ABSTRACT
Up to 49% of certain types of cancer are attributed to obesity, and potential mechanisms include overproduction of hormones, adipokines, and insulin. Cytotoxic immune cells, including natural killer (NK) cells and CD8+ T cells, are important in tumor surveillance, but little is known about the impact of obesity on immunosurveillance. Here, we show that obesity induces robust peroxisome proliferator-activated receptor (PPAR)-driven lipid accumulation in NK cells, causing complete 'paralysis' of their cellular metabolism and trafficking. Fatty acid administration, and PPARα and PPARδ (PPARα/δ) agonists, mimicked obesity and inhibited mechanistic target of rapamycin (mTOR)-mediated glycolysis. This prevented trafficking of the cytotoxic machinery to the NK cell-tumor synapse. Inhibiting PPARα/δ or blocking the transport of lipids into mitochondria reversed NK cell metabolic paralysis and restored cytotoxicity. In vivo, NK cells had blunted antitumor responses and failed to reduce tumor growth in obesity. Our results demonstrate that the lipotoxic obese environment impairs immunosurveillance and suggest that metabolic reprogramming of NK cells may improve cancer outcomes in obesity.
Subject(s)
Immunologic Surveillance/immunology , Killer Cells, Natural/immunology , Killer Cells, Natural/metabolism , Melanoma, Experimental/immunology , Obesity/immunology , Adult , Animals , Female , Humans , Killer Cells, Natural/pathology , Male , Melanoma, Experimental/complications , Mice , Mice, Inbred C57BL , Middle Aged , Obesity/complications , Young AdultABSTRACT
Adipose tissue has a dynamic immune system that adapts to changes in diet and maintains homeostatic tissue remodeling. Adipose type 1 innate lymphoid cells (AT1-ILCs) promote pro-inflammatory macrophages in obesity, but little is known about their functions at steady state. Here we found that human and murine adipose tissue harbor heterogeneous populations of AT1-ILCs. Experiments using parabiotic mice fed a high-fat diet (HFD) showed differential trafficking of AT1-ILCs, particularly in response to short- and long-term HFD and diet restriction. At steady state, AT1-ILCs displayed cytotoxic activity toward adipose tissue macrophages (ATMs). Depletion of AT1-ILCs and perforin deficiency resulted in alterations in the ratio of inflammatory to anti-inflammatory ATMs, and adoptive transfer of AT1-ILCs exacerbated metabolic disorder. Diet-induced obesity impaired AT1-ILC killing ability. Our findings reveal a role for AT1-ILCs in regulating ATM homeostasis through cytotoxicity and suggest that this function is relevant in both homeostasis and metabolic disease.
Subject(s)
Adipose Tissue/immunology , Cytotoxicity, Immunologic/immunology , Homeostasis/immunology , Lymphocytes/immunology , Macrophages/immunology , Obesity/immunology , Adipose Tissue/cytology , Animals , Female , Humans , Immunity, Innate , Male , Mice , Mice, Inbred C57BL , Obesity/pathologyABSTRACT
Mucosal-Associated Invariant T (MAIT) cells are a population of innate T cells that play a critical role in host protection against bacterial and viral pathogens. Upon activation, MAIT cells can rapidly respond via both TCR-dependent and -independent mechanisms, resulting in robust cytokine production. The metabolic and nutritional requirements for optimal MAIT cell effector responses are still emerging. Iron is an important micronutrient and is essential for cellular fitness, in particular cellular metabolism. Iron is also critical for many pathogenic microbes, including those that activate MAIT cells. However, iron has not been investigated with respect to MAIT cell metabolic or functional responses. In this study, we show that human MAIT cells require exogenous iron, transported via CD71 for optimal metabolic activity in MAIT cells, including their production of ATP. We demonstrate that restricting iron availability by either chelating environmental iron or blocking CD71 on MAIT cells results in impaired cytokine production and proliferation. These data collectively highlight the importance of a CD71-iron axis for human MAIT cell metabolism and functionality, an axis that may have implications in conditions where iron availability is limited.
Subject(s)
Antigens, CD , Cytokines , Iron , Lymphocyte Activation , Mucosal-Associated Invariant T Cells , Receptors, Transferrin , Humans , Mucosal-Associated Invariant T Cells/immunology , Iron/metabolism , Receptors, Transferrin/metabolism , Receptors, Transferrin/immunology , Antigens, CD/metabolism , Antigens, CD/immunology , Lymphocyte Activation/immunology , Cytokines/metabolism , Cell Proliferation , Cells, Cultured , Adenosine Triphosphate/metabolismABSTRACT
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.
