RESUMEN
Even though dealing with septic patients, the communication of the Gram stain result of positive blood cultures is postponed in most laboratories outside of conventional working hours. There is little evidence from clinics that this issue is being addressed. This study evaluates the potential benefit of an around-the-clock communication. Therefore, the effect of the communication on the antibiotic treatment and the delay of the communication during our non-office hours were measured. Over a three-month period, all blood cultures which were positive for the first time outside the normal working hours were analyzed. Two standardized telephone calls were used to compare the antibiotic treatment before and after the communication of the Gram stain result. The evaluation of the antibiotic treatment was based on the final testing result. In total, 135 patients were included. The rate of the adequate antibiotic increased by 8 percentage points to 69%. The average delay in the patients adjusted to an adequate treatment was 8:57 h (range 2:16-16:59). This prospective study shows a benefit of the immediate communication. Nevertheless, this benefit seems to be partly the result of suboptimal adherence to the guidelines regarding empirical antibiotic treatment. This prospective study has been registered in the German Clinical Trials Register under the identifier DRKS00014996 ( http://www.drks.de/DRKS00014996 ).
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Antibacterianos/uso terapéutico , Bacteriemia/diagnóstico , Bacteriemia/tratamiento farmacológico , Cultivo de Sangre/estadística & datos numéricos , Comunicación en Salud/métodos , Teléfono/normas , Atención Posterior , Bacteriemia/microbiología , Violeta de Genciana , Comunicación en Salud/normas , Humanos , Laboratorios de Hospital , Microscopía , Fenazinas , Estudios Prospectivos , Coloración y Etiquetado , Factores de Tiempo , Resultado del TratamientoAsunto(s)
Quimiocinas/metabolismo , Colitis/inmunología , Células Epiteliales/metabolismo , Enfermedades Inflamatorias del Intestino/inmunología , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Macrófagos/inmunología , Grasa Abdominal/metabolismo , Animales , Quimiocinas/genética , Quimiotaxis , Colitis/inducido químicamente , Colon/patología , Regulación de la Expresión Génica , Humanos , Inmunidad Mucosa , Péptidos y Proteínas de Señalización Intercelular/genética , Activación de Macrófagos , Ratones , Modelos AnimalesRESUMEN
Obesity is a risk factor for breast cancer development. A recent hypothesis suggests that the adipokines, adiponectin and leptin, are involved in breast cancer development. This prompted us to investigate the role of adiponectin and leptin in mammary carcinogenesis. Adiponectin receptors (AdipoR1 and AdipoR2) and leptin receptor (Ob-Rt, representing all the isoforms of Ob-R) proteins were detected by immunohistochemistry in in situ ductal carcinoma, invasive ductal malignancy, and healthy adjacent tissue. In addition, mRNA expression of adiponectin, AdipoR1, AdipoR2, leptin, Ob-Rt, and Ob-Rl (the long isoform of Ob-R) was observed in MCF-7 breast cancer cells. Interestingly, leptin mRNA expression was 34.7-fold higher than adiponectin mRNA expression in the MCF-7 cell line. Moreover, adiponectin (10 microg/ml) tended to decrease the mRNA expression of leptin (-36%) and Ob-Rl (-28%) and significantly decreased Ob-Rt mRNA level (-26%). In contrast, leptin treatment (1 microg/ml) significantly decreased AdipoR1 mRNA (-23%). Adiponectin treatment (10 microg/ml) inhibited the proliferation of MCF-7 cells, whereas leptin (1 microg/ml) stimulated the growth of cancer cells. In addition, adiponectin inhibited leptin-induced cell proliferation (both 1 microg/ml). Using microarray analysis, we found that adiponectin reduced the mRNA levels of genes involved in cell cycle regulation (mitogen-activated protein kinase 3 and ATM), apoptosis (BAG1, BAG3, and TP53), and potential diagnosis/prognosis markers (ACADS, CYP19A1, DEGS1, and EVL), whereas leptin induced progesterone receptor mRNA expression. In conclusion, the current study indicates an interaction of leptin- and adiponectin-signaling pathways in MCF-7 cancer cells whose proliferation is stimulated by leptin and suppressed by adiponectin.
