RESUMEN
Fibrinogen C domain-containing protein 1 (FIBCD1) is an immune protein proposed to be involved in host recognition of chitin on the surface of pathogens. As FIBCD1 readily binds acetylated molecules, we have determined the high-resolution crystal structures of a recombinant fragment of the FIBCD1 C-terminal domain complexed with small N-acetyl-containing ligands to determine the mode of recognition. All ligands bind at the conserved N-acetyl-binding site (S1) with galactose and glucose-derived ligands rotated 180° relative to each other. One subunit of a native structure derived from protein expressed in mammalian cells binds glycosylation from a neighboring subunit, in an extended binding site. Across the various structures, the primary S1 binding pocket is occupied by N-acetyl-containing ligands or acetate, with N-acetyl, acetate, or sulfate ion in an adjacent pocket S1(2). Inhibition binding studies of N-acetylglucosamine oligomers, (GlcNAc)n, n = 1, 2, 3, 5, 11, via ELISA along with microscale thermophoresis affinity assays indicate a strong preference of FIBCD1 for longer N-acetylchitooligosaccharides. Binding studies of mutant H396A, located beyond the S1(2) site, showed no significant difference from wildtype, but K381L, within the S1(2) pocket, blocked binding to the model ligand acetylated bovine serum albumin, suggesting that S1(2) may have functional importance in ligand binding. The binding studies, alongside structural definition of diverse N-acetyl monosaccharide binding in the primary S1 pocket and of additional, adjacent binding pockets, able to accommodate both carbohydrate and sulfate functional groups, suggest a versatility in FIBCD1 to recognize chitin oligomers and other pathogen-associated carbohydrate motifs across an extended surface.
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Receptores de Superficie Celular , Humanos , Acetatos , Sitios de Unión/fisiología , Carbohidratos/química , Quitina/metabolismo , Hemostáticos , Ligandos , Unión Proteica , Receptores de Superficie Celular/química , Receptores de Superficie Celular/metabolismo , Sulfatos , Modelos Moleculares , Estructura Terciaria de ProteínaRESUMEN
[Figure: see text].
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Cardiomegalia/metabolismo , Proteínas Portadoras/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Glicoproteínas/metabolismo , Angiotensinas/toxicidad , Animales , Aorta/cirugía , Cardiomegalia/etiología , Cardiomegalia/patología , Proteínas Portadoras/genética , Células Cultivadas , Constricción , Proteínas de la Matriz Extracelular/genética , Femenino , Glicoproteínas/genética , Humanos , Integrinas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Miocitos Cardíacos/metabolismo , Comunicación Paracrina , Fenilefrina/toxicidad , Ratas , Estrés Fisiológico , Factor de Crecimiento Transformador beta/metabolismo , Remodelación VentricularRESUMEN
Lung cancer is one of the most common cancers and its incidence is rising around the world. Various studies suggest that miR-330 acts as a tumor-suppressor microRNA (miRNA) in different types of cancers, but precisely how has remained unclear. In this study, we investigate miR-330 expression in lung cancer patient samples, as well as in vitro, by studying how normalization of miR-330 expression affects lung cancer cellular phenotypes such as viability, apoptosis, proliferation, and migration. We establish that low miR-330 expression predicts poor lung cancer prognosis. Stable restoration of reduced miR-330 expression in lung cancer cells reduces cell viability, increases the fraction of apoptotic cells, causes G2/M cell cycle arrest, and inhibits cell migration. These findings are substantiated by increased mRNA and protein expression of markers for apoptosis via the intrinsic pathway, such as caspase 9, and decreased mRNA and protein expression of markers for cell migration, such as vimentin, C-X-C chemokine receptor type 4, and matrix metalloproteinase 9. We showed that reduced miR-330 expression predicts poor lung cancer survival and that stable restoration of miR-330 expression in lung cancer cells has a broad range of tumor-suppressive effects. This indicates that miR-330 is a promising candidate for miRNA replacement therapy for lung cancer patients.
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Movimiento Celular/genética , Proliferación Celular/genética , Supervivencia Celular/genética , Neoplasias Pulmonares/genética , MicroARNs/genética , Células A549 , Apoptosis/genética , Biomarcadores de Tumor/genética , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica/genética , Genes Supresores de Tumor/fisiología , Humanos , Neoplasias Pulmonares/patología , ARN Mensajero/genéticaRESUMEN
BACKGROUND AND AIMS: Hepatic sinusoidal cells are known actors in the fibrogenic response to injury. Activated hepatic stellate cells (HSCs), liver sinusoidal endothelial cells, and Kupffer cells are responsible for sinusoidal capillarization and perisinusoidal matrix deposition, impairing vascular exchange and heightening the risk of advanced fibrosis. While the overall pathogenesis is well understood, functional relations between cellular transitions during fibrogenesis are only beginning to be resolved. At single-cell resolution, we here explored the heterogeneity of individual cell types and dissected their transitions and crosstalk during fibrogenesis. APPROACH AND RESULTS: We applied single-cell transcriptomics to map the heterogeneity of sinusoid-associated cells in healthy and injured livers and reconstructed the single-lineage HSC trajectory from pericyte to myofibroblast. Stratifying each sinusoidal cell population by activation state, we projected shifts in sinusoidal communication upon injury. Weighted gene correlation network analysis of the HSC trajectory led to the identification of core genes whose expression proved highly predictive of advanced fibrosis in patients with nonalcoholic steatohepatitis (NASH). Among the core members of the injury-repressed gene module, we identified plasmalemma vesicle-associated protein (PLVAP) as a protein amply expressed by mouse and human HSCs. PLVAP expression was suppressed in activated HSCs upon injury and may hence define hitherto unknown roles for HSCs in the regulation of microcirculatory exchange and its breakdown in chronic liver disease. CONCLUSIONS: Our study offers a single-cell resolved account of drug-induced injury of the mammalian liver and identifies key genes that may serve important roles in sinusoidal integrity and as markers of advanced fibrosis in human NASH.
