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OBJECTIVE: To evaluate the performance of childhood obesity prediction models in four independent cohorts in the United States, using previously validated variables obtained easily from medical records as measured in different clinical settings. STUDY DESIGN: Data from four prospective cohorts, Latinx, Eating, and Diabetes; Stress in Pregnancy Study; Project Viva; and Center for the Health Assessment of Mothers and Children of Salinas were used to test childhood obesity risk models and predict childhood obesity by ages 4 through 6, using five clinical variables (maternal age, maternal prepregnancy body mass index, birth weight Z-score, weight-for-age Z-score change, and breastfeeding), derived from a previously validated risk model and as measured in each cohort's clinical setting. Multivariable logistic regression was performed within each cohort, and performance of each model was assessed based on discrimination and predictive accuracy. RESULTS: The risk models performed well across all four cohorts, achieving excellent discrimination. The area under the receiver operator curve was 0.79 for Center for the Health Assessment of Mothers and Children of Salinas and Project Viva, 0.83 for Stress in Pregnancy Study, and 0.86 for Latinx, Eating, and Diabetes. At a 50th percentile threshold, the sensitivity of the models ranged from 12% to 53%, and specificity was ≥ 90%. The negative predictive values were ≥ 80% for all cohorts, and the positive predictive values ranged from 62% to 86%. CONCLUSION: All four risk models performed well in each independent and demographically diverse cohort, demonstrating the utility of these five variables for identifying children at high risk for developing early childhood obesity in the United States.
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During infection, cellular resources are allocated toward the metabolically-demanding processes of synthesizing and secreting effector proteins that neutralize and kill invading pathogens. In Drosophila, these effectors are antimicrobial peptides (AMPs) that are produced in the fat body, an organ that also serves as a major lipid storage depot. Here we asked how activation of Toll signaling in the larval fat body perturbs lipid homeostasis to understand how cells meet the metabolic demands of the immune response. We find that genetic or physiological activation of fat body Toll signaling leads to a tissue-autonomous reduction in triglyceride storage that is paralleled by decreased transcript levels of the DGAT homolog midway, which carries out the final step of triglyceride synthesis. In contrast, Kennedy pathway enzymes that synthesize membrane phospholipids are induced. Mass spectrometry analysis revealed elevated levels of major phosphatidylcholine and phosphatidylethanolamine species in fat bodies with active Toll signaling. The ER stress mediator Xbp1 contributed to the Toll-dependent induction of Kennedy pathway enzymes, which was blunted by deleting AMP genes, thereby reducing secretory demand elicited by Toll activation. Consistent with ER stress induction, ER volume is expanded in fat body cells with active Toll signaling, as determined by transmission electron microscopy. A major functional consequence of reduced Kennedy pathway induction is an impaired immune response to bacterial infection. Our results establish that Toll signaling induces a shift in anabolic lipid metabolism to favor phospholipid synthesis and ER expansion that may serve the immediate demand for AMP synthesis and secretion but with the long-term consequence of insufficient nutrient storage.
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Peptídeos Catiônicos Antimicrobianos/metabolismo , Infecções por Bactérias Gram-Positivas/imunologia , Imunidade Inata , Metabolismo dos Lipídeos/imunologia , Animais , Animais Geneticamente Modificados , Peptídeos Catiônicos Antimicrobianos/genética , Colina-Fosfato Citidililtransferase/genética , Colina-Fosfato Citidililtransferase/metabolismo , Proteínas de Ligação a DNA/metabolismo , Diacilglicerol O-Aciltransferase/metabolismo , Modelos Animais de Doenças , Drosophila , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Retículo Endoplasmático/imunologia , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático/imunologia , Enterococcus faecalis/imunologia , Corpo Adiposo/enzimologia , Corpo Adiposo/imunologia , Feminino , Infecções por Bactérias Gram-Positivas/microbiologia , Humanos , Larva/enzimologia , Larva/imunologia , Metabolismo dos Lipídeos/genética , Masculino , Fosfolipídeos/biossíntese , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Receptores Toll-Like/metabolismo , Triglicerídeos/metabolismoRESUMO
Insulin secretion from ß-cells is reduced at the onset of type-1 and during type-2 diabetes. Although inflammation and metabolic dysfunction of ß-cells elicit secretory defects associated with type-1 or type-2 diabetes, accompanying changes to insulin granules have not been established. To address this, we performed detailed functional analyses of insulin granules purified from cells subjected to model treatments that mimic type-1 and type-2 diabetic conditions and discovered striking shifts in calcium affinities and fusion characteristics. We show that this behavior is correlated with two subpopulations of insulin granules whose relative abundance is differentially shifted depending on diabetic model condition. The two types of granules have different release characteristics, distinct lipid and protein compositions, and package different secretory contents alongside insulin. This complexity of ß-cell secretory physiology establishes a direct link between granule subpopulation and type of diabetes and leads to a revised model of secretory changes in the diabetogenic process.