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Adiponectina/metabolismo , Neoplasias de la Mama/metabolismo , Leptina/metabolismo , Receptores de Adiponectina/metabolismo , Receptores de Leptina/metabolismo , Adiponectina/genética , Apoptosis , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Western Blotting , Mama/metabolismo , Mama/patología , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Carcinoma Ductal de Mama/genética , Carcinoma Ductal de Mama/metabolismo , Carcinoma Ductal de Mama/patología , Proliferación Celular , Femenino , Perfilación de la Expresión Génica , Humanos , Técnicas para Inmunoenzimas , Técnicas In Vitro , Leptina/genética , Invasividad Neoplásica , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Adiponectina/genética , Receptores de Leptina/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales CultivadasRESUMEN
BACKGROUND: Increasing data support the hypothesis of a local and systemic crosstalk between adipocytes and monocytes mediated by fatty acids. The aim of this study was to characterize the immunomodulatory effects of a large panel of fatty acids on cytokines and chemokines in monocytic THP-1 cells and primary human monocytes. We tested whether anti-inflammatory fatty acids are able to inhibit the binding of lipopolysaccharide (LPS) to its receptor, toll-like receptor/MD-2 (TLR4/MD-2). MATERIALS AND METHODS: Resistin, monocyte chemoattractant protein-1 (MCP-1) and tumour necrosis factor (TNF) were measured by enzyme-linked immunosorbent assay. Proteins were analysed by Western blot. A designed Flag-tagged TLR4/MD-2 fusion protein (LPS trap) was used to investigate the effect of fatty acids on binding of LPS to its receptor. In 30 patients with type 2 diabetes mellitus (T2D), the correlation of serum triglyceride levels with LPS-induced monocyte activation was analysed. RESULTS: Eleven fatty acids investigated exerted differential effects on the monocytic release of cytokines and chemokines. Eicosapentaenoic acid had potent anti-inflammatory effects on human primary monocytes and THP-1 cells; 100 and 200 microM eicosapentaenoic acid dose-dependently inhibited LPS binding to the LPS trap. LPS-induced release of monocytic MCP-1 and TNF was significantly and positively correlated with serum triglyceride levels in 30 patients with T2D. CONCLUSIONS: Monocytic activation is differentially regulated by fatty acids and depends on triglyceride levels in T2D. The main finding of the present study shows that eicosapentaenoic acid inhibits the specific binding of LPS to TLR4/MD-2. Eicosapentaenoic acid represents a new anti-inflammatory LPS-antagonist.
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Quimiocina CCL2/metabolismo , Diabetes Mellitus Tipo 2/inmunología , Ácidos Grasos/metabolismo , Inmunidad Innata/inmunología , Resistina/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Adipocitos/efectos de los fármacos , Adipocitos/metabolismo , Western Blotting , Quimiocina CCL2/farmacología , Femenino , Humanos , Lipopolisacáridos/metabolismo , Antígeno 96 de los Linfocitos/metabolismo , Masculino , Persona de Mediana Edad , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
BACKGROUND: CD163 is a monocyte/macrophage specific receptor whose soluble form (sCD163) is elevated in inflammatory diseases. Obesity is associated with chronic inflammation and low adiponectin, an anti-inflammatory adipokine. Adiponectin, 5-aminoimidazole-4-carboxamide-1-4-ribofuranoside (AICAR) and metformin activate the AMP-kinase that exerts anti-inflammatory effects, and the influence of adiponectin and these drugs on monocytic CD163 was analysed, and cellular and sCD163 were determined in obesity and type 2 diabetes. MATERIALS AND METHODS: Monocytes were incubated with adiponectin, AICAR or metformin. Furthermore, monocytes and serum were obtained from type 2 diabetic patients (T2D), overweight (defined as a body mass index > or = 25 kg m(-2)) and normal-weight (NW) controls. CD163 was analysed by immunoblot and sCD163 was measured by enzyme-linked immunosorbent assay in the supernatants of the monocytes and in serum. RESULTS: In monocytes, adiponectin reduced cellular and surface CD163, whereas sCD163 was not altered in the corresponding supernatants. Further, metformin and AICAR downregulated CD163. Monocytic CD163 was higher in T2D and obesity, whereas sCD163 in the supernatants was not elevated and neither correlated with serum sCD163 nor systemic adiponectin. There was a positive correlation of monocytic sCD163 with serum but not with monocytic IL-6. In the serum of obese controls and T2D patients, sCD163 was significantly higher compared to NW donors and was positively associated with systemic IL-6. CONCLUSIONS: This study indicates that monocytic CD163 and systemic sCD163 are elevated in T2D and obesity. Adiponectin reduces CD163 in vitro, but additional factors related to obesity like IL-6 may be more relevant in vivo.