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Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Células Endoteliales/patología , Redes Reguladoras de Genes , Cirrosis Hepática/genética , Enfermedad del Hígado Graso no Alcohólico/patología , Animales , Biopsia , Capilares/citología , Capilares/patología , Tetracloruro de Carbono/administración & dosificación , Tetracloruro de Carbono/toxicidad , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Modelos Animales de Enfermedad , Endotelio Vascular/citología , Endotelio Vascular/patología , Femenino , Venas Hepáticas/citología , Venas Hepáticas/patología , Humanos , Hígado/irrigación sanguínea , Hígado/patología , Cirrosis Hepática/patología , Proteínas de la Membrana/genética , Ratones , Ratones Transgénicos , Análisis de Secuencia por Matrices de Oligonucleótidos , RNA-Seq , Análisis de la Célula IndividualRESUMEN
PURPOSE: Chemotherapy-induced gastrointestinal toxicity is a common adverse event during chemotherapeutic treatment. No uniformly applicable strategies exist to predict, prevent, or treat gastrointestinal toxicity. Thus, a goal of mucositis research is to identify targets for therapeutic interventions and individualized risk prediction. Fibrinogen C domain containing 1 (FIBCD1) is a transmembrane protein expressed in human intestinal epithelial cells with functions in the innate immune system. Previous observations have shown that FIBCD1 ameliorates dextran sulfate sodium (DSS)-induced intestinal inflammation in vivo. We evaluated the effect of FIBCD1 in a murine model of chemotherapy-induced gastrointestinal toxicity and inflammation. METHODS: Transgenic (Tg) mice overexpressing FIBCD1 in the intestinal epithelium (Fibcd1Tg) and wild-type (WT) littermates (C57BL/6N) were randomized to receive an intraperitoneal injection of doxorubicin 20 mg/kg or saline and were terminated 2 or 7 days after the injection. Gastrointestinal toxicity was evaluated by weight change, intestinal length, villus height/crypt depth, and histological mucositis score. Expression of inflammatory markers (IL-6, IL-1ß, and Tnfα) was measured by quantitative real-time PCR in intestinal tissue samples. RESULTS: Following doxorubicin treatment, WT mice exhibited an increased weight loss compared with Tg littermates (p < 0.001). No differences between genotypes were seen in mucositis score, intestinal length, villus height/crypt depth, or IL-6, IL-1ß, and Tnfα expression. CONCLUSION: Our findings suggest that FIBCD1 could ameliorate chemotherapy-induced gastrointestinal toxicity by reducing weight loss; however, the mechanism of this possible protective effect remains to be defined warranting additional investigations.
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Antineoplásicos/uso terapéutico , Mucositis/inducido químicamente , Mucositis/tratamiento farmacológico , Receptores de Superficie Celular/uso terapéutico , Pérdida de Peso/efectos de los fármacos , Animales , Modelos Animales de Enfermedad , Genotipo , Inyecciones Intraperitoneales , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
BACKGROUND: Alcoholic liver disease (ALD) is a public health concern that is the cause of half of all cirrhosis-related deaths. Early detection of fibrosis, ideally in the precirrhotic stage, is a key strategy for improving ALD outcomes and for preventing progression to cirrhosis. Previous studies identified the blood-borne marker human microfibrillar-associated protein 4 (MFAP4) as a biomarker for detection of hepatitis C virus (HCV)-related fibrosis. AIM: To evaluate the diagnostic accuracy of MFAP4 to detect ALD-induced fibrosis. METHOD: We performed a prospective, liver biopsy-controlled study involving 266 patients with prior or current alcohol overuse. Patients were split into a training and a validation cohort. RESULTS: MFAP4 was present in fibrotic hepatic tissue and serum MFAP4 levels increased with fibrosis grade. The area under the receiver operating characteristic curve (AUROC) for detection of cirrhosis was 0.91 (95% CI 0.85-0.96) in the training cohort and 0.91 (95% CI 0.79-1.00) in the validation cohort. For detection of advanced fibrosis, the AUROC was 0.88 (95% CI 0.81-0.94) in the training cohort and 0.92 (95% CI 0.83-1.00) in the validation cohort. The diagnostic accuracy did not differ between MFAP4 and the enhanced liver fibrosis (ELF) test or transient elastography (TE) in an intention-to-diagnose analysis. MFAP4 did not predict hepatic decompensation in a time-to-decompensation analysis in a subgroup of patients with cirrhosis. CONCLUSION: MFAP4 is a novel biomarker that can detect ALD-related fibrosis with high accuracy.