Subject(s)
Glycogenolysis , Mucosal-Associated Invariant T Cells , Humans , Cytokines , Glycogen , GlucoseABSTRACT
BACKGROUND: As genomic studies continue to implicate non-coding sequences in disease, testing the roles of these variants requires insights into the cell type(s) in which they are likely to be mediating their effects. Prior methods for associating non-coding variants with cell types have involved approaches using linkage disequilibrium or ontological associations, incurring significant processing requirements. GaiaAssociation is a freely available, open-source software that enables thousands of genomic loci implicated in a phenotype to be tested for enrichment at regulatory loci of multiple cell types in minutes, permitting insights into the cell type(s) mediating the studied phenotype. RESULTS: In this work, we present Regulatory Landscape Enrichment Analysis (RLEA) by GaiaAssociation and demonstrate its capability to test the enrichment of 12,133 variants across the cis-regulatory regions of 44 cell types. This analysis was completed in 134.0 ± 2.3 s, highlighting the efficient processing provided by GaiaAssociation. The intuitive interface requires only four inputs, offers a collection of customizable functions, and visualizes variant enrichment in cell-type regulatory regions through a heatmap matrix. GaiaAssociation is available on PyPi for download as a command line tool or Python package and the source code can also be installed from GitHub at https://github.com/GreallyLab/gaiaAssociation . CONCLUSIONS: GaiaAssociation is a novel package that provides an intuitive and efficient resource to understand the enrichment of non-coding variants across the cis-regulatory regions of different cells, empowering studies seeking to identify disease-mediating cell types.
Subject(s)
Software , Genetic Variation , Humans , Genomics/methods , Computational Biology/methods , Phenotype , Regulatory Sequences, Nucleic Acid/genetics , Linkage DisequilibriumABSTRACT
OBJECTIVES: To develop consensus data statements and clinical recommendations to provide guidance for improving cardiometabolic health outcomes in people with HIV based on the knowledge and experience of an international panel of experts. METHODS: A targeted literature review including 281 conference presentations, peer-reviewed articles, and background references on cardiometabolic health in adults with HIV published between January 2016 and April 2022 was conducted and used to develop draft consensus data statements. Using a modified Delphi method, an international panel of 16 experts convened in workshops and completed surveys to refine consensus data statements and generate clinical recommendations. RESULTS: Overall, 10 data statements, five data gaps and 14 clinical recommendations achieved consensus. In the data statements, the panel describes increased risk of cardiometabolic health concerns in people with HIV compared with the general population, known risk factors, and the potential impact of antiretroviral therapy. The panel also identified data gaps to inform future research in people with HIV. Finally, in the clinical recommendations, the panel emphasizes the need for a holistic approach to comprehensive care that includes regular assessment of cardiometabolic health, access to cardiometabolic health services, counselling on potential changes in weight after initiating or switching antiretroviral therapy and encouraging a healthy lifestyle to lower cardiometabolic health risk. CONCLUSIONS: On the basis of available data and expert consensus, an international panel developed clinical recommendations to address the increased risk of cardiometabolic disorders in people with HIV to ensure appropriate cardiometabolic health management for this population.
Subject(s)
Cardiovascular Diseases , Consensus , HIV Infections , Humans , HIV Infections/complications , HIV Infections/drug therapy , Delphi Technique , Risk Factors , Cardiometabolic Risk FactorsABSTRACT
The outbreak of e-cigarette or vaping use-associated lung injury (EVALI) in the United States in 2019 led to a total of 2807 hospitalizations with 68 deaths. While the exact causes of this vaping-related lung illness are still being debated, laboratory analyses of products from victims of EVALI have shown that vitamin E acetate (VEA), an additive in some tetrahydrocannabinol (THC)-containing products, is strongly linked to the EVALI outbreak. Because of its similar appearance and viscosity to pure THC oil, VEA was used as a diluent agent in cannabis oils in illicit markets. A potential mechanism for EVALI may involve VEA's thermal decomposition product, ketene, a highly poisonous gas, being generated under vaping conditions. In this study, a novel approach was developed to evaluate ketene production from VEA vaping under measurable temperature conditions in real-world devices. Ketene in generated aerosols was captured by two different chemical agents and analyzed by gas chromatography-mass spectrometry (GC-MS) and liquid chromatography with tandem mass spectrometry (LC-MS/MS). The LC-MS/MS method takes advantage of the high sensitivity and specificity of tandem mass spectrometry and appears to be more suitable than GC-MS for the analysis of large batches of samples. Our results confirmed the formation of ketene when VEA was vaped. The production of ketene increased with repeat puffs and showed a correlation to temperatures (200 to 500 °C) measured within vaping devices. Device battery power strength, which affects the heating temperature, plays an important role in ketene formation. In addition to ketene, the organic oxidant duroquinone was also obtained as another thermal degradation product of VEA. Ketene was not detected when vitamin E was vaped under the same conditions, confirming the importance of the acetate group for its generation.