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Adiponectina/administración & dosificación , Aminoimidazol Carboxamida/análogos & derivados , Antígenos CD/efectos de los fármacos , Antígenos de Diferenciación Mielomonocítica/efectos de los fármacos , Hipoglucemiantes/administración & dosificación , Metformina/administración & dosificación , Obesidad/metabolismo , Receptores de Superficie Celular/efectos de los fármacos , Ribonucleótidos/administración & dosificación , Adiponectina/farmacología , Adulto , Aminoimidazol Carboxamida/administración & dosificación , Aminoimidazol Carboxamida/farmacología , Antígenos CD/genética , Antígenos de Diferenciación Mielomonocítica/genética , Diabetes Mellitus Tipo 2 , Regulación hacia Abajo , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Hipoglucemiantes/farmacología , Masculino , Metformina/farmacología , Persona de Mediana Edad , Obesidad/tratamiento farmacológico , Receptores de Superficie Celular/genética , Ribonucleótidos/farmacologíaRESUMEN
BACKGROUND: Visceral adipose tissues secret a variety of adipokines; however, it is not known whether they are present in the peritoneal fluid. It was the aim of this study to investigate peritoneal fluid concentrations of novel (cartonectin, omentin) and classical adipokines (leptin, adiponectin, resistin, visfatin) in patients with ascites. MATERIAL AND METHODS: Ninety-six patients (71 men and 25 women) undergoing paracentesis were included. Of these, 76 suffered from liver cirrhosis. Adipokines were measured by enzyme-linked immunosorbent assay or Western blot. RESULTS: Each adipokine was detected in ascites with a broad range. Serum-ascites ratios (SAR) correlated with clinical and laboratory parameters. The main variables influencing peritoneal fluid adipokine concentrations were body mass index (BMI), local inflammation, systemic inflammation and serum adipokine concentrations. Resistin was significantly higher in patients with peritonitis and showed a positive correlation with peripheral leucocytes (white blood cell count). Leptin was correlated with the underlying disease. Visfatin correlated with peripheral white blood cell and C-reactive protein levels. Omentin expression was correlated with ascitic leucocyte count, ascitic albumin concentration and low albumin SAR. BMI was correlated positively with ascitic leptin levels and cartonectin protein levels. CONCLUSIONS: Peritoneal fluid adipokine concentrations are characterized by individual SARs, depend on the presence of peritonitis, and correlate with underlying disease, BMI and systemic inflammation. The data open a new field of research on the role of the peritoneum and visceral adipokines in gastrointestinal diseases.
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Adipoquinas/sangre , Adiponectina/sangre , Ascitis/diagnóstico , Líquido Ascítico/metabolismo , Cirrosis Hepática/metabolismo , Peritonitis/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Western Blotting/métodos , Ensayo de Inmunoadsorción Enzimática/métodos , Femenino , Humanos , Masculino , Persona de Mediana Edad , Paracentesis/métodos , Análisis de RegresiónRESUMEN
INTRODUCTION: Even diabetic patients with excellent glycemic control can develop diabetic complications very early. Possibly, not only the degree of glycemic control, but other factors as well are responsible for the development of diabetic microangiopathy. Since adiponectin represents an adipocyte-specific secretory protein modulating endothelial cell functions, it was the aim of the present study to investigate the role of adiponectin serum levels as well as adiponectin gene polymorphisms in the development of diabetic retinopathy. METHODS: A population based cohort of caucasian patients (n=523) with type 2 diabetes mellitus was recruited from an epidemiological field survey. Serum adiponectin levels were determined by ELISA. Genotypes of the Tyr111His and the Gly15Gly polymorphism were determined by PCR-based RFLP analysis. Diabetic retinopathy was graded by fundus photography. RESULTS: The data demonstrate, that a) the Tyr111His (T-->C) polymorphism influences adiponectin serum levels, b) adiponectin serum levels do correlate with the prevalence of diabetic retinopathy, and c) patients heterozygous for the +45 T-->G (Gly15Gly) polymorphism show a lower prevalence of diabetic retinopathy. Furthermore, we could generate the proof of principle that adiponectin is detectable in the fluid of the human vitreous body. SUMMARY: Adiponectin gene polymorphisms influence adiponectin serum levels and elevated adiponectin serum levels are associated with diabetic retinopathy in patients with diabetes mellitus type 2. Therefore, endothelial cell modulating adiponectin should be further investigated as a candidate gene in the development and progression of retinopathy associated with type 2 diabetes mellitus.
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Adiponectina/sangre , Adiponectina/genética , Diabetes Mellitus Tipo 2/genética , Retinopatía Diabética/genética , Polimorfismo de Nucleótido Simple , Anciano , Sustitución de Aminoácidos , Índice de Masa Corporal , Retinopatía Diabética/epidemiología , Retinopatía Diabética/fisiopatología , Femenino , Angiografía con Fluoresceína , Hemoglobina Glucada/metabolismo , Humanos , Masculino , Mutación , Población Blanca/genéticaRESUMEN
INTRODUCTION: Cell culture media with high glucose concentration are normally used. Data on the secretion of the adipokines adiponectin and resistin from adipocytes in response to insulin and growth hormone (GH) both under normo- and hyperglycemic conditions are not available. It was the aim of the study to investigate the impact of standard metabolic conditions (normo-/hyperglycemia, normo-/hyperinsulinemia) and of GH on the secretion of adiponectin and resistin. MATERIAL AND METHODS: 3T3-L1 preadipocytes were differentiated into adipocytes and then incubated under normoglycemia (100 mg/dl), hyperglycemia (450 mg/dl), in combination with insulin (0, 0.2, 2.0 nM) and/or GH (1 nM). Adiponectin and resistin secretion was measured by ELISA. RESULTS: Insulin significantly stimulates adiponectin and resistin secretion under normo- and hyperglycemia. Hyperglycemia PER SE stimulates adiponectin and resistin secretion both in the absence and presence of low or high insulin concentrations. GH stimulates adiponectin secretion both under normoglycemic and hyperglycemic conditions. Whereas insulin does not modulate GH-induced adiponectin secretion under normoglycemia, insulin augments adiponectin release under hyperglycemia. GH stimulates resistin secretion only under normoglycemia, but not under hyperglycemic conditions. Since scavenger receptor B-I expression did not change, these effects are specific and not caused by a simple enhancement of adipocyte differentiation. DISCUSSION: Glucose, insulin and growth hormone have significant and interfering effects on the secretion of resistin and adiponectin. Several of the well-known in vivo phenomena such as diurnal variation or effects of re-feeding and weight-loss might be explained by direct effects of these hormones on adipocytes. Finally, when effects of hormones on adipocyte function are investigated, it is a prerequisite to take glucose levels of the cell culture media into account.
