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T-cell acute lymphoblastic leukemia (T-ALL) is a cancer of the immune system. Approximately 20% of paediatric and 50% of adult T-ALL patients have refractory disease or relapse and die from the disease. To improve patient outcome new therapeutics are needed. With the aim to identify new therapeutic targets, we combined the analysis of T-ALL gene expression and metabolism to identify the metabolic adaptations that T-ALL cells exhibit. We found that glutamine uptake is essential for T-ALL proliferation. Isotope tracing experiments showed that glutamine fuels aspartate synthesis through the TCA cycle and that glutamine and glutamine-derived aspartate together supply three nitrogen atoms in purines and all but one atom in pyrimidine rings. We show that the glutamate-aspartate transporter EAAT1 (SLC1A3), which is normally expressed in the central nervous system, is crucial for glutamine conversion to aspartate and nucleotides and that T-ALL cell proliferation depends on EAAT1 function. Through this work, we identify EAAT1 as a novel therapeutic target for T-ALL treatment.
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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.
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Calcitriol , Interferón gamma , Calcitriol/metabolismo , Calcitriol/farmacología , Glucólisis , Interferón gamma/metabolismo , Linfocitos T Colaboradores-Inductores/metabolismo , Vitamina DRESUMEN
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 oralâ±ârectal 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.
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Clostridioides difficile , Infecciones por Clostridium , Adulto , Humanos , Vancomicina/uso terapéutico , Fidaxomicina/uso terapéutico , Metronidazol/uso terapéutico , Infecciones por Clostridium/tratamiento farmacológico , Infecciones por Clostridium/prevención & control , Antibacterianos/uso terapéuticoRESUMEN
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.
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COVID-19 , Tromboembolia Venosa , Anticoagulantes/efectos adversos , Australia/epidemiología , Estudios de Cohortes , Humanos , Estudios Retrospectivos , SARS-CoV-2 , Tromboembolia Venosa/epidemiología , Tromboembolia Venosa/prevención & controlRESUMEN
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 â¼10 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.
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Cilios , Depuración Mucociliar , Epitelio , Humanos , Laboratorios , Programas Informáticos , Estados UnidosRESUMEN
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.
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Hematología , Neoplasias , Australia , Instituciones Oncológicas , Humanos , Pacientes InternosRESUMEN
BACKGROUND: Tigecycline is a third-line therapy for severe Clostridium difficile infection (CDI) in Australasian guidelines. Differences in strain types make it difficult to extrapolate international tigecycline efficacy data with combination or monotherapy to Australian practice, where experience is limited. AIM: To evaluate the efficacy and adverse effects associated with tigecycline combination therapy for severe and severe-complicated CDI in an Australian healthcare setting. METHODS: This was a retrospective observational study at a metropolitan university-affiliated hospital. All patients between February 2013 and October 2016 treated with adjunctive intravenous tigecycline for >48 h for severe or severe-complicated CDI were included. Tigecycline was given in addition to oral vancomycin ± intravenous metronidazole. The primary outcome was all-cause mortality at 30 days from start of tigecycline combination therapy. Secondary outcomes included clinical cure, colectomy, adverse events and recurrence rates. RESULTS: Thirteen patients with median age of 61 years had severe (n = 9) or severe-complicated (n = 4) CDI at tigecycline commencement. In 92% of patients, tigecycline started within 48 h after in-hospital CDI treatment, for median duration of 9 days. All-cause mortality at 30 days was 8% with no mortality in severe CDI and 25% (1/4) in patients with severe-complicated fulminant CDI, comparing favourably with historical rates of 9-38% and 30-80% in similar respective groups. Clinical cure was achieved in 77% of cases. There were no colectomies and one attributable tigecycline adverse reaction. CONCLUSIONS: Tigecycline appears safe and effective as a part of combination therapy in severe CDI, and may be given earlier and for shorter durations than in current guidelines.
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Antibacterianos/administración & dosificación , Infecciones por Clostridium/tratamiento farmacológico , Infección Hospitalaria/tratamiento farmacológico , Minociclina/análogos & derivados , Administración Intravenosa , Anciano , Anciano de 80 o más Años , Australia , Clostridioides difficile/efectos de los fármacos , Infección Hospitalaria/microbiología , Quimioterapia Combinada , Femenino , Humanos , Masculino , Metronidazol/administración & dosificación , Persona de Mediana Edad , Minociclina/administración & dosificación , Estudios Retrospectivos , Tigeciclina , Resultado del Tratamiento , Vancomicina/administración & dosificaciónRESUMEN
Previous research demonstrating that positive episodic simulation enhances future expectancies has relied on explicit expectancy measures. The current study investigated the effects of episodic simulation on implicit expectancies. Using the Future Thinking Implicit Relational Assessment Procedure (FT-IRAP), participants made true/false decisions to indicate whether or not they expected positive/negative outcomes after adopting orientations consistent or inconsistent with an optimistic disposition. The outcome measure, DIRAP, was based on response time differences between consistent and inconsistent blocks. Participants then engaged in either positive simulation training, in which they imagined positive future events, or a neutral visualisation task before repeating the FT-IRAP twice following 10-minute intervals. Positive simulation training increased DIRAP scores for don't-expect-negative trials-boosting participants' readiness to affirm that negative events were unlikely to happen to them. Although findings did not generalise across all trial types, they show potential for positive simulation training to enhance implicit future expectancies.
