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BACKGROUND: Pulmonary Langerhans cell histiocytosis (PLCH) is a rare interstitial lung disease (ILD) associated with smoking, whose definitive diagnosis requires the exclusion of other forms of ILD and a compatible surgical lung biopsy. Bronchoalveolar lavage (BAL) is commonly proposed for the diagnosis of ILD, including PLCH, but the diagnostic value of this technique is limited. Here, we have analyzed the levels of a panel of cytokines and chemokines in BAL from PLCH patients, in order to identify a distinct immune profile to discriminate PLCH from other smoking related-ILD (SR-ILD), and comparing the results with idiopathic pulmonary fibrosis (IPF) as another disease in which smoking is considered a risk factor. METHODS: BAL samples were collected from thirty-six patients with different ILD, including seven patients with PLCH, sixteen with SR-ILD and thirteen with IPF. Inflammatory profiles were analyzed using the Human Cytokine Membrane Antibody Array. Principal component analysis (PCA) was performed to reduce dimensionality and protein-protein interaction (PPI) network analysis using STRING 11.5 database were conducted. Finally, Random forest (RF) method was used to build a prediction model. RESULTS: We have found significant differences (p < 0.05) on thirty-two cytokines/chemokines when comparing BAL from PLCH patients with at least one of the other ILD. Four main groups of similarly regulated cytokines were established, identifying distinct sets of markers for each cluster. Exploratory analysis using PCA (principal component analysis) showed clustering and separation of patients, with the two first components capturing 69.69% of the total variance. Levels of TARC/CCL17, leptin, oncostatin M (OSM) and IP-10/CXCL10 were associated with lung function parameters, showing positive correlation with FVC. Finally, random forest (RF) algorithm demonstrates that PLCH patients can be differentiated from the other ILDs based solely on inflammatory profile (accuracy 96.25%). CONCLUSIONS: Our results show that patients with PLCH exhibit a distinct BAL immune profile to SR-ILD and IPF. PCA analysis and RF model identify a specific immune profile useful for discriminating PLCH.
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Histiocitosis de Células de Langerhans , Fibrosis Pulmonar Idiopática , Enfermedades Pulmonares Intersticiales , Humanos , Líquido del Lavado Bronquioalveolar , Enfermedades Pulmonares Intersticiales/diagnóstico , Enfermedades Pulmonares Intersticiales/etiología , Enfermedades Pulmonares Intersticiales/metabolismo , Histiocitosis de Células de Langerhans/diagnóstico , Histiocitosis de Células de Langerhans/patología , Fumar/efectos adversos , Citocinas , Inmunoglobulinas , QuimiocinasRESUMEN
BACKGROUND: Asthma is a complex, multifactorial disease often linked with sensitization to house dust mites (HDM). There is a subset of patients that does not respond to available treatments, who present a higher number of exacerbations and a worse quality of life. To understand the mechanisms of poor asthma control and disease severity, we aim to elucidate the metabolic and immunologic routes underlying this specific phenotype and the associated clinical features. METHODS: Eighty-seven patients with a clinical history of asthma were recruited and stratified in 4 groups according to their response to treatment: corticosteroid-controlled (ICS), immunotherapy-controlled (IT), biologicals-controlled (BIO) or uncontrolled (UC). Serum samples were analysed by metabolomics and proteomics; and classifiers were built using machine-learning algorithms. RESULTS: Metabolomic analysis showed that ICS and UC groups cluster separately from one another and display the highest number of significantly different metabolites among all comparisons. Metabolite identification and pathway enrichment analysis highlighted increased levels of lysophospholipids related to inflammatory pathways in the UC patients. Likewise, 8 proteins were either upregulated (CCL13, ARG1, IL15 and TNFRSF12A) or downregulated (sCD4, CCL19 and IFNγ) in UC patients compared to ICS, suggesting a significant activation of T cells in these patients. Finally, the machine-learning model built including metabolomic and clinical data was able to classify the patients with an 87.5% accuracy. CONCLUSIONS: UC patients display a unique fingerprint characterized by inflammatory-related metabolites and proteins, suggesting a pro-inflammatory environment. Moreover, the integration of clinical and experimental data led to a deeper understanding of the mechanisms underlying UC phenotype.