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Adipocitos/efectos de los fármacos , Adipocitos/metabolismo , Adipoquinas/metabolismo , Glucosa/administración & dosificación , Hormona del Crecimiento/farmacología , Insulina/farmacología , Células 3T3-L1 , Adipocitos/química , Adiponectina/metabolismo , Animales , Western Blotting , Interacciones Farmacológicas , Ratones , Resistina/metabolismo , Receptores Depuradores de Clase B/análisisRESUMEN
AOX1, a member of the cytosolic molybdenum hydroxylase family, has been identified by us earlier as an ABCA1-interacting protein. AOX1 is well-described as xenobiotic metabolizing enzyme, which upon oxidation of acetaldehyde and retinaldehyde to acetic acid and retinoic acid generates reactive oxygen species. Here we show that knock-down of AOX1 in HepG2 by small interfering RNA significantly reduced ABCA1-dependent lipid efflux and enhanced phagocytic uptake of microspheres similar to ABCA1 deficiency, without affecting ABCA1 mRNA and protein levels. ABCA1 and AOX1 are coexpressed in human hepatocytes, kidney proximal tubular epithelial cells, Leydig, and adrenocortical cells. Expression of ABCA1 and AOX1 was investigated by immunohistochemistry in liver tissue arrays. A strong AOX1 expression was found in normal liver, and in cirrhosis. In contrast, hepatocellular carcinomas showed either a complete loss or reduced expression of AOX1. Significant correlations were found between reduced AOX1 expression and tumor stage, or metastatic or regional lymph node states. Deregulation was also observed for ABCA1 expression but to a lesser extent. Our findings show that the interaction of ABCA1 with AOX1 modulates ABCA1-linked cellular functions such as lipid efflux and phagocytosis in hepatocytes, and the reduced expression of AOX1 in malignant transformed hepatocytes supports the differentiation dependent upregulation of AOX1.
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Transportadoras de Casetes de Unión a ATP/metabolismo , Aldehído Oxidasa/metabolismo , Carcinoma Hepatocelular/enzimología , Hepatocitos/metabolismo , Transportador 1 de Casete de Unión a ATP , Transportadoras de Casetes de Unión a ATP/genética , Aldehído Oxidasa/genética , Línea Celular , Línea Celular Tumoral , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Ácidos Grasos/metabolismo , Biblioteca de Genes , Silenciador del Gen/fisiología , Hepatocitos/enzimología , Hepatocitos/patología , Humanos , ARN Mensajero/análisis , ARN Interferente Pequeño/fisiología , Transducción de Señal/fisiología , Estadísticas no Paramétricas , Distribución TisularRESUMEN
INTRODUCTION: Apolipoprotein A-IV (apoA-IV), an intestinally and cerebrally synthesized satiety factor and anti-atherogenic plasma apolipoprotein, was recently identified as an anti-inflammatory protein. In order to elucidate whether intestinal apoA-IV exerts similar repair function as its hepatic homologue apolipoprotein A-V (apoA-V), apoA-IV-interactive proteins were searched and in vitro functional studies were performed with apoA-IV overexpressing cells. ApoA-IV was also analyzed in the intestinal mucosa of patients with inflammatory bowel diseases (IBD), together with other genes involved in epithelial junctional integrity. METHODS: A yeast-two-hybrid screening was used to identify apoA-IV-interactors. ApoA-IV was overexpressed in Caco-2 and HT-29 mucosal cells for colocalization and in vitro epithelial permeability studies. Mucosal biopsies from quiescent regions of colon transversum and terminal ileum were subjected to DNA-microarray analysis and pathway-related data mining. RESULTS: Four proteins interacting with apoA-IV were identified, including apolipoprotein B-100, alpha1-antichymotrypsin, cyclin C, and the cytosolic adaptor alpha-catenin, thus linking apoA-IV to adherens junctions. Overexpression of apoA-IV was paralleled with a differentiated phenotype of intestinal epithelial cells, upregulation of junctional proteins, and decreased paracellular permeability. Colocalization between alpha-catenin and apoA-IV occurred exclusively in junctional complexes. ApoA-IV was downregulated in quiescent mucosal tissues from patients suffering from IBD. In parallel, only a distinct set of junctional genes was dysregulated in non-inflamed regions of IBD gut. CONCLUSIONS: ApoA-IV may act as a stabilizer of adherens junctions interacting with alpha-catenin, and is likely involved in the maintenance of junctional integrity. ApoA-IV expression is significantly impaired in IBD mucosa, even in non-inflamed regions.