Diabetes is a disease that occurs when sugar levels in the blood can no longer be controlled by a hormone called insulin. People with type 1 diabetes lose the ability to produce insulin after their immune system attacks the ß-cells in their pancreas that make this hormone. People with type 2 diabetes develop the disease when ß-cells become exhausted from increased insulin demand and stop producing insulin. ß-cells store insulin in small compartments called granules. When blood sugar levels rise, these granules fuse with the cell membrane allowing ß-cells to release large quantities of insulin at once. This fusion is disrupted early in type 1 diabetes, but later in type 2: the underlying causes of these disruptions are unclear. In the laboratory, signals that trigger inflammation and molecules called fatty acids can mimic type 1 or type 2 diabetes respectively when applied to insulin-producing cells. Kreutzberger, Kiessling et al. wanted to know whether pro-inflammatory molecules and fatty acids affect insulin granules differently at the molecular level. To do this, insulin-producing cells were grown in the lab and treated with either fatty acids or pro-inflammatory molecules. The insulin granules of these cells were then isolated. Next, the composition of the granules and how they fused to lab-made membranes that mimic the cell membrane was examined. The experiments revealed that healthy ß-cells have two types of granules, each with a different version of a protein called synaptotagmin. Cells treated with molecules mimicking type 1 diabetes lost granules with synaptotagmin-7, while granules with synaptotagmin-9 were lost in cells treated with fatty acids to imitate type 2 diabetes. Each type of granule responded differently to calcium levels in the cell and secreted different molecules, indicating that each elicits a different diabetic response in the body. These findings suggest that understanding how insulin granules are formed and regulated may help find treatments for type 1 and 2 diabetes, possibly leading to therapies that reverse the loss of different types of granules. Additionally, the molecules of these granules may also be used as markers to determine the stage of diabetes. More broadly, these results show how understanding how molecule release changes with disease in different cell types may help diagnose or stage a disease.
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Cálcio/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Exocitose , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Animais , Colesterol/metabolismo , Citocinas/farmacologia , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 2/genética , Exocitose/efeitos dos fármacos , Humanos , Insulina/genética , Células Secretoras de Insulina/efeitos dos fármacos , Células PC12 , Palmitatos/farmacologia , Ratos , Proteínas SNARE/metabolismo , Via Secretória , Esfingomielinas/metabolismo , Sinaptotagminas/metabolismoRESUMO
On April 10, 2020, a treasured cell biologist and ardent champion of the Golgi complex passed away. This has caused deep sadness, and we seek to commemorate her remarkable scientific contributions, her warm and generous personality, and her endearing sense of humor.
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Fisiologia/história , Via Secretória , Feminino , Complexo de Golgi/metabolismo , História do Século XX , História do Século XXI , Humanos , Transporte ProteicoRESUMO
PURPOSE: Circulating tumour cells (CTCs) are a marker of poor prognosis and are associated with increased risk of venous thromboembolism in metastatic breast cancer (MBC). We aimed to determine if the presence of CTCs and plasma markers of hypercoagulability [thrombin-antithrombin III (TAT), fibrinogen and D-dimer] are biomarkers of survival in MBC. METHODS/PATIENTS: In a prospective study of MBC patients, CTC (CellSearch®) enumeration and plasma TAT, fibrinogen and D-dimer measured prior to commencement of treatment for disease progression were correlated to overall survival. RESULTS: At study completion, of 50 MBC patients recruited (median age 59 years, range 36-82), 40 patients had died (median survival 417 days, range 58-2141). CTCs (≥ 1/7.5 ml) were identified in 16 patients (median number of cells per 7.5 ml, 3 (range 1-31) and were associated with systemic hypercoagulability (medians TAT: 8.1 vs. 5.2 ng/ml, p = 0.03; fibrinogen: 4.3 vs. 3.1 g/l, p = 0.03; D-dimer: 1327 vs. 683 ng/ml, p = 0.0001). At 1 year, of 16 patients with ≥ 1 CTC, 7 had died (44%), compared to 5 of 26 (19%) patients in the no-CTC group. The presence of ≥ 1 CTC was associated with a trend for reduced overall survival (median 455 days vs. 614 days, p = 0.15). Plasma TAT inversely correlated with survival and was significantly higher in patients dying within 1 year (median 9.8 vs. 5.2 ng/ml, p = 0.004) whilst D-dimer showed a trend for reduced 1-year survival (median 1211 vs. 817 ng/ml, p = 0.06). MBC patients with combined high D-dimer (≥ 895 ng/ml) and CTC positivity (≥ 1/7.5 ml whole peripheral blood) had significantly reduced survival (p = 0.04). CONCLUSIONS: The correlation between CTCs, hypercoagulability and reduced survival in MBC suggests the coagulation system supports tumour cell metastasis and is, therefore, a potential therapeutic target.