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Asma , Hipersensibilidad , Animales , Antígenos Dermatofagoides , Humanos , Pyroglyphidae , Calidad de VidaRESUMEN
OBJECTIVES: To determine the burden and impact of cardiovascular risk factors (CRF) in antiphospholipid syndrome (APS) patients. METHODS: Analysis of the patients diagnosed with APS identified in the Spanish Hospital Discharge Database between 2016 and 2017. We analysed the admissions due to arterial (ATE) and venous thromboembolic events (VTE) and evaluated the incidence and the attributed risk of each CRF. RESULTS: 5424 admissions in patients diagnosed with APS were identified. 64.6% were women and the mean age was 54.6. The mortality rate was 3.1%. Overall, 35.8% of patients had hypertension, 14% were diabetic, 21.7% hypercholesterolaemic, 9.9% obese and 26.7% smokers. Thromboembolic events (67.9% arterial and 32.1% venous) accounted for 11.9% of admissions and 7.1% of deaths. Male sex (OR 1.83, 95% CI 1.41-2.21), cholesterol (OR 1.25, 95% CI 1.01-1.54) and smoking (OR 1.49, 95% CI 1.22-1.81) were independently associated with thromboembolic events. Meanwhile, patients with ATE were older (57 vs. 54.1 years p=0.033), and presented more secondary APS (17.1% vs. 10.6%, p=0.034), hypertension (47.7% vs. 33.5%, p=0.001), diabetes (16.9% vs. 9.6%, p=0.017), cholesterol (34.3% vs. 17.8%, p<0.001) and smoking habit (41.2% vs. 24%, p<0.001) when compared with VTE. Risk factors independently associated with ATE events were male sex (OR=1.61, 95% CI=1.30-2.03), hypertension (OR=1.30, 95% CI=1.03-1.64), cholesterol (OR=1.51, 95% CI=1.18-1.94) and smoking habit (OR=1.84, 95% CI=1.47-2.32), while VTE events were determined by male sex (OR=2.06, 95% CI=1.53-2.77) and obesity (OR=1.61, CI=1.02-2.52). CONCLUSIONS: Thromboembolic events in APS were in part determined by a high prevalence of CRF. The identification of distinct profiles may allow us to undertake a more personalised approach to reduce thromboembolic events and to individualise anticoagulant and antiplatelet therapy.
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Síndrome Antifosfolípido , Enfermedades Cardiovasculares , Hipertensión , Tromboembolia Venosa , Humanos , Masculino , Femenino , Persona de Mediana Edad , Síndrome Antifosfolípido/diagnóstico , Síndrome Antifosfolípido/epidemiología , Síndrome Antifosfolípido/complicaciones , Tromboembolia Venosa/diagnóstico , Tromboembolia Venosa/epidemiología , Tromboembolia Venosa/etiología , Enfermedades Cardiovasculares/diagnóstico , Enfermedades Cardiovasculares/epidemiología , Enfermedades Cardiovasculares/complicaciones , Factores de Riesgo , Sistema de Registros , Factores de Riesgo de Enfermedad Cardiaca , Hipertensión/epidemiologíaRESUMEN
Familias Unidas del Chamizal is a community organization in El Paso, TX that works to increase awareness around the issues of education, environmental racism, development, and public health. In 2019, the El Paso Independent School District launched an initiative to close several schools due to low enrollment, forcing students in Barrio Chamizal to relocate to Frederick Douglass Elementary, which borders a designated industrial zone and is surrounded by two major recycling plants. In response, Familias Unidas has organized to reverse the decision and call attention to the discriminatory practices of the school district. We take the public advocacy efforts of Familias Unidas as an instrumental case study to trace the intersections of health communication, Latinx communication, and environmental racism along the U.S.-Mexico border. Utilizing an intersectional borderlands health communication approach, we draw on publicly available texts to trace the argumentative strategies employed by Familias Unidas to mobilize community members. Familias Unidas rhetorically constructs a public health crisis in the borderlands by connecting the school closures to larger coalitional struggles concerning the environment, citizenship, race/ethnicity, language, and class. We identify three rhetorical strategies - familia, comunidad, and (in)justicia - employed by Familias Unidas to shape their public argument(s).
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Trastornos Relacionados con Sustancias , Comunicación , Justicia Ambiental , Hispánicos o Latinos , Humanos , Instituciones AcadémicasRESUMEN
Abdominal aortic aneurysm (AAA) evolution is unpredictable and no specific treatment exists for AAA, except surgery to prevent aortic rupture. Galectin-3 has been previously associated with CVD, but its potential role in AAA has not been addressed. Galectin-3 levels were increased in the plasma of AAA patients (n=225) compared with the control group (n=100). In addition, galectin-3 concentrations were associated with the need for surgical repair, independently of potential confounding factors. Galectin-3 mRNA and protein expression were increased in human AAA samples compared with healthy aortas. Experimental AAA in mice was induced via aortic elastase perfusion. Mice were treated intravenously with the galectin-3 inhibitor modified citrus pectin (MCP, 10 mg/kg, every other day) or saline. Similar to humans, galectin-3 serum and aortic mRNA levels were also increased in elastase-induced AAA mice compared with control mice. Mice treated with MCP showed decreased aortic dilation, as well as elastin degradation, vascular smooth muscle cell (VSMC) loss, and macrophage content at day 14 postelastase perfusion compared with control mice. The underlying mechanism(s) of the protective effect of MCP was associated with a decrease in galectin-3 and cytokine (mainly CCL5) mRNA and protein expression. Interestingly, galectin-3 induced CCL5 expression by a mechanism involving STAT3 activation in VSMC. Accordingly, MCP treatment decreased STAT3 phosphorylation in elastase-induced AAA. In conclusion, increased galectin-3 levels are associated with AAA progression, while galectin-3 inhibition decreased experimental AAA development. Our data suggest the potential role of galectin-3 as a therapeutic target in AAA.