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Apolipoproteínas A/metabolismo , Apolipoproteínas A/fisiología , Permeabilidad de la Membrana Celular/genética , Enfermedades Inflamatorias del Intestino/genética , Enfermedades Inflamatorias del Intestino/metabolismo , Mucosa Intestinal/fisiología , alfa Catenina/metabolismo , Apolipoproteínas A/genética , Células CACO-2 , Perfilación de la Expresión Génica , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HT29 , Humanos , Enfermedades Inflamatorias del Intestino/patología , Uniones Intercelulares/metabolismo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Análisis de Secuencia por Matrices de Oligonucleótidos , Unión Proteica , ARN Mensajero/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Saciedad/fisiología , TransfecciónRESUMEN
The recent technological advances in high-throughput gene expression analysis allow the simultaneous investigation of thousands of genes. These technologies represent promising tools for the identification of new drug targets and considerable progress has been achieved in cancer research where microarray data provide a basis to design new drugs and to predict adverse reactions and the efficacy of chemotherapy. The metabolic syndrome represents a cluster of disorders including high blood pressure, insulin resistance/type 2 diabetes mellitus, visceral obesity and dyslipidaemia with fatty liver disease being a common associated complication. High-throughput gene expression analyses using GeneChips, microarrays and serial analysis of gene expression (SAGE) have been applied to study global gene expression in insulin resistance/type 2 diabetes mellitus. Type 2 diabetes mellitus is a multifactorial and polygenic disease by which several organs are affected. Therefore, the identification of both, disease causing and therapeutically relevant target genes is an ambitious challenge. In the present review we focus on genomic approaches that used biopsies from human skeletal muscle, liver and adipose tissue, the main organs affected by insulin resistance. Members of the PPARgamma coactivator-1 (PGC-1) family of transcriptional coactivators are decreased in skeletal muscle in insulin resistance accounting for the reduced expression of genes involved in mitochondrial oxidative phosphorylation. Hepatic steatosis is also linked to alterations in mitochondrial phosphorylation and oxidative metabolism. An up regulation of pro-inflammatory genes can be detected in early stages of fatty liver disease without histological signs of inflammation. Impaired adipogenesis, intra-adipose accumulation of macrophages and a sustained release of inflammatory and acute phase proteins are characteristic features of adipose tissue in obesity and may aggravate systemic insulin resistance.
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Diabetes Mellitus Tipo 2/genética , Sistemas de Liberación de Medicamentos/métodos , Animales , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Diabetes Mellitus Tipo 2/metabolismo , Perfilación de la Expresión Génica , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos/métodosRESUMEN
Using coherent x-ray speckle metrology, we have measured the influence of disorder on major loop return point memory (RPM) and complementary point memory (CPM) for a series of perpendicular anisotropy Co/Pt multilayer films. In the low disorder limit, the domain structures show no memory with field cycling--no RPM and no CPM. With increasing disorder, we observe the onset and the saturation of both the RPM and the CPM. These results provide the first direct ensemble-sensitive experimental study of the effects of varying disorder on microscopic magnetic memory and are compared against the predictions of existing theories.
RESUMEN
Plasminogen activator inhibitor-1 (PAI-1) levels were found to be associated with obesity, indicating that adipocytes might influence PAI-1 plasma levels. In addition, the 4G/5G promoter polymorphism of the PAI-1 gene possibly modulates PAI-1 gene transcription and, as a consequence, PAI-1 plasma levels. Metabolic parameters, diabetes complications, PAI-1 plasma levels, and PAI-1 promoter genotypes were determined and were tested for correlation in 547 Caucasian patients with type 2 diabetes. Genotyping was performed by using allele-specific PCR, and PAI-1 plasma levels were measured in 547 well-characterized subjects with type 2 diabetes. The allelic frequencies of the polymorphism (0.56 for the 4G-genotype, 0.44 for the 5G-genotype) were not different from those observed in nondiabetic controls. The PAI-1 concentration was positively associated with MI, but not with the 4G/5G polymorphism. Statistical analysis of metabolic parameters, diabetic complications, and the 4G/5G polymorphism revealed that serum fibrinogen levels were significantly higher in the 4G/4G subgroup compared with the 4G/5G and 5G/5G subgroups. The correlation between serum fibrinogen and 4G allele remained significant, even when additional variables, such as gender, age, BMI, duration of diabetes, and HbA1c, were controlled. In patients with type 2 diabetes mellitus, the PAI-1 4G/5G promoter polymorphism does not predict PAI-1 plasma levels and is not associated with common metabolic parameters besides fibrinogen levels.