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Neoplasias da Mama/sangue , Neoplasias da Mama/mortalidade , Células Neoplásicas Circulantes/metabolismo , Trombofilia/sangue , Adulto , Idoso , Idoso de 80 Anos ou mais , Antitrombina III , Biomarcadores Tumorais/sangue , Neoplasias da Mama/patologia , Feminino , Produtos de Degradação da Fibrina e do Fibrinogênio/metabolismo , Fibrinogênio/metabolismo , Humanos , Pessoa de Meia-Idade , Metástase Neoplásica , Peptídeo Hidrolases/sangue , Prognóstico , Taxa de SobrevidaRESUMO
The diagnosis of prostate cancer has changed. Improved magnetic resonance imaging (MRI) technology with diffusion-weighted imaging has led to the use of multiparametric MRI (mpMRI) before biopsy in patients suspected of having prostate cancer. This has the advantage that patients with a negative mpMRI may not need biopsy, therefore avoiding the risk of complications. Those in whom mpMRI is positive can have targeted biopsies with a higher probability of diagnosing clinically significant cancer. Prostate Cancer UK (PCUK) and the British Society of Urogenital Radiology submitted a Freedom of Information (FOI) request in 2016 to UK health areas in order to assess the use of mpMRI before biopsy as part of the initial diagnosis pathway. Another request was submitted by PCUK in 2018 to assess the progress made in the UK between these two dates. Both requests had the secondary aim of identifying barriers to the implementation of mpMRI. The FOI requests showed an increase in the use of mpMRI before biopsy with 59% of areas reporting improvement between the two requests. There has been a reduction in the percentage of areas not providing any form of pre-biopsy MRI from 25% in 2016 to 13% in 2018. There remains, however, geographical variation in implementation across the UK nations. Imaging practice also shows variation with some areas performing scans without dynamic contrast enhancement (DCE) and using the findings to guide referral decisions for biopsy. Eligibility criteria for pre-biopsy MRI also vary leading to some restrictive practices. Reported barriers to implementation included scanner capacity and staffing levels. Recent guidelines and recommendations by the National Institute for Health and Care Excellence (NICE) and NHS England for men aged 50-69 years with a prostate-specific antigen (PSA) level between >3 and <30 ng/ml to receive mpMRI before biopsy put further pressure on already understaffed and under-resourced radiology departments.
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Imageamento por Ressonância Magnética Multiparamétrica/estatística & dados numéricos , Neoplasias da Próstata/patologia , Idoso , Biópsia/estatística & dados numéricos , Detecção Precoce de Câncer , Utilização de Instalações e Serviços , Acessibilidade aos Serviços de Saúde/estatística & dados numéricos , Humanos , Masculino , Pessoa de Meia-Idade , Encaminhamento e Consulta , Projetos de Pesquisa/estatística & dados numéricos , Reino UnidoRESUMO
Regulated exocytosis of synaptic vesicles is substantially faster than of endocrine dense core vesicles despite similar molecular machineries. The reasons for this difference are unknown and could be due to different regulatory proteins, different spatial arrangements, different vesicle sizes, or other factors. To address these questions, we take a reconstitution approach and compare regulated SNARE-mediated fusion of purified synaptic and dense core chromaffin and insulin vesicles using a single vesicle-supported membrane fusion assay. In all cases, Munc18 and complexin are required to restrict fusion in the absence of calcium. Calcium triggers fusion of all docked vesicles. Munc13 (C1C2MUN domain) is required for synaptic and enhanced insulin vesicle fusion, but not for chromaffin vesicles, correlating inversely with the presence of CAPS protein on purified vesicles. Striking disparities in calcium-triggered fusion rates are observed, increasing with curvature with time constants 0.23 s (synaptic vesicles), 3.3 s (chromaffin vesicles), and 9.1 s (insulin vesicles) and correlating with rate differences in cells.