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Diagnóstico por Imagen de Elasticidad , Hepatopatías Alcohólicas , Biopsia , Proteínas Portadoras , Proteínas de la Matriz Extracelular , Glicoproteínas , Humanos , Hígado/patología , Cirrosis Hepática/diagnóstico , Cirrosis Hepática/patología , Hepatopatías Alcohólicas/diagnóstico , Hepatopatías Alcohólicas/patología , Estudios Prospectivos , Curva ROCRESUMEN
BACKGROUND: Symptomatic peripheral artery disease (PAD) is an atherosclerotic occlusive disease affecting the lower extremities. The cause of symptomatic PAD is atherosclerosis, vascular dysfunctions, impaired angiogenesis and neointima formation. Microfibrillar-associated protein 4 (MFAP4) is an extracellular matrix protein, which is highly expressed in the heart and arteries and recently introduced as a potential mediator of pathological vascular remodeling and neointima formation. We aimed to investigate the relationship between serum MFAP4 (sMFAP4) and symptomatic PAD outcomes. METHODS: A total of 286 PAD patients were analyzed if they had either intermittent claudication or critical lower-extremity ischemia (CLI) and followed for 7 years. The level of serum MFAP4 (sMFAP4) was measured by alphaLISA. Kaplan-Meier, Cox proportional hazard and logistic regression analysis were used to analyze the associations between upper tertile sMFAP4 and symptomatic PAD outcomes. RESULTS: Patients with upper tertile sMFAP4 had an odds ratio (OR) of 2.65 (p < 0.001) for having CLI diagnosis. Further analysis indicated that patients with upper tertile sMFAP4 had a hazard ratio (HR) of 1.97 (p = 0.04) for cardiovascular death during the 7-years follow-up. However, analysis of 2-year primary patency showed that patients with upper tertile sMFAP4 had decreased risk of vascular occlusion after reconstructive surgery with HR of 0.15 (p = 0.02). CONCLUSIONS: sMFAP4 has potential as a prognostic marker for cardiovascular death, primary patency of reconstructed vessels and CLI diagnosis in symptomatic PAD patients. Confirmation of observations in larger cohorts is warranted.
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Proteínas Portadoras/genética , Proteínas de la Matriz Extracelular/genética , Variación Genética , Glicoproteínas/genética , Enfermedad Arterial Periférica/genética , Anciano , Proteínas Portadoras/sangre , Determinación de Punto Final , Proteínas de la Matriz Extracelular/sangre , Femenino , Estudios de Seguimiento , Predisposición Genética a la Enfermedad , Glicoproteínas/sangre , Humanos , Estimación de Kaplan-Meier , Modelos Logísticos , Masculino , Persona de Mediana Edad , Enfermedad Arterial Periférica/sangre , Enfermedad Arterial Periférica/cirugía , Factores de RiesgoRESUMEN
Objectives: Surfactant protein-D (SP-D), an innate immune defence molecule of the collectin family, is expressed in lungs and additional extrapulmonary epithelia. SP-D has immune modulatory and anti-microbial effects depending on its oligomerization. The ratio of high molecular weight (HMW): low molecular weight (LMW) SP-D in serum is mainly determined by the Met11Thr polymorphism (SNP rs721917). We aimed to study the SP-D serum level and the molecular size distribution in patients with untreated axial spondyloarthritis (axSpA) as compared with control subjects. Methods: Thirty-four patients with disease modifier untreated axSpA according to the ASAS criteria, age 19-63 years, disease duration 3.9 (2.2-5.6) years were included. Demographics, smoking habits, HLA-B27 status, ASDAS, BASDAI, BASFI, BASMI and visual analogue scale scores were recorded. SP-D in serum was measured by ELISA. DNA was isolated from whole blood and single nucleotide polymorphism rs721917 was genotyped. SP-D molecular size distribution was determined using gel filtration chromatography. Results: SP-D in serum did not differ between patients with axSpA and healthy controls, 1177 (869, 1536) vs 910 (494, 1682) (P = 0.35) and SP-D did not correlate with disease activity. However, the HMW/LMW ratio of SP-D in serum was significantly lower in axSpA, 0.38 (0.18, 0.53) compared with controls 1.49 (0.37, 3.24) when adjusting for the Met11Thr polymorphism, gender, age, BMI and smoking (P = 0.0004). There was no correlation between HMW/LMW ratio and CRP or composite diseases outcome measures. Conclusion: We suggest that predominance of LMW oligomeric variants of SP-D may enhance local or systemic inflammatory responses in axSpA.
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Mediadores de Inflamación/sangre , Proteína D Asociada a Surfactante Pulmonar/sangre , Proteína D Asociada a Surfactante Pulmonar/genética , Espondiloartritis/sangre , Espondiloartritis/genética , Adulto , Estudios de Casos y Controles , Femenino , Genotipo , Antígeno HLA-B27 , Humanos , Masculino , Persona de Mediana Edad , Polimorfismo de Nucleótido Simple , Multimerización de Proteína , Adulto JovenRESUMEN
BACKGROUND AND OBJECTIVE: A structural single nucleotide polymorphism rs721917 in the surfactant protein D (SP-D) gene, known as Met11Thr, was reported to influence the circulating levels and degree of multimerization of SP-D and was associated with both COPD and atopy in asthma. Moreover, disease-related processes are known to degrade multimerized SP-D, however, the degree of the protein degradation in these diseases is not clarified. We aimed to determine the distribution of multimerized (high molecular weight (HMW)) and non-multimerized (low molecular weight (LMW)) species of serum SP-D and their correlation with genetic polymorphisms and presence of disease in Lebanese COPD and asthmatic patients. METHODS: Serum SP-D levels were measured by ELISA in 88 COPD, 121 asthmatic patients and 223 controls. Randomly selected subjects were chosen for genotyping of rs721917 and multimerization studies. HMW and LMW SP-D were separated by gel permeation chromatography. RESULTS: Serum SP-D levels were significantly increased in patients with COPD, but not in asthmatic patients, when compared to controls. Met11Thr variation strongly affected serum SP-D levels and the degree of multimerization, but was not associated with COPD and asthma in the study. Remarkably, HMW/LMW serum SP-D ratio was significantly lower in Met11/Met11 COPD and asthmatic patients compared to controls. CONCLUSION: Collectively, non-multimerized species of serum SP-D were dominant in COPD and asthmatic patients suggesting that degradation of SP-D takes place to a significant degree in pulmonary disease. Assays that can separate SP-D proteolytic breakdown products or modified forms from naturally occurring SP-D trimers may result in optimal disease markers for pulmonary inflammatory diseases.