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Enfermedad Coronaria/genética , Diabetes Mellitus Tipo 2/genética , Retinopatía Diabética/genética , Inhibidor 1 de Activador Plasminogénico/genética , Regiones Promotoras Genéticas/genética , Anciano , Enfermedad Coronaria/sangre , Enfermedad Coronaria/complicaciones , Diabetes Mellitus Tipo 2/sangre , Diabetes Mellitus Tipo 2/complicaciones , Retinopatía Diabética/sangre , Retinopatía Diabética/etiología , Femenino , Frecuencia de los Genes , Genotipo , Humanos , Masculino , Persona de Mediana Edad , Inhibidor 1 de Activador Plasminogénico/sangre , Polimorfismo Genético , Estadísticas no ParamétricasRESUMEN
The collagenous repeat-containing sequence of 26-kDa protein (CORS-26) was recently described as a new gene that is induced during adipocyte differentiation. Since the transcription factors specificity protein-1 (SP-1) and PPARgamma have been demonstrated to modulate transcriptional activation of adipocytic genes, we investigated the putative role of SP-1 and PPARgamma in the regulation of the murine CORS-26 promoter. Computer-based sequence analysis revealed two putative SP-1 binding sites and binding sites for PPARgamma and Pit-1 within the TATA-box containing promoter. Electrophoretic mobility shift assays (EMSA) with nuclear extracts from 3T3-L1 adipocytes and appropriate promoter fragments demonstrated that SP-1 binds specifically to both SP-1 binding sites. Specificity was demonstrated by (i) the appearance of supershift bands, (ii) competition experiments and, (iii) by using oligonucleotides carrying mutated SP-1 binding sites. Functional promoter activity was analyzed by Luciferase reporter gene assays and SP-1 was shown to exert inhibitory effects on the transcriptional activation of the murine CORS-26 gene. Additionally, specific binding activity of PPARgamma and Pit-1 to the CORS-26 promoter was demonstrated. Taken together, the present data demonstrate the functionality of the proximal murine CORS-26 promoter, which is regulated specifically by two SP-1 binding sites via SP-3-independent repressive effects of SP-1 on transcriptional activation. Pit-1 and PPARgamma can bind specifically to the promoter and might play an additive functional role in gene regulation of murine CORS-26.
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Proteínas de Unión al ADN/fisiología , Regulación de la Expresión Génica , Regiones Promotoras Genéticas , Proteínas/fisiología , Receptores Citoplasmáticos y Nucleares/fisiología , Factor de Transcripción Sp1/fisiología , Factores de Transcripción/fisiología , Células 3T3-L1 , Adipocitos/metabolismo , Adipoquinas , Animales , Sitios de Unión , Unión Competitiva , Diferenciación Celular , Núcleo Celular/metabolismo , Células Cultivadas , Genes Reporteros , Luciferasas/metabolismo , Ratones , Mutación , Células 3T3 NIH , Unión Proteica , Estructura Terciaria de Proteína , Programas Informáticos , Factor de Transcripción Pit-1 , Activación TranscripcionalRESUMEN
Plasminogen activator inhibitor-1 (PAI-1) levels were found to be associated with obesity indicating that adipocytes influence PAI-1 plasma levels. In addition, the 4 G/5 G promoter polymorphism of the PAI-1 gene may modulate PAI-1 transcription. We investigated the transcriptional regulation of the human PAI-1 gene in adipocytes and analyzed the genetic contribution of the 4 G/5 G polymorphism. The PAI-1 promoter was analyzed using electrophoretic mobility shift assays (EMSAs) and luciferase reporter gene assays. A putative binding site for the upstream stimulatory factor-1/2 (USF-1/2) at the polymorphic region of the PAI-1 promoter was identified. The binding of USF-1/2 was studied using nuclear extracts prepared from adipocytes and was similar in all the promoter variants as analyzed by EMSA. A 257 bp PAI-1 promoter fragment including the 4 G/5 G site was transcriptionally active in adipocytes and was not influenced by the polymorphism. The present data indicate for the first time that USF-1/2 is transcriptionally active in differentiated adipocytes. However, USF-1/2 binding activity and PAI-1 transcription are not influenced by the 4 G/5 G-allele. These data possibly explain the observation that PAI-1 secretion from adipose tissue is not influenced by the PAI-1 promoter polymorphism.