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Fusão de Membrana/fisiologia , Proteínas SNARE/metabolismo , Vesículas Secretórias/metabolismo , Vesículas Sinápticas/metabolismo , Animais , Transporte Biológico , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Membrana Celular/metabolismo , Exocitose , Humanos , Insulina , Proteínas Munc18/metabolismo , Proteínas do Tecido Nervoso , Células PC12 , RatosRESUMO
The regulated exocytotic release of neurotransmitter and hormones is accomplished by a complex protein machinery whose core consists of SNARE proteins and the calcium sensor synaptotagmin-1. We propose a mechanism in which the lipid membrane is intimately involved in coupling calcium sensing to release. We found that fusion of dense core vesicles, derived from rat PC12 cells, was strongly linked to the angle between the cytoplasmic domain of the SNARE complex and the plane of the target membrane. We propose that, as this tilt angle increases, force is exerted on the SNARE transmembrane domains to drive the merger of the two bilayers. The tilt angle markedly increased following calcium-mediated binding of synaptotagmin to membranes, strongly depended on the surface electrostatics of the membrane, and was strictly coupled to the lipid order of the target membrane.
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Exocitose , Modelos Moleculares , Sinaptotagminas/fisiologia , Vesículas Transportadoras/química , Animais , Sinalização do Cálcio , Metabolismo dos Lipídeos/fisiologia , Células PC12 , Domínios Proteicos , Proteínas Qa-SNARE/química , Proteínas Qa-SNARE/metabolismo , Proteínas Qa-SNARE/fisiologia , Ratos , Proteínas SNARE/química , Proteínas SNARE/metabolismo , Proteínas SNARE/fisiologia , Sinaptotagminas/química , Sinaptotagminas/metabolismo , Vesículas Transportadoras/metabolismo , Vesículas Transportadoras/fisiologiaRESUMO
The ABC transporter ABCG1 contributes to the regulation of cholesterol efflux from cells and to the distribution of cholesterol within cells. We showed previously that ABCG1 deficiency inhibits insulin secretion by pancreatic beta cells and, based on its immunolocalization to insulin granules, proposed its essential role in forming granule membranes that are enriched in cholesterol. While we confirm elsewhere that ABCG1, alongside ABCA1 and oxysterol binding protein OSBP, supports insulin granule formation, the aim here is to clarify the localization of ABCG1 within insulin-secreting cells and to provide added insight regarding ABCG1's trafficking and sites of function. We show that stably expressed GFP-tagged ABCG1 closely mimics the distribution of endogenous ABCG1 in pancreatic INS1 cells and accumulates in the trans-Golgi network (TGN), endosomal recycling compartment (ERC) and on the cell surface but not on insulin granules, early or late endosomes. Notably, ABCG1 is short-lived, and proteasomal and lysosomal inhibitors both decrease its degradation. Following blockade of protein synthesis, GFP-tagged ABCG1 first disappears from the ER and TGN and later from the ERC and plasma membrane. In addition to aiding granule formation, our findings raise the prospect that ABCG1 may act beyond the TGN to regulate activities involving the endocytic pathway, especially as the amount of transferrin receptor is increased in ABCG1-deficient cells. Thus, ABCG1 may function at multiple intracellular sites and the plasma membrane as a roving sensor and modulator of cholesterol distribution, membrane trafficking and cholesterol efflux.
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Membro 1 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/análise , Membro 1 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Células Secretoras de Insulina/metabolismo , Animais , Linhagem Celular , Citoplasma/metabolismo , Citoplasma/ultraestrutura , Degradação Associada com o Retículo Endoplasmático , Endossomos/metabolismo , Endossomos/ultraestrutura , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/ultraestrutura , Lisossomos/metabolismo , Lisossomos/ultraestrutura , Mesotelina , Camundongos , Microscopia Confocal , Transporte Proteico , Proteólise , Ratos , Rede trans-Golgi/metabolismo , Rede trans-Golgi/ultraestruturaRESUMO
In pancreatic ß-cells, insulin granule membranes are enriched in cholesterol and are both recycled and newly generated. Cholesterol's role in supporting granule membrane formation and function is poorly understood. ATP binding cassette transporters ABCG1 and ABCA1 regulate intracellular cholesterol and are important for insulin secretion. RNAi inter-ference-induced depletion in cultured pancreatic ß-cells shows that ABCG1 is needed to stabilize newly made insulin granules against lysosomal degradation; ABCA1 is also involved but to a lesser extent. Both transporters are also required for optimum glucose-stimulated insulin secretion, likely via complementary roles. Exogenous cholesterol addition rescues knockdown-induced granule loss (ABCG1) and reduced secretion (both transporters). Another cholesterol transport protein, oxysterol binding protein (OSBP), appears to act proximally as a source of endogenous cholesterol for granule formation. Its knockdown caused similar defective stability of young granules and glucose-stimulated insulin secretion, neither of which were rescued with exogenous cholesterol. Dual knockdowns of OSBP and ABC transporters support their serial function in supplying and concentrating cholesterol for granule formation. OSBP knockdown also decreased proinsulin synthesis consistent with a proximal endoplasmic reticulum defect. Thus, membrane cholesterol distribution contributes to insulin homeostasis at production, packaging, and export levels through the actions of OSBP and ABCs G1 and A1.