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Pensamiento , Humanos , Femenino , Masculino , Pensamiento/fisiología , Adulto Joven , Adulto , Tiempo de Reacción/fisiología , Imaginación , AdolescenteRESUMEN
'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 20â¯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.
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Nicotina , Tabaco sin Humo , Ratones , Animales , Nicotina/análisis , Tabaco sin Humo/toxicidad , Mutágenos/análisis , Estrés OxidativoRESUMEN
Cigarette smoking is a risk factor for several diseases such as cancer, cardiovascular disease (CVD), and chronic obstructive pulmonary diseases (COPD), however, the underlying mechanisms are not fully understood. Alternative nicotine products with reduced risk potential (RRPs) including tobacco heating products (THPs), and e-cigarettes have recently emerged as viable alternatives to cigarettes that may contribute to the overall strategy of tobacco harm reduction due to the significantly lower levels of toxicants in these products' emissions as compared to cigarette smoke. Assessing the effects of RRPs on biological responses is important to demonstrate the potential value of RRPs towards tobacco harm reduction. Here, we evaluated the inflammatory and signaling responses of human lung epithelial cells to aqueous aerosol extracts (AqE) generated from the 1R6F reference cigarette, the glo™ THP, and the Vype ePen 3.0 e-cigarette using multiplex analysis of 37 inflammatory and phosphoprotein markers. Cellular exposure to the different RRPs and 1R6F AqEs resulted in distinct response profiles with 1R6F being the most biologically active followed by glo™ and ePen 3.0. 1R6F activated stress-related and pro-survival markers c-JUN, CREB1, p38 MAPK and MEK1 and led to the release of IL-1α. glo™ activated MEK1 and decreased IL-1ß levels, whilst ePen 3.0 affected IL-1ß levels but had no effect on the signaling activity compared to untreated cells. Our results demonstrated the reduced biological effect of RRPs and suggest that targeted analysis of inflammatory and cell signaling mediators is a valuable tool for the routine assessment of RRPs.
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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.
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Artritis Reumatoide , Linfocitos T CD4-Positivos , Proteínas Proto-Oncogénicas c-akt , Transducción de Señal , Serina-Treonina Quinasas TOR , Factor de Necrosis Tumoral alfa , Humanos , Artritis Reumatoide/metabolismo , Artritis Reumatoide/patología , Artritis Reumatoide/inmunología , Artritis Reumatoide/genética , Serina-Treonina Quinasas TOR/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Factor de Necrosis Tumoral alfa/metabolismo , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/inmunología , Diferenciación Celular , Mitocondrias/metabolismo , Reprogramación MetabólicaRESUMEN
Dengue neurological disease is an uncommon yet severe complication of dengue infection. It can manifest as encephalitis, encephalopathy, neuro-ophthalmic complications, or neuromuscular disorders. Severe infection can result in viral shedding across multiple body sites. We describe a case of severe neuro-ophthalmic dengue infection in an otherwise healthy returned traveller, presenting with prolonged multiple-body-site viral detections by PCR. The dengue virus (DENV) dynamics and serological response support a direct DENV neuropathogenicity. A retrospective review of the laboratory data at the Victorian Infectious Diseases Reference Laboratory (VIDRL) suggests that blood is the most frequent sample type with DENV detection (92% of all DENV-positive samples). Genotype variation is seen across different sample types. The similarity of CSF and nasopharyngeal DENV subtypes (genotype 1 and 3) suggests a possible correlation between nasopharyngeal replication and neurological complications. The case presented highlights the direct neuropathogenicity of DENV early in the course of infection, and a potential correlation between nasopharyngeal replication and neurological disease.
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Virus del Dengue , Dengue , Humanos , Dengue/virología , Dengue/diagnóstico , Virus del Dengue/genética , Virus del Dengue/aislamiento & purificación , Masculino , Genotipo , Adulto , Estudios RetrospectivosRESUMEN
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.
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Sistemas Electrónicos de Liberación de Nicotina , Productos de Tabaco , Humanos , Aerosoles , Técnicas In VitroRESUMEN
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.
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Inteligencia Artificial , Diabetes Mellitus , Humanos , Colombia , Mejoramiento de la Calidad , Diabetes Mellitus/terapiaRESUMEN
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.
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Mieloma Múltiple , Humanos , Mieloma Múltiple/terapia , Médula Ósea , Linfocitos T CD8-positivos , Microambiente TumoralRESUMEN
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.