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Aorta Abdominal/efectos de los fármacos , Aneurisma de la Aorta Abdominal/prevención & control , Galectina 3/antagonistas & inhibidores , Galectina 3/sangre , Elastasa Pancreática , Pectinas/farmacología , Animales , Aorta Abdominal/enzimología , Aorta Abdominal/patología , Aneurisma de la Aorta Abdominal/sangre , Aneurisma de la Aorta Abdominal/enzimología , Aneurisma de la Aorta Abdominal/patología , Proteínas Sanguíneas , Estudios de Casos y Controles , Células Cultivadas , Quimiocina CCL5/genética , Quimiocina CCL5/metabolismo , Dilatación Patológica , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Galectina 3/genética , Galectina 3/metabolismo , Galectinas , Humanos , Ratones Endogámicos C57BL , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , Fosforilación , ARN Mensajero/sangre , ARN Mensajero/genética , Factor de Transcripción STAT3/metabolismo , Transducción de Señal/efectos de los fármacos , Factores de Tiempo , Regulación hacia ArribaRESUMEN
Abdominal aortic aneurysm (AAA) is a permanent dilation of the aorta due to excessive proteolytic, oxidative and inflammatory injury of the aortic wall. We aimed to identify novel mediators involved in AAA pathophysiology, which could lead to novel therapeutic approaches. For that purpose, plasma from four AAA patients and four controls were analysed by a label-free proteomic approach. Among identified proteins, paraoxonase-1 (PON1) was decreased in plasma of AAA patients compared with controls, which was further validated in a bigger cohort of samples by ELISA. The phenylesterase enzymatic activity of PON1 was also decreased in serum of AAA patients compared with controls. To address the potential role of PON1 as a mediator of AAA, experimental AAA was induced by aortic elastase perfusion in wild-type (WT) mice and human transgenic PON1 (HuTgPON1) mice. Similar to humans, PON1 activity was also decreased in serum of elastase-induced AAA mice compared with healthy mice. Interestingly, overexpression of PON1 was accompanied by smaller aortic dilation and higher elastin and vascular smooth muscle cell (VSMC) content in the AAA of HuTgPON1 compared with WT mice. Moreover, HuTgPON1 mice display decreased oxidative stress and apoptosis, as well as macrophage infiltration and monocyte chemoattractant protein-1 (MCP1) expression, in elastase-induced AAA. In conclusion, decreased circulating PON1 activity is associated with human and experimental AAA. PON1 overexpression in mice protects against AAA progression by reducing oxidative stress, apoptosis and inflammation, suggesting that strategies aimed at increasing PON1 activity could prevent AAA.
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Aneurisma de la Aorta Abdominal/metabolismo , Arildialquilfosfatasa/metabolismo , Animales , Aneurisma de la Aorta Abdominal/prevención & control , Apoptosis/efectos de los fármacos , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Humanos , Inflamación/metabolismo , Macrófagos/metabolismo , Masculino , Ratones , Ratones Transgénicos , Proteómica/métodosRESUMEN
OBJECTIVE: To identify proteins related to intraluminal thrombus biological activities that could help to find novel pathological mechanisms and therapeutic targets for human abdominal aortic aneurysm (AAA). APPROACH AND RESULTS: Tissue-conditioned media from patients with AAA were analyzed by a mass spectrometry-based strategy using liquid chromatography coupled to tandem mass spectrometry. Global pathway analysis by Ingenuity software highlighted the presence of several circulating proteins, among them were proteins from the complement system. Complement C3 concentration and activation were assessed in plasma from AAA patients (small AAA, AAA diameter=3-5 cm and large AAA, AAA diameter >5 cm), showing decreased C3 levels and activation in large AAA patients. No association of a combination of single-nucleotide polymorphisms in complement genes between large and small AAA patients was observed. Intense extracellular C3 inmunostaining, along with C9, was observed in AAA thrombus. Analysis of C3 in AAA tissue homogenates and tissue-conditioned media showed increased levels of C3 in AAA thrombus, as well as proteolytic fragments (C3a/C3c/C3dg), suggesting its local deposition and activation. Finally, the functional role of local complement activation in polymorphonuclear (PMN) cell activation was tested, showing that C3 blockade by anti-C3 antibody was able to decrease thrombus-induced neutrophil chemotaxis and reactive oxygen species production. CONCLUSIONS: A decrease of systemic C3 concentration and activity in the later stages of AAA associated with local complement retention, consumption, and proteolysis in the thrombus could induce PMN chemotaxis and activation, playing a detrimental role in AAA progression.