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Transportador 1 de Cassete de Ligação de ATP/metabolismo , Membro 1 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Grânulos Citoplasmáticos/metabolismo , Insulina/metabolismo , Receptores de Esteroides/metabolismo , Transportador 1 de Cassete de Ligação de ATP/deficiência , Membro 1 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/deficiência , Animais , Colesterol/farmacologia , Grânulos Citoplasmáticos/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Exocitose/efeitos dos fármacos , Fluorescência , Técnicas de Silenciamento de Genes , Glucose/farmacologia , Proteínas de Fluorescência Verde/metabolismo , Humanos , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Camundongos , Modelos Biológicos , Interferência de RNA , Ratos , Via Secretória/efeitos dos fármacos , beta-Ciclodextrinas/farmacologiaRESUMO
Little attention has been given to how the asymmetric lipid distribution of the plasma membrane might facilitate fusion pore formation during exocytosis. Phosphatidylethanolamine (PE), a cone-shaped phospholipid, is predominantly located in the inner leaflet of the plasma membrane and has been proposed to promote membrane deformation and stabilize fusion pores during exocytotic events. To explore this possibility, we modeled exocytosis using plasma membrane SNARE-containing planar-supported bilayers and purified neuroendocrine dense core vesicles (DCVs) as fusion partners, and we examined how different PE distributions between the two leaflets of the supported bilayers affected SNARE-mediated fusion. Using total internal reflection fluorescence microscopy, the fusion of single DCVs with the planar-supported bilayer was monitored by observing DCV-associated neuropeptide Y tagged with a fluorescent protein. The time-dependent line shape of the fluorescent signal enables detection of DCV docking, fusion-pore opening, and vesicle collapse into the planar membrane. Four different distributions of PE in the planar bilayer mimicking the plasma membrane were examined: exclusively in the leaflet facing the DCVs; exclusively in the opposite leaflet; equally distributed in both leaflets; and absent from both leaflets. With PE in the leaflet facing the DCVs, overall fusion was most efficient and the extended fusion pore lifetime (0.7 s) enabled notable detection of content release preceding vesicle collapse. All other PE distributions decreased fusion efficiency, altered pore lifetime, and reduced content release. With PE exclusively in the opposite leaflet, resolution of pore opening and content release was lost.
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Membrana Celular/metabolismo , Fusão de Membrana , Fosfatidiletanolaminas/metabolismo , Membrana Celular/química , Fosfatidiletanolaminas/química , Porosidade , ProbabilidadeRESUMO
Regulated exocytosis is a process by which neurotransmitters, hormones, and secretory proteins are released from the cell in response to elevated levels of calcium. In cells, secretory vesicles are targeted to the plasma membrane, where they dock, undergo priming, and then fuse with the plasma membrane in response to calcium. The specific roles of essential proteins and how calcium regulates progression through these sequential steps are currently incompletely resolved. We have used purified neuroendocrine dense-core vesicles and artificial membranes to reconstruct in vitro the serial events that mimic SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)-dependent membrane docking and fusion during exocytosis. Calcium recruits these vesicles to the target membrane aided by the protein CAPS (calcium-dependent activator protein for secretion), whereas synaptotagmin catalyzes calcium-dependent fusion; both processes are dependent on phosphatidylinositol 4,5-bisphosphate. The soluble proteins Munc18 and complexin-1 are necessary to arrest vesicles in a docked state in the absence of calcium, whereas CAPS and/or Munc13 are involved in priming the system for an efficient fusion reaction.
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Cálcio/metabolismo , Exocitose , Vesículas Secretórias/metabolismo , Transporte Biológico , Linhagem Celular , Membrana Celular/metabolismo , Humanos , Bicamadas Lipídicas , Fusão de Membrana , Lipídeos de Membrana/metabolismo , Modelos BiológicosRESUMO
AIMS: To test whether adjusting insulin and glucagon in response to exercise within a dual-hormone artificial pancreas (AP) reduces exercise-related hypoglycaemia. MATERIALS AND METHODS: In random order, 21 adults with type 1 diabetes (T1D) underwent three 22-hour experimental sessions: AP with exercise dosing adjustment (APX); AP with no exercise dosing adjustment (APN); and sensor-augmented pump (SAP) therapy. After an overnight stay and 2 hours after breakfast, participants exercised for 45 minutes at 60% of their maximum heart rate, with no snack given before exercise. During APX, insulin was decreased and glucagon was increased at exercise onset, while during SAP therapy, subjects could adjust dosing before exercise. The two primary outcomes were percentage of time spent in hypoglycaemia (<3.9 mmol/L) and percentage of time spent in euglycaemia (3.9-10 mmol/L) from the start of exercise to the end of the study. RESULTS: The mean (95% confidence interval) times spent in hypoglycaemia (<3.9 mmol/L) after the start of exercise were 0.3% (-0.1, 0.7) for APX, 3.1% (0.8, 5.3) for APN, and 0.8% (0.1, 1.4) for SAP therapy. There was an absolute difference of 2.8% less time spent in hypoglycaemia for APX versus APN (p = .001) and 0.5% less time spent in hypoglycaemia for APX versus SAP therapy (p = .16). Mean time spent in euglycaemia was similar across the different sessions. CONCLUSIONS: Adjusting insulin and glucagon delivery at exercise onset within a dual-hormone AP significantly reduces hypoglycaemia compared with no adjustment and performs similarly to SAP therapy when insulin is adjusted before exercise.