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Adipocitos/metabolismo , Proteínas de Unión al ADN/genética , Inhibidor 1 de Activador Plasminogénico/metabolismo , Polimorfismo Genético/genética , Regiones Promotoras Genéticas/genética , Factores de Transcripción/genética , Células 3T3-L1 , Adipocitos/citología , Animales , Sitios de Unión/genética , Diferenciación Celular/fisiología , Proteínas de Unión al ADN/metabolismo , Ensayo de Cambio de Movilidad Electroforética , Regulación de la Expresión Génica , Humanos , Ratones , Factores de Transcripción/metabolismo , Factores Estimuladores hacia 5'RESUMEN
Adipophilin, a marker of lipid accumulation initially described in adipocytes, was recently shown to be induced in macrophage foam cells. We found that even freshly isolated blood monocytes express adipophilin and that the amount of adipophilin protein is variable in monocytes from different healthy individuals. However, the physiological expression of adipophilin does not correlate with the levels of free fatty acids, cholesterylesters or free cholesterol. Enzymatically modified low-density lipoprotein (E-LDL) induces rapid foam cell formation in monocytes and upregulates adipophilin mRNA and protein within 2 h of incubation. This rapid induction of adipophilin is accompanied by a significant increase of free fatty acids in monocytes incubated with E-LDL. Adipophilin facilitates the uptake of free fatty acids, and here we demonstrate that free fatty acids increase is related to the early upregulation of adipophilin expression in blood monocytes. Fatty acids are ligands for peroxisome proliferator-activated receptor-gamma (PPARgamma), and the upregulation of adipophilin mRNA by PPARgamma agonists like 15d-PGJ(2) and ciglitazone indicates that PPARgamma may mediate the induction of adipophilin expression in human blood monocytes.
Asunto(s)
Lípidos/sangre , Monocitos/metabolismo , Péptidos/metabolismo , Adulto , Secuencia de Bases , Northern Blotting , Western Blotting , Cartilla de ADN , Citometría de Flujo , Humanos , Lipoproteínas LDL/metabolismo , Proteínas de la Membrana , Persona de Mediana Edad , Péptidos/genética , Perilipina-2 , Prostaglandina D2/análogos & derivados , Prostaglandina D2/farmacología , ARN Mensajero/genética , Receptores Citoplasmáticos y Nucleares/agonistas , Factores de Transcripción/agonistas , Regulación hacia ArribaRESUMEN
CD163 is a recently identified member of the scavenger receptor cysteine-rich superfamily, which is expressed on peripheral blood monocytes and most tissue macrophages and is thought to play an important role in the regulation of the inflammatory response of these cells. Cross-linking of CD163 on glucocorticoid-stimulated macrophages results in the secretion of several proinflammatory cytokines, but the precise mechanism of CD163 mediated signal transduction is not understood. The existence of several CD163 isoforms, which differ in the structure of their cytoplasmic domains and putative phosphorylation sites, suggests that these isoforms also differ in their signaling mechanism. Using the Yeast Two-Hybrid system and further in vitro and in vivo studies, we identified the regulatory beta-subunit of casein kinase II (CKII), which specifically binds to the cytoplasmic domain of CD163 and its isoforms. We also found, that in vitro the CD163 isoforms differ in their association with the CKII holoenzyme and in the phosphorylation by CKII. Furthermore, we demonstrated that the cytoplasmic domains of CD163 variants are phosphorylated by PKC-alpha in vitro. Inhibition studies using specific kinase inhibitors reveal that both CKII and PKC are involved in the CD163 signaling mechanism resulting in the secretion of proinflammatory cytokines.
Asunto(s)
Antígenos CD , Antígenos de Diferenciación Mielomonocítica/metabolismo , Isoenzimas/metabolismo , Proteína Quinasa C/metabolismo , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismo , Receptores de Superficie Celular/metabolismo , Transducción de Señal , 2,3-Difosfoglicerato/farmacología , Secuencia de Aminoácidos , Anticuerpos Monoclonales , Antígenos de Diferenciación Mielomonocítica/química , Antígenos de Diferenciación Mielomonocítica/genética , Quinasa de la Caseína II , Células Cultivadas , Dexametasona/farmacología , Diclororribofuranosil Benzoimidazol/farmacología , Genisteína/farmacología , Holoenzimas/química , Holoenzimas/metabolismo , Humanos , Indoles/farmacología , Interleucina-1/biosíntesis , Interleucina-6/biosíntesis , Isoenzimas/antagonistas & inhibidores , Macrófagos/efectos de los fármacos , Macrófagos/enzimología , Macrófagos/metabolismo , Maleimidas/farmacología , Datos de Secuencia Molecular , Fosforilación/efectos de los fármacos , Unión Proteica , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C-alfa , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , Estructura Terciaria de Proteína , Subunidades de Proteína , Receptores de Superficie Celular/química , Receptores de Superficie Celular/genética , Transducción de Señal/efectos de los fármacos , Especificidad por Sustrato , Técnicas del Sistema de Dos HíbridosRESUMEN
Elevated plasma lipoprotein (a) (Lp[a]) and cardiac events show a modest but significant association in various clinical studies. However, the influence of high Lp(a) on the gene expression in blood monocytes as a major cell involved in atherogenesis is poorly described. To identify genes influenced by elevated serum Lp(a), the gene expression was analyzed on a complementary DNA microarray comparing monocytes from a patient with isolated Lp(a) hyperlipidemia and coronary heart disease with monocytes from a healthy blood donor with low Lp(a). By using this approach, numerous genes were found differentially expressed in patient-versus-control monocytes. Verification of these candidates by Northern blot analysis or semiquantitative polymerase chain reaction in monocytes from additional patients with Lp(a) hyperlipidemia and healthy blood donors with elevated Lp(a) confirmed a significant induction of plasminogen activator inhibitor type 2 (PAI-2) messenger RNA (mRNA) in monocytes from male, but not from female, individuals with high Lp(a), indicating that this observation is gender specific. This led also to increased intracellular and secreted PAI-2 protein in monocytes from male probands with Lp(a) hyperlipidemia. Plasminogen activator inhibitor type 1 (PAI-1) mRNA was found suppressed only in the patients' monocytes and not in healthy probands with high Lp(a) levels. Purified Lp(a) induced PAI-2 mRNA and protein and reduced PAI-1 expression in monocytes isolated from various controls. The finding that PAI-2 is elevated in monocytes from male patients with isolated Lp(a) hyperlipidemia and male healthy probands with high Lp(a) and that purified Lp(a) up-regulates PAI-2 in control monocytes in vitro indicate a direct, but gender-specific, effect of Lp(a) for the induction of PAI-2 expression.