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Enfermedades Autoinmunes , Diabetes Mellitus Tipo 2 , Inhibidores del Cotransportador de Sodio-Glucosa 2 , Humanos , Canagliflozina/farmacología , Canagliflozina/uso terapéutico , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Autoinmunidad , Linfocitos T , Inhibidores del Cotransportador de Sodio-Glucosa 2/uso terapéutico , Enfermedades Autoinmunes/tratamiento farmacológico , Hipoglucemiantes/farmacologíaRESUMEN
BACKGROUND: Cigarette smoking is a leading cause of mortality and morbidity and is associated with cardiovascular disease via contributory processes such as endothelial dysfunction, inflammation and thrombosis. Cigarette smoke both contains and stimulates the production of cellular oxidants and it may also promote vascular inflammation. Osteopontin is a non-collagenous matrix protein first identified in bone and there is increasing evidence for its role in inflammation and cardiovascular disease via its action as a soluble cytokine. METHODS: In this study we have examined the mechanisms underlying the expression of osteopontin in human vascular endothelial cells in vitro following exposure to cigarette smoke particulate matter (PM), using PCR, electrochemiluminescence, immunostaining and Western blotting. We further determined if serum osteopontin levels changed in humans who quit smoking. RESULTS: Non-cytotoxic concentrations of PM increased osteopontin levels in cultured human endothelial cells and this effect was reduced in the presence of ascorbate, suggesting a role for oxidants in the response to PM. However, oxidant production played no role in the PM-evoked induction MMP-3, an enzyme which cleaves osteopontin. In smokers who quit smoking for 5 days, serum osteopontin levels were significantly lowered compared to those measured prior to smoking cessation. CONCLUSIONS: In vitro cigarette smoke extract exposure induced osteopontin expression in human endothelial cells in an oxidative stress-dependent manner, which may involve MMP-3 cleavage. In humans, serum osteopontin was decreased with short-term smoking cessation. Endothelial-derived osteopontin may contribute to inflammation in smokers, and may also contribute to atherosclerosis and cardiovascular disease-related processes.
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Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Osteopontina/metabolismo , Humo/efectos adversos , Fumar/efectos adversos , Adulto , Antioxidantes/farmacología , Ácido Ascórbico/farmacología , Western Blotting , Células Cultivadas , Relación Dosis-Respuesta a Droga , Técnicas Electroquímicas , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Inmunohistoquímica , Metaloproteinasa 3 de la Matriz/metabolismo , Persona de Mediana Edad , Osteopontina/sangre , Osteopontina/genética , Reacción en Cadena de la Polimerasa , Fumar/sangre , Fumar/genética , Cese del Hábito de Fumar , Prevención del Hábito de Fumar , Factores de Tiempo , Regulación hacia Arriba , Adulto JovenRESUMEN
Endothelial damage plays a key role in atherosclerosis and this is impacted upon by numerous risk factors including cigarette smoking. A potential measure to reduce the cardiovascular burden associated with smoking is to reduce smoke toxicant exposure. In an in vitro endothelial damage repair assay, endothelial cell migration was inhibited by cigarette smoke particulate matter (PM) generated from several cigarette types. This inhibition was reduced when cells were exposed to PM from an experimental cigarette with reduced smoke toxicant levels. As a number of toxicants induce oxidative stress and since oxidative stress may link cigarette smoke and endothelial damage, we hypothesized that PM effects were dependent on elevated cellular oxidants. However, although PM-induced cellular oxidant production could be inhibited by ascorbic acid or n-acetylcysteine, both these antioxidants were without effect on migration responses to PM. Furthermore, reactive oxygen species production, as indicated by dihydroethidium fluorescence, was not different in cells exposed to smoke from cigarettes with different toxicant levels. In summary, our data demonstrate that a cardiovascular disease-related biological response may be modified when cells are exposed to smoke containing different levels of toxicants. This appeared independent of the induction of oxidative stress.
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Endotelio Vascular/efectos de los fármacos , Nicotiana , Material Particulado/farmacología , Humo/efectos adversos , Acetilcisteína/farmacología , Antioxidantes/farmacología , Ácido Ascórbico/farmacología , Enfermedades Cardiovasculares/etiología , Enfermedades Cardiovasculares/metabolismo , Enfermedades Cardiovasculares/prevención & control , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Interacciones Farmacológicas , Endotelio Vascular/patología , Endotelio Vascular/fisiología , Radicales Libres/metabolismo , Células Endoteliales de la Vena Umbilical Humana , Humanos , Procesamiento de Imagen Asistido por Computador , Estrés Oxidativo/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Humo/análisis , Factor A de Crecimiento Endotelial Vascular/farmacologíaRESUMEN
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.
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Neoplasias de las Glándulas Suprarrenales , Paraganglioma , Feocromocitoma , Neoplasias de las Glándulas Suprarrenales/genética , Linfocitos T CD8-positivos , Citocinas , Glucosa , Humanos , Paraganglioma/genética , Feocromocitoma/genética , Succinatos , Ácido Succínico , Microambiente TumoralRESUMEN
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.