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Aneurisma de la Aorta Abdominal/metabolismo , Aneurisma de la Aorta Abdominal/patología , Proteómica/métodos , Trombosis/metabolismo , Trombosis/patología , Anciano , Anciano de 80 o más Años , Aneurisma de la Aorta Abdominal/epidemiología , Autoanticuerpos/metabolismo , Quimiotaxis/fisiología , Cromatografía Liquida/métodos , Complemento C3/genética , Complemento C3/metabolismo , Complemento C9/genética , Complemento C9/metabolismo , Medios de Cultivo Condicionados/farmacología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Neutrófilos/citología , Neutrófilos/metabolismo , Polimorfismo de Nucleótido Simple , Especies Reactivas de Oxígeno/metabolismo , Factores de Riesgo , Espectrometría de Masas en Tándem/métodos , Trombosis/epidemiologíaRESUMEN
The reasons behind the onset and continuation of chronic inflammation in individuals with severe allergies are still not understood. Earlier findings indicated that there is a connection between severe allergic inflammation, systemic metabolic alterations and impairment of regulatory functions. Here, we aimed to identify transcriptomic alterations in T cells associated with the degree of severity in allergic asthmatic patients. T cells were isolated from severe (n = 7) and mild (n = 9) allergic asthmatic patients, and control (non-allergic, non-asthmatic healthy) subjects (n = 8) to perform RNA analysis by Affymetrix gene expression. Compromised biological pathways in the severe phenotype were identified using significant transcripts. T cells' transcriptome of severe allergic asthmatic patients was distinct from that of mild and control subjects. A higher count of differentially expressed genes (DEGs) was observed in the group of individuals with severe allergic asthma vs. control (4,924 genes) and vs. mild (4,232 genes) groups. Mild group also had 1,102 DEGs vs. controls. Pathway analysis revealed alterations in metabolism and immune response in the severe phenotype. Severe allergic asthmatic patients presented downregulation in genes related to oxidative phosphorylation, fatty acid oxidation and glycolysis together with increased expression of genes coding inflammatory cytokines (e.g. IL-19, IL-23A and IL-31). Moreover, the downregulation of genes involved in TGFß pathway together with a decreased tendency on the percentage of T regulatory cell (CD4 + CD25+), suggest a compromised regulatory function in severe allergic asthmatic patients. This study demonstrates a transcriptional downregulation of metabolic and cell signalling pathways in T cells of severe allergic asthmatic patients associated with diminished regulatory T cell function. These findings support a link between energy metabolism of T cells and allergic asthmatic inflammation.
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Cardiovascular diseases are the first leading cause of death and morbidity in developed countries. The use of animal models have contributed to increase our knowledge, providing new approaches focused to improve the diagnostic and the treatment of these pathologies. Several models have been developed to address cardiovascular complications, including atherothrombotic and cardiac diseases, and the same pathology have been successfully recreated in different species, including small and big animal models of disease. However, genetic and environmental factors play a significant role in cardiovascular pathophysiology, making difficult to match a particular disease, with a single experimental model. Therefore, no exclusive method perfectly recreates the human complication, and depending on the model, additional considerations of cost, infrastructure, and the requirement for specialized personnel, should also have in mind. Considering all these facts, and depending on the budgets available, models should be selected that best reproduce the disease being investigated. Here we will describe models of atherothrombotic diseases, including expanding and occlusive animal models, as well as models of heart failure. Given the wide range of models available, today it is possible to devise the best strategy, which may help us to find more efficient and reliable solutions against human cardiovascular diseases.
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Aneurisma de la Aorta , Aterosclerosis , Modelos Animales de Enfermedad , Insuficiencia Cardíaca , Trombosis , Animales , Aneurisma de la Aorta/etiología , Aneurisma de la Aorta/genética , Aneurisma de la Aorta/fisiopatología , Apolipoproteínas E/genética , Aterosclerosis/etiología , Aterosclerosis/genética , Aterosclerosis/fisiopatología , Perros , Insuficiencia Cardíaca/etiología , Insuficiencia Cardíaca/genética , Insuficiencia Cardíaca/fisiopatología , Humanos , Ratones , Conejos , Ratas , Receptores de LDL/genética , Porcinos , Trombosis/etiología , Trombosis/genética , Trombosis/fisiopatologíaRESUMEN
BACKGROUND: the admission and death causes of SLE patients might have changed over the last years. METHODS: Analysis of the Spanish National Hospital Discharge database. All individuals admitted with SLE, according to ICD-9, were selected. The following five admission categories were considered: SLE, cardiovascular disease (CVD), neoplasm, infection, and venous-thromboembolic disease (VTED), along four periods of time (1997-2000, 2001-2005, 2006-2010, and 2011-2015). RESULTS: The admissions (99,859) from 43.432 patients with SLE were included. The absolute number of admissions increased from 15,807 in 1997-2000 to 31,977 in 2011-2015. SLE decreased as a cause of admission (from 47.1% to 20.8%, p < 0.001), while other categories increased over the time, as follows: 5% to 8.6% for CVD, 8.2% to 13% for infection, and 1.4% to 5.5% for neoplasm (p < 0.001 for all). The admission mortality rate rose from 2.22% to 3.06% (p < 0.001) and the causes of death evolved in parallel with the admission categories. A significant trend to older age was observed over time in the overall population and deceased patients (p < 0.001). CONCLUSIONS: Better control of SLE over the past two decades has led to a decrease in early admissions, and disease chronification. As a counterpart, CVD, infections, and neoplasm have become the main causes of admissions and mortality.