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Asma/genética , ADN/genética , Polimorfismo de Nucleótido Simple , Multimerización de Proteína/genética , Enfermedad Pulmonar Obstructiva Crónica/genética , Proteína D Asociada a Surfactante Pulmonar/genética , Adulto , Anciano , Asma/metabolismo , Ensayo de Inmunoadsorción Enzimática , Femenino , Genotipo , Humanos , Masculino , Persona de Mediana Edad , Enfermedad Pulmonar Obstructiva Crónica/sangre , Proteína D Asociada a Surfactante Pulmonar/sangre , Adulto JovenRESUMEN
MFAP4 (microfibrillar-associated protein 4) is an extracellular glycoprotein found in elastic fibers without a clearly defined role in elastic fiber assembly. In the present study, we characterized molecular interactions between MFAP4 and elastic fiber components. We established that MFAP4 primarily assembles into trimeric and hexameric structures of homodimers. Binding analysis revealed that MFAP4 specifically binds tropoelastin and fibrillin-1 and -2, as well as the elastin cross-linking amino acid desmosine, and that it co-localizes with fibrillin-1-positive fibers in vivo. Site-directed mutagenesis disclosed residues Phe(241) and Ser(203) in MFAP4 as being crucial for type I collagen, elastin, and tropoelastin binding. Furthermore, we found that MFAP4 actively promotes tropoelastin self-assembly. In conclusion, our data identify MFAP4 as a new ligand of microfibrils and tropoelastin involved in proper elastic fiber organization.
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Proteínas Portadoras/metabolismo , Desmosina/metabolismo , Tejido Elástico/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Glicoproteínas/metabolismo , Microfibrillas/metabolismo , Proteínas de Microfilamentos/metabolismo , Modelos Moleculares , Tropoelastina/metabolismo , Sustitución de Aminoácidos , Animales , Proteínas Portadoras/química , Proteínas Portadoras/genética , Proteínas de la Matriz Extracelular/química , Proteínas de la Matriz Extracelular/genética , Fibrilina-1 , Fibrilinas , Glicoproteínas/química , Glicoproteínas/genética , Humanos , Ligandos , Masculino , Ratones Endogámicos C57BL , Ratones Mutantes , Proteínas de Microfilamentos/química , Proteínas de Microfilamentos/genética , Mutación , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Dominios y Motivos de Interacción de Proteínas , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Multimerización de Proteína , Transporte de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Tropoelastina/química , Tropoelastina/genéticaRESUMEN
OBJECTIVE: Arterial injury stimulates remodeling responses that, when excessive, lead to stenosis. These responses are influenced by integrin signaling in vascular smooth muscle cells (VSMCs). Microfibrillar-associated protein 4 (MFAP4) is an integrin ligand localized to extracellular matrix fibers in the vascular wall. The role of MFAP4 in vascular biology is unknown. We aimed to test the hypothesis that MFAP4 would enhance integrin-dependent VSMC activation. APPROACH AND RESULTS: We produced Mfap4-deficient (Mfap4(-/-)) mice and performed carotid artery ligation to explore the role of MFAP4 in vascular biology in vivo. Furthermore, we investigated the effects of MFAP4 in neointimal formation ex vivo and in primary VSMC and monocyte cultures in vitro. When challenged with carotid artery ligation, Mfap4(-/-) mice exhibited delayed neointimal formation, accompanied by early reduction in the number of proliferating medial and neointimal cells, as well as infiltrating leukocytes. Delayed neointimal formation was associated with decreased cross-sectional area of ligated Mfap4(-/-) carotid arteries resulting in lumen narrowing 28 days after ligation. MFAP4 blockade prohibited the formation of neointimal hyperplasia ex vivo. Moreover, we demonstrated that MFAP4 is a ligand for integrin αVß3 and mediates VSMC phosphorylation of focal adhesion kinase, migration, and proliferation in vitro. MFAP4-dependent VSMC activation was reversible by treatment with MFAP4-blocking antibodies and inhibitors of focal adhesion kinase and downstream kinases. In addition, we showed that MFAP4 promotes monocyte chemotaxis in integrin αVß3-dependent manner. CONCLUSIONS: MFAP4 regulates integrin αVß3-induced VSMC proliferation and migration, as well as monocyte chemotaxis, and accelerates neointimal hyperplasia after vascular injury.