ABSTRACT
Flow chemistry has emerged as an integral process within the chemical sector permitting energy efficient synthetic scale-up while improving safety and minimising solvent usage. Herein, we report the first applications of the photoactivated, radical-mediated thiol-ene reaction for peptide bioconjugation under continuous flow. Bioconjugation reactions employing deep eutectic solvents, bio-based solvents and fully aqueous systems are reported here for a range of biologically relevant peptide substrates. The use of a water soluble photoinitiator, Irgacure 2959, permitted synthesis of glycosylated peptides in fully aqueous conditions, obviating the need for addition of organic solvents and enhancing the green credentials of these rapid, photoactivated, bioconjugation reactions.
Subject(s)
Peptides , Sulfhydryl Compounds , Solvents , WaterABSTRACT
PURPOSE: To explore profiles of fractional O2 extraction (using near-infrared spectroscopy) during ramp incremental cycling in older individuals with type 2 diabetes (T2D). METHODS: Twelve individuals with T2D (mean ± SD, age: 63 ± 3 years) and 12 healthy controls (mean age: 65 ± 3 years) completed a ramp cycling exercise. Rates of muscle deoxygenation (i.e., deoxygenated haemoglobin and myoglobin, Δ[HHb + Mb]) profiles of the vastus lateralis muscle were normalised to 100% of the response, plotted against absolute (W) and relative (%peak) power output (PO) and fitted with a double linear regression model. RESULTS: Peak oxygen uptake (VÌO2peak) was significantly (P < 0.01) reduced in T2D (23.0 ± 4.2 ml.kg-1.min-1) compared with controls (28.3 ± 5.3 ml.kg-1.min-1). The slope of the first linear segment of the model was greater (median (interquartile range)) in T2D (1.06 (1.50)) than controls (0.79 (1.06)) when Δ%[HHb + Mb] was plotted as a function of PO. In addition, the onset of the second linear segment of the Δ%[HHb + Mb]/PO model occurred at a lower exercise intensity in T2D (101 ± 35 W) than controls (140 ± 34 W) and it displayed a near-plateau response in both groups. When the relationship of the Δ%[HHb + Mb] profile was expressed as a function of %PO no differences were observed in any parameters of the double linear model. CONCLUSIONS: These findings suggest that older individuals with uncomplicated T2D demonstrate greater fractional oxygen extraction for a given absolute PO compared with older controls. Thus, the reductions in VÌO2peak in older people with T2D are likely influenced by impairments in microvascular O2 delivery.
Subject(s)
Diabetes Mellitus, Type 2 , Humans , Aged , Middle Aged , Muscle, Skeletal/metabolism , Oxygen Consumption/physiology , Exercise/physiology , Hemoglobins/metabolism , Oxygen/metabolism , Exercise Test/methodsABSTRACT
INTRODUCTION: The purpose of this study was to identify the common factors that help and hinder transgender and nonbinary youth accessing gender-specific health care in Ireland and to identify how these factors may be perceived differently by young people seeking gender-affirming care, their parents, and health-care providers. DESIGN: Qualitative investigation utilizing framework analysis (FA). METHODS: In-depth one-one interviews were conducted with transgender and nonbinary youth (n = 10), parents of youth (n = 10), and gender-specific health-care providers (n = 10). Maximum variation and snowball sampling were used to recruit participants across Ireland. An interview guide codesigned with an expert panel of gender-diverse youth was utilized. Interviews were audio-recorded and transcribed verbatim. FA was used to code the data and identify key issues and recommendations. RESULTS: Four themes were derived: (1) "Needing bricks to build" (structural factors); (2) "Enduring and convincing" (diagnostic factors); (3) "Being me, hiding me"; (personal factors); and (4) "It takes a tribe" (interpersonal factors). Each stakeholder group perceived different factors as help or hindrance in accessing care with varying intensities. CONCLUSIONS: Paramount to the future of gender services in Ireland is the investment of resources for children and young adults. Assessment is likely to remain a component of gender care, but youth recommend distinct revisions to the assessment process. Additional research would be useful in exploring the intersection of neurodiversity and gender as it pertains to health-care navigation. Family and peer support is a strong protective factor and enabler of health-care access among youth. CLINICAL RELEVANCE: Access to gender-specific health care remains difficult for transgender and non-binary youth. An understanding of the complexity of this healthcare navigation by healthcare professionals may help to mitigate future negative experiences. This study explores some of the clinical considerations that arise for this population from provider perspectives while elucidating the experiences of youth and parents attempting to access care. Further research is needed on longitudinal outcomes following medical and surgical interventions for transgender youth, including nonbinary identities.