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BACKGROUND: Immunotherapy is being tested in early-stage non-small cell lung cancer (NSCLC), and achieving higher rates of complete pathological responses (CPR) as compared to standard of care. Early identification of CPR patients has vital clinical implications. In this study, we focused on basal peripheral immune cells and their treatment-related changes to find biomarkers associated to CPR. METHODS: Blood from 29 stage IIIA NSCLC patients participating in the NADIM trial (NCT03081689) was collected at diagnosis and post neoadjuvant treatment. More than 400 parameters of peripheral blood mononuclear cells (PBMCs) phenotype and plasma soluble factors were analyzed. RESULTS: Neoadjuvant chemoimmunotherapy altered more than 150 immune parameters. At diagnosis, 11 biomarkers associated to CPR were described, with an area under the ROC curve >0.70 and p-value <.05. CPR patients had significantly higher levels of CD4+ PD-1+ cells, NKG2D, and CD56 expression on T CD56 cells, intensity of CD25 expression on CD4+ CD25hi+ cells and CD69 expression on intermediate monocytes; but lower levels of CD3+ CD56- CTLA-4+ cells, CD14++ CD16+ CTLA-4+ cells, CTLA-4 expression on T CD56 cells and lower levels of b-NGF, NT-3, and VEGF-D in plasma compared to non-CPR. Post treatment, CPR patients had significantly higher levels of CD19 expression on B cells, BCMA, 4-1BB, MCSF, and PARC and lower levels of MPIF-1 and Flt-3L in plasma compared to non-CPR. CONCLUSIONS: Patients achieving CPR seem to have a distinctive peripheral blood immune status at diagnosis, even showing different immune response to treatment. These results reinforce the different biology behind CPR and non-CPR responses.
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Biomarcadores de Tumor/sangre , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Neoplasias Pulmonares/terapia , Anciano , Antígenos CD19/metabolismo , Antineoplásicos/uso terapéutico , Área Bajo la Curva , Antígeno de Maduración de Linfocitos B/sangre , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Femenino , Humanos , Inmunoterapia , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/metabolismo , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Masculino , Persona de Mediana Edad , Terapia Neoadyuvante , Estadificación de Neoplasias , Factor de Crecimiento Nervioso/sangre , Neurotrofina 3/sangre , Curva ROC , Factor D de Crecimiento Endotelial Vascular/sangreRESUMEN
Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease whose prognosis is associated with clinical features, cell-of-origin and genetic aberrations. Recent integrative, multi-omic analyses had led to identifying overlapping genetic DLBCL subtypes. We used targeted massive sequencing to analyze 84 diagnostic samples from a multicenter cohort of patients with DLBCL treated with rituximab-containing therapies and a median follow-up of 6 years. The most frequently mutated genes were IGLL5 (43%), KMT2D (33.3%), CREBBP (28.6%), PIM1 (26.2%), and CARD11 (22.6%). Mutations in CD79B were associated with a higher risk of relapse after treatment, whereas patients with mutations in CD79B, ETS1, and CD58 had a significantly shorter survival. Based on the new genetic DLBCL classifications, we tested and validated a simplified method to classify samples in five genetic subtypes analyzing the mutational status of 26 genes and BCL2 and BCL6 translocations. We propose a two-step genetic DLBCL classifier (2-S), integrating the most significant features from previous algorithms, to classify the samples as N12-S, EZB2-S, MCD2-S, BN22-S, and ST22-S groups. We determined its sensitivity and specificity, compared with the other established algorithms, and evaluated its clinical impact. The results showed that ST22-S is the group with the best clinical outcome and N12-S, the more aggressive one. EZB2-S identified a subgroup with a worse prognosis among GCB-DLBLC cases.