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Enfermedades de las Arterias Carótidas/metabolismo , Proteínas Portadoras/metabolismo , Movimiento Celular , Proliferación Celular , Proteínas de la Matriz Extracelular/metabolismo , Glicoproteínas/metabolismo , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Neointima , Animales , Apoptosis , Arterias Carótidas/metabolismo , Arterias Carótidas/patología , Enfermedades de las Arterias Carótidas/genética , Enfermedades de las Arterias Carótidas/patología , Proteínas Portadoras/genética , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Quimiotaxis de Leucocito , Modelos Animales de Enfermedad , Proteínas de la Matriz Extracelular/deficiencia , Proteínas de la Matriz Extracelular/genética , Quinasa 1 de Adhesión Focal/antagonistas & inhibidores , Quinasa 1 de Adhesión Focal/metabolismo , Genotipo , Glicoproteínas/deficiencia , Glicoproteínas/genética , Humanos , Hiperplasia , Integrina alfaVbeta3/metabolismo , Ligandos , Masculino , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Monocitos/metabolismo , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/patología , Fenotipo , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Transducción de Señal , Factores de Tiempo , Remodelación VascularRESUMEN
BACKGROUND: Surfactant protein D (SP-D) is a host defense molecule of the innate immune system that enhances pathogen clearance and modulates inflammatory responses. We hypothesized that circulating SP-D levels are associated with chemotherapy-induced mucositis and infectious morbidity in children with acute lymphoblastic leukemia (ALL). PROCEDURE: In a prospective study, 43 children receiving treatment for ALL were monitored for mucosal toxicity from diagnosis through the induction phase of treatment. Serial blood draws were taken to determine the levels of SP-D, interleukin-6 (IL-6), C-reactive protein, and white blood cells. Data on fever, antibiotics, and bacteremia were collected. Baseline levels of circulating SP-D were compared with healthy controls. RESULTS: Baseline values of circulating SP-D were similar to levels in healthy controls (median: 829 ng/ml vs. 657 ng/ml, respectively, P > 0.05). After initiation of chemotherapy, a significant reduction in SP-D levels was observed at all time points: 704 ng/ml at day 8, 413 ng/ml at day 15, 395 ng/ml at day 22, and 520 ng/ml at day 29 (all, P < 0.05). No significant associations between SP-D values, the occurrence of mucosal toxicity, or infectious morbidity were observed. However, loss of circulating SP-D from days 8 to 15 was associated with more systemic inflammation, and lower SP-D values at day 15 were associated with elevated intestinal mucositis scores (P < 0.05). CONCLUSIONS: The current study supports the hypothesis that the detrimental effect of chemotherapy on patients' immune functions includes decreased circulating levels of innate mucosal molecules such as SP-D, potentially aggravating mucosal and systemic inflammatory responses.
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Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Mucositis/diagnóstico , Neumonía/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras/sangre , Proteína D Asociada a Surfactante Pulmonar/sangre , Adolescente , Proteína C-Reactiva/análisis , Niño , Preescolar , Femenino , Estudios de Seguimiento , Humanos , Lactante , Interleucina-6/sangre , Masculino , Mucositis/sangre , Mucositis/inducido químicamente , Membrana Mucosa/efectos de los fármacos , Estadificación de Neoplasias , Neumonía/sangre , Neumonía/inducido químicamente , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Pronóstico , Estudios ProspectivosRESUMEN
BACKGROUND: The human microfibrillar-associated protein 4 (MFAP4) is located to extracellular matrix fibers and plays a role in disease-related tissue remodeling. Previously, we identified MFAP4 as a serum biomarker candidate for hepatic fibrosis and cirrhosis in hepatitis C patients. The aim of the present study was to elucidate the potential of MFAP4 as biomarker for hepatic fibrosis with a focus on the differentiation of no to moderate (F0-F2) and severe fibrosis stages and cirrhosis (F3 and F4, Desmet-Scheuer scoring system). METHODS: MFAP4 levels were measured using an AlphaLISA immunoassay in a retrospective study including n = 542 hepatitis C patients. We applied a univariate logistic regression model based on MFAP4 serum levels and furthermore derived a multivariate model including also age and gender. Youden-optimal cutoffs for binary classification were determined for both models without restrictions and considering a lower limit of 80 % sensitivity (correct classification of F3 and F4), respectively. To assess the generalization error, leave-one-out cross validation (LOOCV) was performed. RESULTS: MFAP4 levels were shown to differ between no to moderate fibrosis stages F0-F2 and severe stages (F3 and F4) with high statistical significance (t test on log scale, p value <2.2·10(-16)). In the LOOCV, the univariate classification resulted in 85.8 % sensitivity and 54.9 % specificity while the multivariate model yielded 81.3 % sensitivity and 61.5 % specificity (restricted approaches). CONCLUSIONS: We confirmed the applicability of MFAP4 as a novel serum biomarker for assessment of hepatic fibrosis and identification of high-risk patients with severe fibrosis stages in hepatitis C. The combination of MFAP4 with existing tests might lead to a more accurate non-invasive diagnosis of hepatic fibrosis and allow a cost-effective disease management in the era of new direct acting antivirals.