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Técnicas Biossensoriais/instrumentação , Diabetes Mellitus Tipo 1/tratamento farmacológico , Exercício Físico/fisiologia , Glucagon/administração & dosagem , Hipoglicemiantes/administração & dosagem , Sistemas de Infusão de Insulina , Insulina/administração & dosagem , Pâncreas Artificial , Adolescente , Adulto , Técnicas Biossensoriais/métodos , Glicemia/efeitos dos fármacos , Glicemia/metabolismo , Estudos Cross-Over , Diabetes Mellitus Tipo 1/sangue , Relação Dose-Resposta a Droga , Feminino , Glucagon/efeitos adversos , Humanos , Hipoglicemia/induzido quimicamente , Hipoglicemia/prevenção & controle , Hipoglicemiantes/efeitos adversos , Insulina/efeitos adversos , Masculino , Pessoa de Meia-Idade , Pâncreas Artificial/efeitos adversos , Adulto JovemRESUMO
INTRODUCTION: Cancer stem-like cells (CSCs) are tumour initiating, resistant to chemotherapy and play a role in metastasis. We have previously shown thrombin pathway signalling regulates CSC activity: thrombin pathway activation with FVIIa or through TF overexpression increases CSC activity whilst TF siRNA knockdown decreases it. We hypothesise that NOACs, by targeting specific factors in the thrombin pathway, may inhibit CSC activity. AIM: In breast cancer cell lines, to determine the effect of i) thrombin and ii) dabigatran (Boehringer Ingelheim), a direct thrombin inhibitor on cancer stem cell activity. MATERIALS AND METHODS: MDA-MB-231, MCF-7, SKBR3 and MDA-MB-157 cell lines representing the spectrum of breast cancer subtypes were cultured with: i) 0.1 NIH Units/ml human thrombin (Sigma) ii) 0.5µM Dabigatran (clinically relevant plasma concentration). Mammosphere culture is an established in vitro assay to measure CSC activity. Mammosphere forming efficiency (MFE) was calculated from the proportion of plated cells forming mammospheres in non-adherent culture, (each experiment, n=≥4, two-tailed independent t-test). RESULTS: Thrombin increased MFE in the high PAR-1 expressing MDA-MB-231 and MDA-MB-157 cell lines, but not the low PAR-1 expressing MCF-7/SKBR3 cell lines as compared to untreated controls (MDA-MB-231 mean (range): thrombin treated: 0.91 (0.70-1.11) vs control: 0.73 (0.51-0.93)%, p=<0.04; MDA-MB-157: thrombin treated: 0.58 (0.45-0.69) vs control: 0.37 (0.31-0.47)%, p=<0.001). Dabigatran abrogated the stimulatory effect of thrombin on MFE in thrombin-treated MDA-MB-231 and MDA-MB-157 cells (MDA-MB-231 mean (range) thrombin+Dabigatran: 1.10 (0.83-1.41) vs thrombin only: 1.45 (1.32-1.66)%, p=<0.01); MDA-MB-157 thrombin+Dabigatran: 0.48 (0.39-0.56) vs thrombin only: 0.58 (0.45-0.69)%, p=<0.05). CONCLUSIONS: The stimulation of mammosphere formation by thrombin and reduction by thrombin inhibitor treatment indicates a functional relationship between cancer stem-like cells and coagulation. This suggests possible clinical utility of novel oral anticoagulants in targeting this critical cancer cell subpopulation.