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Algoritmos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Linfoma de Células B Grandes Difuso/tratamiento farmacológico , Linfoma de Células B Grandes Difuso/genética , Mutación , Adulto , Anciano , Antígenos CD79/genética , Proteínas de Unión al ADN/genética , Femenino , Humanos , Hibridación Fluorescente in Situ , Linfoma de Células B Grandes Difuso/clasificación , Masculino , Persona de Mediana Edad , Proteínas de Neoplasias/genética , Evaluación de Resultado en la Atención de Salud/métodos , Evaluación de Resultado en la Atención de Salud/estadística & datos numéricos , Pronóstico , Receptor Notch1/genética , Reproducibilidad de los Resultados , Rituximab/administración & dosificaciónRESUMEN
Nitric Oxide (NO) is involved in the development and progression of abdominal aortic aneurysms (AAA). We found that inhibition of inducible NO synthase (iNOS) protects mice in an elastase-induced AAA model, significantly inhibiting the production of matrix metalloproteinase-13 (MMP-13). The extracellular MMP inducer (EMMPRIN; CD147) was increased in human AAA biopsies and in wild-type murine AAA but not in AAA from iNOS null mice. In cells overexpressing ectopic EMMPRIN, MMP-13 secretion was stimulated, whereas silencing of EMMPRIN by RNA interference led to significant inhibition of MMP-13 expression. In addition, elastase infusion of MMP-13 null mouse aortas induced a significant increase of EMMPRIN but reduced aortic dilatation when compared with wild-type mice, suggesting that NO-mediated AAA may be mediated through EMMPRIN induction of MMP-13. These findings were further verified in elastase-infused iNOS null mice, in which daily administration of NO caused a significant aortic dilatation and the expression of EMMPRIN and MMP-13. By contrast, in iNOS wild-type mice, pharmacological inhibition of iNOS by administration of 1400 W induced a reduction of aortic diameter and inhibition of MMP-13 and EMMPRIN expression when compared with control mice. Our results suggest that NO may regulate the development of AAA in part by inducing the expression of EMMPRIN and modulating the activity of MMP-13 in murine and human aneurysms.
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Aneurisma de la Aorta Abdominal/enzimología , Aneurisma de la Aorta Abdominal/patología , Basigina/metabolismo , Metaloproteinasa 13 de la Matriz/biosíntesis , Óxido Nítrico/metabolismo , Animales , Progresión de la Enfermedad , Regulación hacia Abajo , Endotelio Vascular/enzimología , Endotelio Vascular/patología , Inducción Enzimática , Espacio Extracelular/enzimología , Humanos , Ratones , Ratones Endogámicos C57BL , Óxido Nítrico Sintasa de Tipo II/deficiencia , Óxido Nítrico Sintasa de Tipo II/metabolismo , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMEN
OBJECTIVE: Lack of endothelial nitric oxide synthase worsens atherosclerosis at least by increasing monocyte adhesion to endothelial cells. The purpose of this study was to elucidate the molecular mechanism elicited by NO. METHODS AND RESULTS: We evaluated atherosclerosis in apoE and NOS3/apoE-deficient mice fed with high-cholesterol diet. We found significant increase in aortic lesion size, and infiltration of macrophages in NOS3/apoE-null mice when compared to apoE-deficient animals. To test the relevance of cellular adhesion as well as extracellular matrix degradation, we evaluated ICAM-1, VCAM-1, PECAM-1, MMP-2, MMP-9, MMP-12, MT1-MMP, and MMP-13 levels in mouse aortas. Lack of NO inhibits MMP-13 and increases ICAM-1 levels in atherosclerosis as compared to apoE-null mice. Ectopically expression of ICAM-1 in eukaryotic cells revealed that extracellular domain of ICAM-1 harbors a substrate recognized by MMP-13. Incubation of COS-7 cells expressing ectopic ICAM-1 in the presence of active MMP-13 induced inhibition of RAW 264.7 cell adhesion to COS-7 monolayers. MALDI-TOF MS analysis combined to Liquid chromatography coupled to Ion Trap MS on ICAM-1 incubated with MMP-13 allowed us to determine the cleavage sites of MMP-13 at positions E61 and G98 of ICAM-1. G98 is part of a PDGQS moiety, which shows homology with the consensus PDGLS substrate located at the MMP-13 cleaved site of type II collagen I-alpha. CONCLUSIONS: Taking together, these results point toward MMP-13 as a mechanism for the NO-mediated protection of atherosclerosis.
