to defective cellular metabolism. In the current study, we investigated if cellular metabolism is required during the initial period of NK cell cytokine training and if NK cells from people with obesity (PWO) can be effectively trained. We show that increased flux through glycolysis and oxidative phosphorylation during the initial cytokine activation period is essential for NK cell training, as is the metabolic signaling factor Srebp. We show that NK cells from PWO, which are metabolically defective, display impaired NK cell training, which may have implications for immunotherapy in this particularly vulnerable group.

Competition for nutrients and its role in controlling immune responses.

Changes in cellular metabolism are associated with the activation of diverse immune subsets. These changes are fuelled by nutrients including glucose, amino acids and fatty acids, and are closely linked to immune cell fate and function. An emerging concept is that nutrients are not equally available to all immune cells, suggesting that the regulation of nutrient utility through competitive uptake and use is important for controlling immune responses. This review considers immune microenvironments where nutrients become limiting, the signalling alterations caused by insufficient nutrients, and the importance of nutrient availability in the regulation of immune responses.

Natural Killer Cells Integrate Signals Received from Tumour Interactions and IL2 to Induce Robust and Prolonged Anti-Tumour and Metabolic Responses.

Natural Killer (NK) cells are lymphocytes with an important role in anti-tumour responses. NK cells bridge the innate and adaptive arms of the immune system; they are primed for immediate anti-tumour function but can also have prolonged actions alongside the adaptive T cell response. However, the key signals and cellular processes that are required for extended NK cell responses are not fully known. Herein we show that murine NK cell interaction with tumour cells induces the expression of CD25, the high affinity IL2 receptor, rendering these NK cells highly sensitive to the T cell-derived cytokine IL2. In response to IL2, CD25high NK cells show robust increases in metabolic signalling pathways (mTORC1, cMyc), nutrient transporter expression (CD71, CD98), cellular growth and in NK cell effector functions (IFNγ, granzyme B). Specific ligation of an individual activating NK cell receptor, NK1.1, showed similar increases in CD25 expression and IL2-induced responses. NK cell receptor ligation and IL2 collaborate to induce mTORC1/cMyc signalling leading to high rates of glycolysis and oxidative phosphorylation (OXPHOS) and prolonged NK cell survival. Disrupting mTORC1 and cMyc signalling in CD25high tumour interacting NK cells prevents IL2-induced cell growth and function and compromises NK cell viability. This study reveals that tumour cell interactions and T cell-derived IL2 cooperate to promote robust and prolonged NK cell anti-tumour metabolic responses.

Platelet C4d is associated with acute ischemic stroke and stroke severity.

BACKGROUND AND PURPOSE: Platelets bearing complement C4d were recently reported to be 99% specific for a diagnosis of systemic lupus erythematosus (SLE) and associated with neuropsychiatric lupus. We compared the prevalence of platelet C4d and investigated the clinical associations of platelet C4d in patients with acute ischemic stroke. METHODS: We recruited 80 patients hospitalized for acute ischemic stroke. Stroke severity was measured by the National Institutes of Health Stroke Scale (NIH-SS). Infarct volume was determined by MRI. Platelet C4d was measured by flow cytometry. RESULTS: Mean age was 57.9 years (range: 24.6 to 86.8 years), 58% were male, and 91% were white. Eight patients (10%) with acute ischemic stroke were platelet C4d-positive, which was significantly higher in prevalence compared to healthy controls (0%, P<0.0001) and non-SLE patients with immune/inflammatory disease (2%, P=0.004). The median NIH-SS score and infarct volume for acute stroke patients were 6 (interquartile range [IQR]: 2 to 13) and 3.4 cc (IQR: 1.1 to 16.6), respectively. Platelet C4d-positive patients were more likely to have a severe stroke compared to those with negative platelet C4d (NIH-SS median: 17.5 versus 5, P=0.003). Positive platelet C4d was independently associated with stroke severity (P=0.03) after controlling for age, anticardiolipin antibody (aCL) status, and total anterior circulation of stroke involvement, and also with infarct volume (P=0.005) after controlling for age, aCL status, and old stroke by MRI. CONCLUSIONS: Platelet C4d is associated with severe acute ischemic stroke. Platelet C4d may be a biomarker as well as pathogenic clue that links cerebrovascular inflammation and thrombosis.

Selection of Appropriate Autoinducer Analogues for the Modulation of Quorum Sensing at the Host-Bacterium Interface.