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INTRODUCTION: Ductal carcinoma in-situ (DCIS) is a preinvasive breast cancer where cancer cells remain confined within the ductal basement membrane. However, genotypic changes have been identified in stroma surrounding DCIS, outside the basement membrane. Stromal fibroblasts undergo phenotypic change in cancer to promote tumour angiogenesis, proliferation, immunosuppression and metastasis and in vivo can induce invasion of DCIS. Phenotypic changes in DCIS stromal fibroblasts may potentially act as a precursor for invasion. AIM: To determine if stromal fibroblasts in DCIS have procoagulant changes similar to those seen in cancer-associated fibroblasts in invasive breast cancer. MATERIALS AND METHODS: As part of the prospective cohort study CHAMPion (Cancer induced Hypercoagulabulity as a Marker of Prognosis), patients with DCIS (n=72) and invasive breast cancer (n=292) were recruited. Stromal fibroblasts in tumour and corresponding normal breast tissue (distant from the cancer) were quantified (percentage IHC stained) for tissue factor (TF), thrombin, PAR1 and PAR2. Fibroblasts were identified morphologically, at a minimum distance of 0.2mm from ductal tissue, to avoid myoepithelial scoring. Scoring was performed in duplicate by two independent pathologists. RESULTS: Fibroblast TF expression was present in normal breast tissue (mean 43% ([SD 27%]) but markedly increased in DCIS (mean 62% [SD 27%], p=0.002). Fibroblast TF expression was further increased in invasive breast cancer (mean 74% [SD 23%], normal vs invasion, p<0.001; DCIS vs invasion, p=0.03). Fibroblast thrombin and PAR2, but not PAR1, expression was increased in DCIS compared to normal (thrombin: 60% vs 42%, p<0.001; PAR2: 58% vs 41%, p=0.002), however no further significant increase was seen in invasive cancer (thrombin 63%, PAR2 61%). Fibroblast tissue factor correlated with fibroblast thrombin expression (p<0.001, r=0.4) and fibroblast PAR2 expression (p<0.001, r=0.5), with thrombin and PAR2 expression also correlating (p<0.001, r=0.4). CONCLUSIONS: Procoagulant phenotypic changes, in terms of increased TF, thrombin and PAR2 expression, occur in stromal fibroblasts at the preinvasive stage. It needs to be determined if this change is functional and therefore a potential therapeutic target for preventing transition to invasion.
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INTRODUCTION: Circulating tumour cells (CTCs) are a marker of poor prognosis and are associated with increased risk of venous thromboembolism in metastatic breast cancer. AIM: We aimed to correlate presence of CTCs and markers of hyper-coagulability (D-dimer, fibrinogen and thrombin-antithrombin [TAT]) with survival in metastatic breast cancer. MATERIALS AND METHODS: In a prospective study, enumeration of CTCs (CellSearch) and D-dimer, fibrinogen and TAT (ELISA) were measured at a single timepoint in 50 MBC (median age 59, range 36-82) patients undergoing active treatment. Survival data was determined at a median follow-up of 366days (range 58-986). RESULTS: To date, 25 patients have died (median survival 566days, range 135-978). CTCs (>1/7.5ml) were identified in 13 patients (range 2-31) and were associated with increased markers of hypercoagulability [D-dimer: median 1814 (IQR 2700) vs 755 (IQR 735) ng/ml, p=0.004; fibrinogen: median 4.2 (IQR 1.9) vs 3.2 (1.3) g/l, p=0.05; TAT: median 6.2(IQR 6.3) vs 4.7 (5.2) ng/ml, p=0.1]. CTCs were associated with visceral compared to just bony metastases (p=0.03) and their presence was associated with a trend for reduced survival (295days (CI: 0-652) vs 737days (CI: 186-1288), p=0.1). There was no correlation between CTCs /markers of hypercoagulability and age, oestrogen receptor, progesterone receptor or Her2 status. D-dimer, fibrinogen and TAT all inversely correlated with survival and were all significantly higher in patients dying within 1year (D-dimer: 1098 (IQR 1122) vs 723 (IQR 735) ng/ml, p=0.03; fibrinogen: 4.4 (1.1) vs 3.2 (0.8) g/l, p=0.004; TAT: 8.1 (6.3) vs 4.7(3.1) ng/ml, p=0.03 [analysis excludes patients with <1year follow-up, n=13]). D-dimer >1,500ng/ml was associated with significantly reduced survival (295days [CI: 0-615] vs 836days [404-1267], p=0.05). On Cox regression, D-dimer, but not fibrinogen or TAT was associated with an increased risk of death (HR 1.3 per 1,000ng/ml D-dimer, p=0.07). CONCLUSIONS: The correlation between CTCs, hypercoagulability and reduced survival in metastatic breast cancer suggests a possible role for the coagulation system in supporting tumour cell metastasis and is therefore a potential therapeutic target.