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Aorta Torácica/patología , Apolipoproteínas E/deficiencia , Aterosclerosis/patología , Aterosclerosis/fisiopatología , Endotelio Vascular/enzimología , Molécula 1 de Adhesión Intercelular/genética , Metaloproteinasa 13 de la Matriz/metabolismo , Óxido Nítrico Sintasa de Tipo III/deficiencia , Alimentación Animal , Animales , Aterosclerosis/genética , Colesterol en la Dieta , Dieta , Modelos Animales de Enfermedad , Endotelio Vascular/patología , Matriz Extracelular/patología , Macrófagos/fisiología , Metaloproteinasa 13 de la Matriz/genética , Inhibidores de la Metaloproteinasa de la Matriz , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía ConfocalRESUMEN
Bone tissue renovation is a dynamic event in which osteoblasts and osteoclasts are responsible for the turnover between bone formation and bone resorption, respectively. During bone development, extracellular matrix remodeling is required for osteoblast differentiation and the process is largely mediated by the proteolytic activity of extracellular matrix metalloproteinases (MMPs), which play a fundamental role in osteoblast migration, unmineralized matrix degradation, and cell invasion. The recent advances towards investigation in osteogenesis have provided significant information about the transcriptional regulation of several genes, including MMPs, by the expression of crucial transcription factors like NFAT, ATF4, osterix, TAZ, and Cbfa-1-responsive elements. Evidence from gene knock-out studies have shown that bone formation is, at least in part, mediated by nitric oxide (NO), since mice deficient in endothelial nitric oxide synthase (eNOS) and mice deficient in the eNOS downstream effector (cGMP)-dependent protein kinase (PKG) show bone abnormalities, while inducible NOS (iNOS) null mice also show imbalances in bone osteogenesis and abnormalities in bone healing. Recently, in vitro data showed that Cbfa-1 and the MAPK pathways were crucial for osteoblastic cell differentiation, and NO was found to play a significant role. This article sheds light on some of the mechanisms that may influence NO-mediated actions in bone development.
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Desarrollo Óseo/fisiología , Diferenciación Celular/fisiología , Proliferación Celular , Óxido Nítrico/fisiología , Osteoblastos/citología , Animales , Ratones , Osteoblastos/metabolismo , Factores de Transcripción/metabolismoRESUMEN
The crosstalk between cancer cells and the tumor microenvironment has been implicated in cancer progression and metastasis. Fibroblasts and immune cells are widely known to be attracted to and modified by cancer cells. However, the role of pericytes in the tumor microenvironment beyond endothelium stabilization is poorly understood. Here, we report that pericytes promoted colorectal cancer (CRC) cell proliferation, migration, invasion, stemness, and chemoresistance in vitro, as well as tumor growth in a xenograft CRC model. We demonstrate that coculture with human CRC cells induced broad transcriptomic changes in pericytes, mostly associated with TGF-ß receptor activation. The prognostic value of a TGF-ß response signature in pericytes was analyzed in CRC patient data sets. This signature was found to be a good predictor of CRC relapse. Moreover, in response to stimulation by CRC cells, pericytes expressed high levels of TGF-ß1, initiating an autocrine activation loop. Investigation of secreted mediators and underlying molecular mechanisms revealed that IGFBP-3 is a key paracrine factor from activated pericytes affecting CRC cell migration and invasion. In summary, we demonstrate that the interplay between pericytes and CRC cells triggers a vicious cycle that stimulates pericyte cytokine secretion, in turn increasing CRC cell tumorigenic properties. Overall, we provide another example of how cancer cells can manipulate the tumor microenvironment.
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Movimiento Celular , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Comunicación Paracrina , Pericitos/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Animales , Carcinogénesis/patología , Línea Celular Tumoral , Proliferación Celular , Resistencia a Antineoplásicos , Femenino , Humanos , Ratones Desnudos , Invasividad Neoplásica , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Fenotipo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de SeñalRESUMEN
Protein S-nitrosylation is a reversible post-translational modification of protein cysteines that is increasingly being considered as a signal transduction mechanism. The "biotin switch" technique marked the beginning of the study of the S-nitrosoproteome, based on the specific replacement of the labile S-nitrosylation by a more stable biotinylation that allowed further detection and purification. However, its application for proteomic studies is limited by its relatively low sensitivity. Thus, typical proteomic experiments require high quantities of protein extracts, which precludes the use of this method in a number of biological settings. We have developed a "fluorescence switch" technique that, when coupled to 2-DE proteomic methodologies, allows the detection and identification of S-nitrosylated proteins by using limited amounts of starting material, thus significantly improving the sensitivity. We have applied this methodology to detect proteins that become S-nitrosylated in endothelial cells when exposed to S-nitroso-L-cysteine, a physiological S-nitrosothiol, identifying already known S-nitrosylation targets, as well as proteins that are novel targets. This "fluorescence switch" approach also allowed us to identify several proteins that are denitrosylated by thioredoxin in cytokine-activated RAW264.7 (murine macrophage) cells. We believe that this method represents an improvement in order to approach the identification of S-nitrosylated proteins in physiological conditions.