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Proteínas Portadoras/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Glicoproteínas/metabolismo , Hepatitis C/complicaciones , Hepatitis C/metabolismo , Cirrosis Hepática/complicaciones , Cirrosis Hepática/metabolismo , Adulto , Análisis de Varianza , Biomarcadores/metabolismo , Estudios de Cohortes , Femenino , Hepatitis C/diagnóstico , Humanos , Cirrosis Hepática/diagnóstico , Modelos Logísticos , Masculino , Persona de Mediana Edad , Análisis Multivariante , Curva ROC , Sensibilidad y EspecificidadRESUMEN
The high resolution crystal structures of a recombinant fragment of the C-terminal fibrinogen-like recognition domain of FIBCD1, a vertebrate receptor that binds chitin, have been determined. The overall tetrameric structure shows similarity in structure and aggregation to the horseshoe crab innate immune protein tachylectin 5A. The high affinity ligand N-acetylmannosamine (ManNAc) binds in the S1 site, predominantly via the acetyl group with the oxygen and acetamide nitrogen hydrogen-bonded to the protein and the methyl group inserted into a hydrophobic pocket. The binding of the ManNAc pyranose ring differs markedly between the two independent subunits, but in all structures the binding of the N-acetyl group is conserved. In the native structure, a crystal contact results in one of the independent protomers binding the first GlcNAc of the Asn(340) N-linked glycan on the other independent protomer. In the ligand-bound structure this GlcNAc is replaced by the higher affinity ligand ManNAc. In addition, a sulfate ion has been modeled into the electron density at a location similar to the S3 binding site in L-ficolin, whereas in the native structure an acetate ion has been placed in the S1 N-acetyl binding site, and a sulfate ion has been placed adjacent to this site. These ion binding sites are ideally placed to receive the N-acetyl and sulfate groups of sulfated GalNAc residues of glycosaminoglycans such as chondroitin and dermatan sulfate. Together, these structures give insight into important determinants of ligand selectivity, demonstrating versatility in recognition and binding while maintaining conservation in N-acetyl and calcium binding.
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Quitina/metabolismo , Receptores de Superficie Celular/química , Receptores de Superficie Celular/metabolismo , Acetilación , Secuencia de Aminoácidos , Animales , Sitios de Unión , Calcio/metabolismo , Línea Celular , Quitina/química , Cristalografía por Rayos X , Fibrinógeno/química , Humanos , Insectos/citología , Ligandos , Datos de Secuencia Molecular , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Receptores de Superficie Celular/genética , Relación Estructura-ActividadRESUMEN
Surfactant protein D (SP-D) is a pulmonary collectin important in lung immunity. SP-D-deficient mice (Sftpd(-/-)) are reported to be susceptible to ovalbumin (OVA)- and fungal allergen-induced pulmonary inflammation, while treatment with exogenous SP-D has therapeutic effects in such disease models. ß-Glucans are a diverse group of polysaccharides previously suggested to serve as fungal ligands for SP-D. We set out to investigate if SP-D could interact with 1,3-ß-glucan and attenuate allergic pulmonary inflammation in the presence of 1,3-ß-glucan. Allergic airway disease was induced in Sftpd(-/-) and Sftpd(+/+) mice by OVA sensitization and subsequent challenge with OVA, 1,3-ß-glucan, or OVA/1,3-ß-glucan together. Mice in the combined treatment group were further treated with a high dose of recombinant fragment of human SP-D (rfhSP-D). We demonstrated direct interaction between SP-D and 1,3-ß-glucan. OVA-induced mucous cell metaplasia was increased in Sftpd(-/-) mice, supporting previously reported protective effects of endogenous SP-D in allergy. OVA-induced parenchymal CCL11 levels and eosinophilic infiltration in bronchoalveolar lavage were unaffected by 1,3-ß-glucan, but were reversed with rfhSP-D treatment. 1,3-ß-Glucan treatment did, however, induce pulmonary neutrophilic infiltration and increased TNF-α levels in bronchoalveolar lavage, independently of OVA-induced allergy. This infiltration was also reversed by treatment with rfhSP-D. 1,3-ß-Glucan reduced OVA-induced mucous cell metaplasia, T helper 2 cytokines, and IFN-γ production. rfhSP-D treatment further reduced mucous metaplasia and T helper 2 cytokine secretion to background levels. In summary, rfhSP-D treatment resulted in attenuation of both allergic inflammation and 1,3-ß-glucan-mediated neutrophilic inflammation. Our data suggest that treatment with high-dose SP-D protects from mold-induced exacerbations of allergic asthma.
Asunto(s)
Hipersensibilidad/complicaciones , Hipersensibilidad/tratamiento farmacológico , Inflamación/complicaciones , Inflamación/tratamiento farmacológico , Sustancias Protectoras/uso terapéutico , Proteína D Asociada a Surfactante Pulmonar/uso terapéutico , beta-Glucanos/metabolismo , Animales , Quimiocina CCL11/metabolismo , Citocinas/metabolismo , Femenino , Humanos , Hipersensibilidad/patología , Inmunoglobulina E/metabolismo , Inflamación/patología , Ligandos , Metaplasia , Ratones Endogámicos C57BL , Microbiota/efectos de los fármacos , Ovalbúmina , Sustancias Protectoras/farmacología , Proteoglicanos , Alveolos Pulmonares/efectos de los fármacos , Alveolos Pulmonares/patología , Proteína D Asociada a Surfactante Pulmonar/farmacología , Hipersensibilidad Respiratoria/complicacionesRESUMEN
BACKGROUND: Recently, several proteins of the extracellular matrix have been characterised as active contributors to allergic airway disease. Microfibrillar-associated protein 4 (MFAP4) is an extracellular matrix protein abundant in the lung, whose biological functions remain poorly understood. In the current study we investigated the role of MFAP4 in experimental allergic asthma. METHODS: MFAP4-deficient mice were subjected to alum/ovalbumin and house dust mite induced models of allergic airway disease. In addition, human healthy and asthmatic primary bronchial smooth muscle cell cultures were used to evaluate MFAP4-dependent airway smooth muscle responses. RESULTS: MFAP4 deficiency attenuated classical hallmarks of asthma, such as eosinophilic inflammation, eotaxin production, airway remodelling and hyperresponsiveness. In wild-type mice, serum MFAP4 was increased after disease development and correlated with local eotaxin levels. MFAP4 was expressed in human bronchial smooth muscle cells and its expression was upregulated in asthmatic cells. Regarding the underlying mechanism, we showed that MFAP4 interacted with integrin αvß5 and promoted asthmatic bronchial smooth muscle cell proliferation and CCL11 release dependent on phosphatidyloinositol-3-kinase but not extracellular signal-regulated kinase pathway. CONCLUSIONS: MFAP4 promoted the development of asthmatic airway disease in vivo and pro-asthmatic functions of bronchial smooth muscle cells in vitro. Collectively, our results identify MFAP4 as a novel contributor to experimental asthma, acting through modulation of airway smooth muscle cells.