Subject(s)
Transgender Persons , Young Adult , Child , Humans , Adolescent , Ireland , Qualitative Research , Gender Identity , Health Services AccessibilityABSTRACT
Molecular probes with the ability to differentiate between subcellular variations in acidity levels remain important for the investigation of dynamic cellular processes and functions. In this context, a series of cyclic peptide and PEG bio-conjugated dual near-infrared emissive BF2-azadipyrromethene fluorophores with maxima emissions at 720 nm (at pH > 6) and 790 nm (at pH < 5) have been developed and their aqueous solution photophysical properties determined. Their inter-converting emissions and fluorescence lifetime characteristics were exploited to track their spatial and temporal progression from first contact with the plasma membrane to subcellular locales to their release within extracellular vesicles. A pH-dependent reversible phenolate/phenol interconversion on the fluorophore controlled the dynamic changes in dual emission responses and corresponding lifetime changes. Live-cell confocal microscopy experiments in the metastatic breast cancer cell line MDA-MB-231 confirmed the usability of the dual emissive properties for imaging over prolonged periods. All three derivatives performed as probes capable of real-time continuous imaging of fundamental cellular processes such as plasma membrane interaction, tracking endocytosis, lysosomal/large acidic vesicle accumulation, and efflux within extracellular vesicles without perturbing cellular function. Furthermore, fluorescence lifetime imaging microscopy provided valuable insights regarding fluorophore progression through intracellular microenvironments over time. Overall, the unique photophysical properties of these fluorophores show excellent potential for their use as information-rich probes.
Subject(s)
Cell Membrane , Fluorescent Dyes , Humans , Fluorescent Dyes/chemistry , Cell Membrane/metabolism , Cell Membrane/chemistry , Cell Line, Tumor , Microscopy, Confocal , Hydrogen-Ion Concentration , Microscopy, Fluorescence/methods , Endocytosis , Peptides, Cyclic/chemistryABSTRACT
Natural killer (NK) cells are critical in protecting the body against infection and cancer. NK cells can rapidly respond to these threats by directly targeting the infected or transformed cell using their cytotoxic machinery or by initiating and amplifying the immune response via their production of cytokines. Additionally, NK cells are resident across many tissues including adipose, were their role extends from host protection to tissue homeostasis. Adipose resident NK cells can control macrophage polarization via cytokine production, whilst also regulating stressed adipocyte fate using their cytotoxic machinery. Obesity is strongly associated with increased rates of cancer and a heightened susceptibility to severe infections. This is in part due to significant obesity-related immune dysregulation, including defects in both peripheral and adipose tissue NK cells. In this review, we detail the literature to date on NK cells in the setting of obesity - outlining the consequences, mechanisms and therapeutic interventions.
Subject(s)
Killer Cells, Natural , Neoplasms , Humans , Obesity , CytokinesABSTRACT
BACKGROUND/OBJECTIVES: People with obesity (PWO) face an increased risk of severe outcomes from COVID-19, including hospitalisation, ICU admission and death. Obesity has been seen to impair immune memory following vaccination against influenza, hepatitis B, tetanus, and rabies. Little is known regarding immune memory in PWO following COVID-19 adenovirus vector vaccination. SUBJECTS/METHODS: We investigated SARS-CoV-2 specific T cell responses in 50 subjects, five months following a two-dose primary course of ChAdOx1 nCoV-19 (AZD1222) vaccination. We further divided our cohort into PWO (n = 30) and matched controls (n = 20). T cell (CD4+, CD8+) cytokine responses (IFNγ, TNFα) to SARS-CoV-2 spike peptide pools were determined using multicolour flow cytometry. RESULTS: Circulating T cells specific for SARS-CoV-2 were readily detected across our cohort, with robust responses to spike peptide stimulation across both T cell lines. PWO and controls had comparable levels of both CD4+ and CD8+ SARS-CoV-2 spike specific T cells. Polyfunctional T cells - associated with enhanced protection against viral infection - were detected at similar frequencies in both PWO and controls. CONCLUSIONS: These data indicate that PWO who have completed a primary course of ChAdOx1 COVID-19 vaccination have robust, durable, and functional antigen specific T cell immunity that is comparable to that seen in people without obesity.