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Cisteína/análogos & derivados , Procesamiento Proteico-Postraduccional , Proteínas/análisis , Proteínas/metabolismo , Proteómica/métodos , S-Nitrosotioles/metabolismo , Animales , Línea Celular , Células Cultivadas , Cisteína/análisis , Cisteína/metabolismo , Células Endoteliales/citología , Fluorescencia , Humanos , Macrófagos/citología , Ratones , Nitrosación , S-Nitrosotioles/análisis , Sensibilidad y EspecificidadRESUMEN
Nitric oxide (NO) plays a critical role in wound healing, in part by promoting angiogenesis. However, the precise repair pathways affected by NO are not well defined. We now show that NO regulates matrix metalloproteinase-13 (MMP-13) release during wound repair. We find that normally MMP-13 is kept inside endothelial cells by an association with caveolin-1. However, nitration of MMP-13 on tyrosine residue Y338 causes it to dissociate from caveolin-1 and be released from endothelial cells. We next explored the functional significance of MMP-13 nitration in vivo. Skin injury increases nitration of MMP-13 in mice. Skin wounds in inducible nitric oxide synthase knockout mice release less MMP-13 and heal more slowly than skin wounds in wild-type mice. Conversely, skin wounds in caveolin-1 knockout mice have increased NO production, increased MMP-13 nitration, and accelerated wound healing. Collectively, our data reveal a new pathway through which NO modulates wound repair: nitration of MMP-13 promotes its release from endothelial cells, where it accelerates angiogenesis and wound healing.
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Metaloproteinasa 13 de la Matriz/metabolismo , Óxido Nítrico/farmacología , Cicatrización de Heridas/fisiología , Secuencia de Aminoácidos , Animales , Caveolina 1/deficiencia , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Óxido Nítrico Sintasa de Tipo II/fisiología , Tirosina/metabolismoRESUMEN
Macrophages play an important role in rhabdomyolysis-acute kidney injury (AKI), although the molecular mechanisms involved in macrophage differentiation are poorly understood. We analyzed the expression and regulation of CD163, a membrane receptor mainly expressed by anti-inflammatory M2 macrophages, in rhabdomyolysis-AKI and developed targeted probes for its specific detection in vivo by MRI. Intramuscular injection of glycerol in mice promoted an early inflammatory response, with elevated proportion of M1 macrophages, and partial differentiation towards a M2 phenotype in later stages, where increased CD163 expression was observed. Immunohistological studies confirmed the presence of CD163-macrophages in human rhabdomyolysis-AKI. In cultured macrophages, myoglobin upregulated CD163 expression via HO-1/IL-10 axis. Moreover, we developed gold-coated iron oxide nanoparticles vectorized with an anti-CD163 antibody that specifically targeted CD163 in kidneys from glycerol-injected mice, as determined by MRI studies, and confirmed by electron microscopy and immunological analysis. Our findings are the first to demonstrate that CD163 is present in both human and experimental rhabdomyolysis-induced AKI, suggesting an important role of this molecule in this pathological condition. Therefore, the use of probes targeting CD163-macrophages by MRI may provide important information about the cellular composition of renal lesion in rhabdomyolysis.
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Lesión Renal Aguda/diagnóstico por imagen , Antígenos CD/análisis , Antígenos de Diferenciación Mielomonocítica/análisis , Macrófagos/química , Imagen por Resonancia Magnética/métodos , Receptores de Superficie Celular/análisis , Rabdomiólisis/complicaciones , Coloración y Etiquetado/métodos , Lesión Renal Aguda/fisiopatología , Animales , Anticuerpos , Compuestos Férricos , Oro , Humanos , Macrófagos/inmunología , Ratones , NanopartículasRESUMEN
AIMS: To study the role of lipocalin-2 (Lcn2) and the effect of Lcn2 blockade via anti-Lcn2 antibody in the development of abdominal aortic aneurysm (AAA). METHODS AND RESULTS: Expression mRNA and protein levels of Lcn2 and its human orthologue neutrophil gelatinase-associated lipocalin (NGAL) in aortic wall samples from experimental mouse and human AAA samples, respectively, were analysed by real-time PCR and immunohistochemistry. Experimental AAA was induced by aortic elastase perfusion in wild-type mice (WT) and Lcn2-deficient mice (Lcn2-/-). NGAL/Lcn2 mRNA and protein levels in human and murine AAA samples were increased compared with healthy aortas. Decreased AAA incidence and reduced aortic expansion were observed in Lcn2-/- mice or mice preoperative treated with a polyclonal anti-Lcn2 antibody compared with WT mice or mice treated with control IgG, respectively, at Day 14 after elastase perfusion. Moreover, immunohistochemical analysis of AAA tissues from Lcn2-/- or anti-Lcn2-treated mice showed diminished elastin damage, reduced microvessels and polymorphonuclear neutrophil (PMN) infiltration, and enhanced preservation of vascular smooth muscle cells compared with WT aortas. Fluorescent molecular tomography revealed decreased MMP activity in AAA of Lcn2-/- mice compared with WT controls. Therapeutic administration of anti-Lcn2 antibody to WT mice 3 days after elastase perfusion decreased aortic dilatation and PMN infiltration compared with WT mice treated with control IgG. CONCLUSION: Either Lcn2 deficiency or anti-Lcn2 antibody blockade limits AAA expansion in mice by decreasing PMN infiltration in the aorta. Lcn2 modulation may therefore be a viable new therapeutic option for the treatment of AAA.