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INTRODUCTION: Breast cancer is associated with a 3-4 fold increased risk of VTE. These patients have a 4-fold lower survival than those remaining free of VTE, implying VTE is a surrogate marker for aggressive cancer. Tumour expression of thrombin pathway markers are increased in the oestrogen receptor negative (ER-), high Ki67, more aggressive breast cancer subtypes. In in vitro and in vivo studies, the thrombin pathway promotes cancer growth and metastases, highlighting the potential role of the thrombin pathway as a therapeutic target in cancer. AIM: To determine whether 14days of a preoperative oral Factor Xa inhibitor (Rivaroxaban) in oestrogen receptor negative early breast cancer patients results in inhibition of tumour proliferation as determined by a reduction in tumour Ki67 from baseline (pre-treatment) to 14days post treatment start (at time of surgical excision). RATIONALE: The TF-VIIa-FXa complex activates Protease Activated Receptor (PAR)2 to induce angiogensis, growth factors and tumour cell migration. Thrombin, in part via PAR1, induces angiogenesis, tumour cell proliferation as well as in vivo metastasis. In early breast cancer, TF and PAR2 expression is increased in the stroma, particularly in the more aggressive ER-, high Ki67 (proliferation) cancers. Rivaroxaban is an orally active direct Factor Xa inhibitor. Through inhibition of the TF-FVIIa-FXa complex, it can downregulate TF-FVIIa-FXa activation of PAR2, and inhibit conversion of prothrombin to thrombin. We hypothesise that 14days of Rivaroxaban will reduce breast cancer proliferation, as a surrogate marker for anti-cancer efficacy, in early breast cancer patients awaiting resectional surgery. RESULTS: Trial methodology: A multi-centre phase II preoperative 'Window-of Opportunity' randomised controlled trial of Rivaroxaban compared to no treatment in ER-, stage I-III early breast cancer patients. Patients will be randomised 1:1:1 (Rivaroxaban 20mg od: Rivaroxaban 10mg od: no treatment) and receive 14 (+/-3) days of treatment in the window between diagnosis and surgery. Randomisation will be blinded to pathologists, but not to patients or clinicians. Primary analysis will be based on the two Rivaroxaban arms being combined to form a Rivaroxaban: no treatment, 2:1 trial design, with change in Ki67 from baseline (pre) to post Rivaroxaban/no treatment (post) being the primary endpoint, and the no treatment arm acting as a reference group. Subgroup analysis of the Rivaroxaban arm (20mg od:10mg od) will allow assessment of dose-response. ACKNOWLEDGEMENT: Funder: National Institute for Health Research Eudract No: 2014-004909-33 REC Number: 15/NW/0406 UKCRN ID: 19731 Expected commencement: January 2016. For further information please contact Chief Investigator: cliona.kirwan@manchester.ac.uk.
Assuntos
Boca/lesões , Síndrome de Munchausen/diagnóstico , Esquizofrenia Paranoide/diagnóstico , Esquizofrenia/diagnóstico , Pele/lesões , Adulto , Antipsicóticos/uso terapêutico , Biópsia , Diagnóstico Diferencial , Humanos , Masculino , Militares , Síndrome de Munchausen/tratamento farmacológico , Esquizofrenia/tratamento farmacológico , Esquizofrenia Paranoide/tratamento farmacológicoRESUMO
BACKGROUND: In bi-hormonal closed-loop systems for treatment of diabetes, glucagon sometimes fails to prevent hypoglycemia. We evaluated glucagon responses during several closed-loop studies to determine factors, such as gain factors, responsible for glucagon success and failure. METHODS: We extracted data from four closed-loop studies, examining blood glucose excursions over the 50min after each glucagon dose and defining hypoglycemic failure as glucose values<60 mg/dl. Secondly, we evaluated hyperglycemic excursions within the same period, where glucose was>180 mg/dl. We evaluated several factors for association with rates of hypoglycemic failure or hyperglycemic excursion. These factors included age, weight, HbA1c, duration of diabetes, gender, automation of glucagon delivery, glucagon dose, proportional and derivative errors (PE and DE), insulin on board (IOB), night vs. day delivery, and point sensor accuracy. RESULTS: We analyzed a total of 251 glucagon deliveries during 59 closed-loop experiments performed on 48 subjects. Glucagon successfully maintained glucose within target (60-180 mg/dl) in 195 (78%) of instances with 40 (16%) hypoglycemic failures and 16 (6%) hyperglycemic excursions. A multivariate logistic regression model identified PE (p<0.001), DE (p<0.001), and IOB (p<0.001) as significant determinants of success in terms of avoiding hypoglycemia. Using a model of glucagon absorption and action, simulations suggested that the success rate for glucagon would be improved by giving an additional 0.8µg/kg. CONCLUSION: We conclude that glucagon fails to prevent hypoglycemia when it is given at a low glucose threshold and when glucose is falling steeply. We also confirm that high IOB significantly increases the risk for glucagon failures. Tuning of glucagon subsystem parameters may help reduce this risk.