Subject(s)
COVID-19 Vaccines , COVID-19 , Humans , ChAdOx1 nCoV-19 , COVID-19/prevention & control , SARS-CoV-2 , T-Lymphocytes , Obesity , Vaccination , Antibodies, ViralABSTRACT
Diet-induced obesity can induce low-level inflammation and insulin resistance. Interleukin-1ß (IL-1ß) is one of the key proinflammatory cytokines that contributes to the generation of insulin resistance and diabetes, but the mechanisms that regulate obesity-driven inflammation are ill defined. Here we found reduced expression of the E3 ubiquitin ligase Pellino3 in human abdominal adipose tissue from obese subjects and in adipose tissue of mice fed a high-fat diet and showing signs of insulin resistance. Pellino3-deficient mice demonstrated exacerbated high-fat-diet-induced inflammation, IL-1ß expression, and insulin resistance. Mechanistically, Pellino3 negatively regulated TNF receptor associated 6 (TRAF6)-mediated ubiquitination and stabilization of hypoxia-inducible factor 1α (HIF1α), resulting in reduced HIF1α-induced expression of IL-1ß. Our studies identify a regulatory mechanism controlling diet-induced insulin resistance by highlighting a critical role for Pellino3 in regulating IL-1ß expression with implications for diseases like type 2 diabetes.
Subject(s)
Inflammation/immunology , Macrophages/physiology , Obesity/immunology , Ubiquitin-Protein Ligases/metabolism , Abdominal Fat/metabolism , Abdominal Fat/pathology , Adult , Aged , Animals , Cell Differentiation/genetics , Cells, Cultured , Diet, High-Fat , Female , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Inflammation/etiology , Insulin Resistance/genetics , Interleukin-1beta/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Middle Aged , Obesity/complications , TNF Receptor-Associated Factor 6/metabolism , Ubiquitin-Protein Ligases/genetics , Ubiquitination/genetics , Young AdultABSTRACT
Gene set analysis (GSA) remains a common step in genome-scale studies because it can reveal insights that are not apparent from results obtained for individual genes. Many different computational tools are applied for GSA, which may be sensitive to different types of signals; however, most methods implicitly test whether there are differences in the distribution of the effect of some experimental condition between genes in gene sets of interest. We have developed a unifying framework for GSA that first fits effect size distributions, and then tests for differences in these distributions between gene sets. These differences can be in the proportions of genes that are perturbed or in the sign or size of the effects. Inspired by statistical meta-analysis, we take into account the uncertainty in effect size estimates by reducing the influence of genes with greater uncertainty on the estimation of distribution parameters. We demonstrate, using simulation and by application to real data, that this approach provides significant gains in performance over existing methods. Furthermore, the statistical tests carried out are defined in terms of effect sizes, rather than the results of prior statistical tests measuring these changes, which leads to improved interpretability and greater robustness to variation in sample sizes.
Subject(s)
Genome , Research Design , Computer Simulation , Sample SizeABSTRACT
A combined analytical, theoretical, and experimental study has shown that the vaping of vitamin E acetate has the potential to produce exceptionally toxic ketene gas, which may be a contributing factor to the upsurge in pulmonary injuries associated with using e-cigarette/vaping products. Additionally, the pyrolysis of vitamin E acetate also produces carcinogen alkenes and benzene for which the negative long-term medical effects are well recognized. As temperatures reached in vaping devices can be equivalent to a laboratory pyrolysis apparatus, the potential for unexpected chemistries to take place on individual components within a vape mixture is high. Educational programs to inform of the danger are now required, as public perception has grown that vaping is not harmful.