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Asma/metabolismo , Proteínas Portadoras/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Glicoproteínas/metabolismo , Pulmón/metabolismo , Miocitos del Músculo Liso/metabolismo , Animales , Western Blotting , Adhesión Celular , Técnicas de Cultivo de Célula , Proliferación Celular , Modelos Animales de Enfermedad , Femenino , Humanos , Ratones , Fenotipo , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Variation in surfactant protein D (SP-D) is associated with lung function in tobacco smoke-induced chronic respiratory disease. We hypothesized that the same association exists in the general population and could be used to identify individuals sensitive to smoke-induced lung damage. The association between serum SP-D (sSP-D) and expiratory lung function was assessed in a cross-sectional design in a Danish twin population (n = 1,512, 18-72 yr old). The adjusted heritability estimates for expiratory lung function, associations between SP-D gene (SFTPD) single-nucleotide polymorphisms or haplotypes, and expiratory lung function were assessed using twin study methodology and mixed-effects models. Significant inverse associations were evident between sSP-D and the forced expiratory volume in 1 s and forced vital capacity in the presence of current tobacco smoking but not in nonsmokers. The two SFTPD single-nucleotide polymorphisms, rs1923536 and rs721917, and haplotypes, including these single-nucleotide polymorphisms or rs2243539, were inversely associated with expiratory lung function in interaction with smoking. In conclusion, SP-D is phenotypically and genetically associated with lung function measures in interaction with tobacco smoking. The obtained data suggest sSP-D as a candidate biomarker in risk assessments for subclinical tobacco smoke-induced lung damage. The data and derived conclusion warrant confirmation in a longitudinal population following chronic obstructive pulmonary disease initiation and development.
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Biomarcadores/análisis , Haplotipos/genética , Enfermedades Pulmonares/diagnóstico , Polimorfismo de Nucleótido Simple/genética , Proteína D Asociada a Surfactante Pulmonar/genética , Fumar/efectos adversos , Adolescente , Adulto , Anciano , Estudios Transversales , Dinamarca , Femenino , Volumen Espiratorio Forzado , Estudios de Asociación Genética , Humanos , Enfermedades Pulmonares/inducido químicamente , Enfermedades Pulmonares/genética , Masculino , Persona de Mediana Edad , Pronóstico , Surfactantes Pulmonares/metabolismo , Capacidad Vital , Adulto JovenRESUMEN
BACKGROUND: Crosstalk between the immune system in the brain and the periphery may contribute to the long-term outcome both in experimental and clinical stroke. Although, the immune defense collectin surfactant protein-D (SP-D) is best known for its role in pulmonary innate immunity, SP-D is also known to be involved in extrapulmonary modulation of inflammation in mice. We investigated whether SP-D affected cerebral ischemic infarction and ischemia-induced inflammatory responses in mice. METHODS: The effect of SP-D was studied by comparing the size of ischemic infarction and the inflammatory and astroglial responses in SP-D knock out (KO) and wild type (WT) mice subjected to permanent middle cerebral artery occlusion. SP-D mRNA production was assessed in isolated cerebral arteries and in the whole brain by PCR, and SP-D protein in normal appearing and ischemic human brain by immunohistochemistry. Changes in plasma SP-D and TNF were assessed by ELISA and proximity ligation assay, respectively. RESULTS: Infarct volumetric analysis showed that ablation of SP-D had no effect on ischemic infarction one and five days after induction of ischemia. Further, ablation of SP-D had no effect on the ischemia-induced increase in TNF mRNA production one day after induction of ischemia; however the TNF response to the ischemic insult was affected at five days. SP-D mRNA was not detected in parenchymal brain cells in either naïve mice or in mice subjected to focal cerebral ischemia. However, SP-D mRNA was detected in middle cerebral artery cells in WT mice and SP-D protein in vascular cells both in normal appearing and ischemic human brain tissue. Measurements of the levels of SP-D and TNF in plasma in mice suggested that levels were unaffected by the ischemic insult. Microglial-leukocyte and astroglial responses were comparable in SP-D KO and WT mice. CONCLUSIONS: SP-D synthesis in middle cerebral artery cells is consistent with SP-D conceivably leaking into the infarcted area and affecting local cytokine production. However, there was no SP-D synthesis in parenchymal brain cells and ablation of SP-D had no effect on ischemic cerebral infarction.