Bacteria regulate a variety of phenotypes in response to their population density using quorum sensing (QS). This phenomenon is regulated by small molecule or peptide signals, the best characterized of which are the N-acyl l-homoserine lactones (AHLs) utilized by Gram-negative bacteria. As many QS-controlled phenotypes, notably pathogenicity and symbiosis, can profoundly impact host eukaryotes, there is significant interest in developing methods for modulating QS signaling and either ameliorating or augmenting these phenotypes. One strategy has been the use of non-native AHL analogues to agonize or antagonize specific AHL receptors. This approach is complicated, however, by the potential for prospective hosts to respond to both native AHLs and synthetic analogues. Accordingly, identifying AHL analogues with little or no activity toward eukaryotes is important in developing QS modulation as a strategy for the regulation of prokaryotic behaviors. Herein, we utilize the model plant Arabidopsis thaliana to characterize eukaryotic responses to a variety of synthetic AHL analogues to identify structural elements of existing scaffolds that may elicit responses in prospective hosts. Our results indicate that, while many of these compounds have no discernible effect on A. thaliana, some elicit strong phenotypes similar to those produced by auxin, a hormone involved in almost all aspects of plant development. We outline concentrations and chemical scaffolds that are ideal for deployment on plant hosts for the regulation of QS. This approach should be exportable to other eukaryotes for the selection of optimal AHL tools for the study of QS at the host-microbe interface.

Amino acid-dependent cMyc expression is essential for NK cell metabolic and functional responses in mice.

Natural killer (NK) cells are lymphocytes with important anti-tumour functions. Cytokine activation of NK cell glycolysis and oxidative phosphorylation (OXPHOS) are essential for robust NK cell responses. However, the mechanisms leading to this metabolic phenotype are unclear. Here we show that the transcription factor cMyc is essential for IL-2/IL-12-induced metabolic and functional responses in mice. cMyc protein levels are acutely regulated by amino acids; cMyc protein is lost rapidly when glutamine is withdrawn or when system L-amino acid transport is blocked. We identify SLC7A5 as the predominant system L-amino acid transporter in activated NK cells. Unlike other lymphocyte subsets, glutaminolysis and the tricarboxylic acid cycle do not sustain OXPHOS in activated NK cells. Glutamine withdrawal, but not the inhibition of glutaminolysis, results in the loss of cMyc protein, reduced cell growth and impaired NK cell responses. These data identify an essential role for amino acid-controlled cMyc for NK cell metabolism and function.

Glucose represses dendritic cell-induced T cell responses.

Glucose and glycolysis are important for the proinflammatory functions of many immune cells, and depletion of glucose in pathological microenvironments is associated with defective immune responses. Here we show a contrasting function for glucose in dendritic cells (DCs), as glucose represses the proinflammatory output of LPS-stimulated DCs and inhibits DC-induced T-cell responses. A glucose-sensitive signal transduction circuit involving the mTOR complex 1 (mTORC1), HIF1α and inducible nitric oxide synthase (iNOS) coordinates DC metabolism and function to limit DC-stimulated T-cell responses. When multiple T cells interact with a DC, they compete for nutrients, which can limit glucose availability to the DCs. In such DCs, glucose-dependent signalling is inhibited, altering DC outputs and enhancing T-cell responses. These data reveal a mechanism by which T cells regulate the DC microenvironment to control DC-induced T-cell responses and indicate that glucose is an important signal for shaping immune responses.

Focused Cardiac Ultrasound in Place of Repeat Echocardiography: Reliability and Cost Implications.

BACKGROUND: Health care systems are increasingly moving toward models that emphasize the delivery of high-quality health care at lower costs. Rates of repeat echocardiography (two or more transthoracic echocardiographic studies performed within a short interval) are high and can contribute substantially to the cost of providing cardiovascular care. Certain findings from handheld ultrasound scans performed by echocardiographers have been shown to correlate well with findings on transthoracic echocardiography (TTE). It therefore may be feasible and cost effective to use expert focused cardiac ultrasound (eFCU) in place of repeat TTE for highly selected indications in certain settings. The aim of this study was to determine the reliability and cost implications of using eFCU in place of repeat TTE in selected inpatients. METHODS: Inpatients who underwent repeat TTE (prior TTE within 30 days) ordered for the assessment of ventricular function, pericardial effusion, or inferior vena cava collapse were prospectively enrolled. Subjects underwent eFCU in addition to TTE, and results were compared for correlation using the weighted κ statistic. The potential cost savings of using eFCU in place of TTE were modeled from the provider perspective (i.e., physicians and hospitals). RESULTS: Over 45 days, 105 patients were enrolled. The majority of scans were performed for assessment of left ventricular function and pericardial effusions. eFCU showed excellent correlation with TTE for most parameters, including left ventricular systolic function (κ = 0.80) and the presence and size of pericardial effusions (κ = 0.81) (P < .001 for both). Adoption of this eFCU protocol could save between $41 and $64 per study, or between $34,512 and $53,871 annually at the authors' institution. CONCLUSIONS: Findings from eFCU correlate well with those from TTE when used in the setting of repeat testing for assessment of ventricular function, pericardial effusion, and inferior vena cava collapse. The judicious use of eFCU in place of repeat inpatient TTE has the potential to deliver quality cardiac imaging at reduced cost.