Subject(s)
Ethylenes/analysis , Ketones/analysis , Lung Injury , Vaping , Vitamin E/chemistry , Acetates/analysis , Acetates/chemistry , Electronic Nicotine Delivery Systems , Ethylenes/toxicity , Ketones/toxicity , Lung Injury/chemically induced , Molecular Structure , Phenols/analysis , Phenols/chemistry , Pyrolysis , Vaping/adverse effects , Vitamin E/analysisABSTRACT
BF2-azadipyrromethenes are highly versatile fluorophores used for cellular and in vivo imaging in the near-infrared and far-red regions of the spectrum. As of yet, their use in conjunction with super-resolution imaging methodologies has not been explored. In this report, a series of structurally related BF2-azadipyrromethenes has been examined for their suitability for use with stimulated emission depletion (STED) nanoscopy. The potential for STED imaging was initially evaluated using aqueous solutions of fluorophores as an effective predictor of fluorophore suitability. For live cell STED imaging in both 2D and 3D, several far-red emitting BF2-azadipyrromethenes were successfully employed. Image resolution below the diffraction limit of a confocal microscope was demonstrated through measurement of distinct intracellular features including the nuclear membrane, nuclear lamina invaginations, the endoplasmic reticulum, and vacuoles. As the STED ability of BF2-azadipyrromethene fluorophores has now been established, their use with this super-resolution method may be expected to increase in the future.
Subject(s)
Fluorescent Dyes , Vacuoles , Microscopy, Fluorescence/methodsABSTRACT
A series of mono- and bis-polyethylene glycol (PEG)-substituted BF2-azadipyrromethene fluorophores have been synthesized with emissions in the near-infrared region (700-800 nm) for the purpose of fluorescence guided intraoperative imaging; chiefly ureter imaging. The Bis-PEGylation of fluorophores resulted in higher aqueous fluorescence quantum yields, with PEG chain lengths of 2.9 to 4.6 kDa being optimal. Fluorescence ureter identification was possible in a rodent model with the preference for renal excretion notable through comparative fluorescence intensities from the ureters, kidneys and liver. Ureteral identification was also successfully performed in a larger animal porcine model under abdominal surgical conditions. Three tested doses of 0.5, 0.25 and 0.1 mg/kg all successfully identified fluorescent ureters within 20 min of administration which was sustained up to 120 min. 3-D emission heat map imaging allowed the spatial and temporal changes in intensity due to the distinctive peristaltic waves of urine being transferred from the kidneys to the bladder to be identified. As the emission of these fluorophores could be spectrally distinguished from the clinically-used perfusion dye indocyanine green, it is envisaged that their combined use could be a step towards intraoperative colour coding of different tissues.
Subject(s)
Spectroscopy, Near-Infrared , Ureter , Swine , Animals , Spectroscopy, Near-Infrared/methods , Fluorescent Dyes/chemistry , Kidney , Urinary Bladder , Polyethylene Glycols/chemistry , Optical Imaging/methodsABSTRACT
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.
Subject(s)
Insulin Resistance , Mucosal-Associated Invariant T Cells , Pediatric Obesity , Adult , Child , Cross-Sectional Studies , Glucose/metabolism , Humans , Insulin/metabolism , Interleukin-17/metabolism , Lymphocyte Activation , Pediatric Obesity/metabolism , Tumor Necrosis Factor-alpha/metabolismABSTRACT
The importance of bioconjugation reactions continues to grow for cell specific targeting and dual therapeutic plus diagnostic medical applications. This necessitates the development of new bioconjugation chemistries, in-flow synthetic and analytical methods. With this goal, continuous flow bioconjugations were readily achieved with short residence times for strained alkyne substituted carbohydrate and therapeutic peptide biomolecules in reaction with azide and tetrazine substituted fluorophores. The strained alkyne substrates included substituted 2-amino-2-deoxy-α-D-glucopyranose, and the linear and cyclic peptide sequences QIRQQPRDPPTETLELEVSPDPAS-OH and c(RGDfK) respectively. The catalyst and reagent-free inverse electron demand tetrazine cycloadditions proved more favourable than the azide 1,3-dipolar cycloadditions. Reaction completion was achieved with residence times of 5â min at 40 °C for tetrazine versus 10â min at 80 °C for azide cycloadditions. The use of a fluorogenic tetrazine fluorophore, in a glass channelled reactor chip, allowed for intra-chip reaction monitoring by recording fluorescence intensities at various positions throughout the chip. As the Diels-Alder reactions proceeded through the chip, the fluorescence intensity increased accordingly in real-time. The application of continuous flow fluorogenic bioconjugations could offer an efficient translational access to theranostic agents.