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Infarto Cerebral/metabolismo , Proteína D Asociada a Surfactante Pulmonar/sangre , Proteína D Asociada a Surfactante Pulmonar/deficiencia , Animales , Encéfalo/metabolismo , Encéfalo/patología , Antígeno CD11b/metabolismo , Arterias Cerebrales/metabolismo , Arterias Cerebrales/patología , Infarto Cerebral/etiología , Infarto Cerebral/patología , Femenino , Proteína Ácida Fibrilar de la Glía/metabolismo , Humanos , Infarto de la Arteria Cerebral Media/complicaciones , Antígenos Comunes de Leucocito/metabolismo , Leucocitos/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microglía/efectos de los fármacos , Microglía/metabolismo , Proteína D Asociada a Surfactante Pulmonar/genética , Factores de Tiempo , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
CD163-L1 belongs to the group B scavenger receptor cysteine-rich family of proteins, where the CD163-L1 gene arose by duplication of the gene encoding the hemoglobin scavenger receptor CD163 in late evolution. The current data demonstrate that CD163-L1 is highly expressed and colocalizes with CD163 on large subsets of macrophages, but in contrast to CD163 the expression is low or absent in monocytes and in alveolar macrophages, glia, and Kupffer cells. The expression of CD163-L1 increases when cultured monocytes are M-CSF stimulated to macrophages, and the expression is further increased by the acute-phase mediator IL-6 and the anti-inflammatory mediator IL-10 but is suppressed by the proinflammatory mediators IL-4, IL-13, TNF-α, and LPS/IFN-γ. Furthermore, we show that CD163-L1 is an endocytic receptor, which internalizes independently of cross-linking through a clathrin-mediated pathway. Two cytoplasmic splice variants of CD163-L1 are differentially expressed and have different subcellular distribution patterns. Despite its many similarities to CD163, CD163-L1 does not possess measurable affinity for CD163 ligands such as the haptoglobin-hemoglobin complex or various bacteria. In conclusion, CD163-L1 exhibits similarity to CD163 in terms of structure and regulated expression in cultured monocytes but shows clear differences compared with the known CD163 ligand preferences and expression pattern in the pool of tissue macrophages. We postulate that CD163-L1 functions as a scavenger receptor for one or several ligands that might have a role in resolution of inflammation.
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Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/metabolismo , Endocitosis/inmunología , Mediadores de Inflamación/fisiología , Macrófagos/inmunología , Macrófagos/patología , Receptores de Superficie Celular/metabolismo , Animales , Antígenos CD/biosíntesis , Antígenos CD/fisiología , Antígenos de Diferenciación Mielomonocítica/biosíntesis , Antígenos de Diferenciación Mielomonocítica/fisiología , Diferenciación Celular/inmunología , Células Cultivadas , Células HEK293 , Células HL-60 , Humanos , Inflamación/inmunología , Inflamación/metabolismo , Inflamación/patología , Mediadores de Inflamación/metabolismo , Células Jurkat , Macrófagos del Hígado/inmunología , Macrófagos del Hígado/patología , Tejido Linfoide/inmunología , Tejido Linfoide/metabolismo , Tejido Linfoide/patología , Macrófagos/metabolismo , Macrófagos Alveolares/inmunología , Macrófagos Alveolares/patología , Ratones , Monocitos/inmunología , Monocitos/metabolismo , Monocitos/patología , Neuroglía/inmunología , Neuroglía/patología , Receptores de Superficie Celular/biosíntesis , Receptores de Superficie Celular/fisiología , Células U937RESUMEN
BACKGROUND: Abnormalities of resistance arteries may play essential roles in the pathophysiology of aging and hypertension. Deficiency of the vascular extracellular matrix protein MFAP4 (microfibrillar-associated protein 4) has previously been observed as protective against aberrant arterial remodeling. We hypothesized that MFAP4-deficiency would reduce age- and hypertension-dependent arterial changes in extracellular matrix composition and stiffening. METHODS: Mesenteric arteries were isolated from old (20-23 months) littermate Mfap4+/+ and Mfap4-/- mice, and 2-photon excitation microscopy imaging was used to quantify elastin and collagen volumes and dimensions in the vascular wall. Ten-week-old littermate Mfap4+/+ and Mfap4-/- mice were subjected to 20 days of continuous Ang II (angiotensin II) infusion and hypertension was monitored using invasive blood pressure measurements. Arterial stiffness, responses to vascular constrictors, and myogenic tone were monitored using wire- or pressure-myography. Collagen contents were assessed by Western blotting. RESULTS: MFAP4-deficiency significantly increased collagen volume and elastin fragmentation in aged mesenteric arteries without affecting arterial stiffness. MFAP4-deficient mice exhibited reduced diastolic pressure in Ang II-induced hypertension. There was no significant effect of MFAP4-deficiency on mesenteric artery structural remodeling or myogenic tone, although collagen content in mesenteric arteries was tendentially increased in hypertensive Mfap4+/+ mice relative to Mfap4-/- mice. Increased efficacy of vasoconstrictors (phenylephrine, thromboxane) and reduced stiffness were observed in Ang II-treated Mfap4-/- mouse mesenteric arteries in ex vivo myography recordings. CONCLUSIONS: MFAP4-deficiency reduces the elastin/collagen ratio in the aging resistance artery without affecting arterial stiffness. In contrast, MFAP4-deficiency reduces the stiffness of resistance arteries and ameliorates Ang II-induced hypertension.