Asunto(s)
Hiperlipoproteinemias/sangre , Lipoproteína(a)/fisiología , Monocitos/metabolismo , Inhibidor 2 de Activador Plasminogénico/biosíntesis , Adulto , Anciano , Northern Blotting , Células Cultivadas , Enfermedad Coronaria/sangre , Femenino , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Variación Genética , Humanos , Hiperlipoproteinemias/genética , Inflamación , Lipoproteína(a)/farmacología , Masculino , Persona de Mediana Edad , Infarto del Miocardio/sangre , Análisis de Secuencia por Matrices de Oligonucleótidos , Inhibidor 1 de Activador Plasminogénico/biosíntesis , Inhibidor 1 de Activador Plasminogénico/genética , Inhibidor 2 de Activador Plasminogénico/genética , Reacción en Cadena de la Polimerasa , ARN Mensajero/biosíntesis , Receptores de Superficie Celular/biosíntesis , Receptores de Superficie Celular/genética , Receptores del Activador de Plasminógeno Tipo Uroquinasa , Caracteres SexualesRESUMEN
CD163, also referred to as M130, a member of the scavenger receptor cysteine-rich family (SRCR) is exclusively expressed on cells of the monocyte lineage. In freshly isolated monocytes the CD14bright CD16+ monocyte subset revealed the highest expression of CD163 among all monocyte subsets. CD163 mRNA and protein expression is up-regulated during macrophage colony-stimulating factor (M-CSF)-dependent phagocytic differentiation of human blood monocytes. In contrast, monocytic cells treated with GM-CSF and interleukin-4 (IL-4) for dendritic differentiation down-regulate this antigen. CD163 expression is also suppressed by proinflammatory mediators like lipopolysaccharide (LPS), interferon-gamma (IFN-gamma), and tumor necrosis factor alpha, whereas IL-6 and the antiinflammatory cytokine interleukin-10 (IL-10) strongly up-regulate CD163 mRNA in monocytes and macrophages. The effects of the immunosuppressants dexamethasone, cyclosporin A (CA), and cortisol differ in their capacity to influence CD163 mRNA levels. Our results demonstrate that CD163 expression in monocytes/macrophages is regulated by proinflammatory and antiinflammatory mediators. This expression pattern implies a functional role of CD 163 in the antiinflammatory response of monocytes.
Asunto(s)
Antígenos CD , Antígenos de Diferenciación Mielomonocítica/metabolismo , Inflamación/metabolismo , Macrófagos/metabolismo , Proteínas de la Membrana , Monocitos/metabolismo , Receptores de Superficie Celular , Receptores de Lipoproteína , Células Cultivadas , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Humanos , Inflamación/patología , Interferón gamma/farmacología , Interleucinas/farmacología , Lipopolisacáridos/farmacología , Factor Estimulante de Colonias de Macrófagos/farmacología , Macrófagos/patología , Monocitos/patología , Fagocitosis , Receptores Inmunológicos/metabolismo , Receptores Depuradores , Receptores Depuradores de Clase B , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Human Cla-1 is the likely homologue of the murine scavenger receptor class B type I (SR-BI). SR-BI mediates selective transfer of cholesterol to high-density lipoprotein (HDL) and the efflux of endogenously synthesized and plasma membrane sterols to HDL. HDL protects against atherosclerosis but also reduces endotoxic activity by complexation and neutralization of LPS. We found that Cla-1 is upregulated during phagocytic as well as dendritic differentiation of monocytes, indicating a function of this receptor for cholesterol homeostasis in phagocytes and antigen-presenting cells. Cla-1 expression is suppressed by the proinflammatory stimuli lipopolysaccharide, interferon-gamma, and tumor necrosis factor alpha in monocytes and macrophages. Downregulation of Cla-1 mRNA by LPS is likely due to a modification and subsequent destabilization of the mRNA. We propose that suppression of Cla-1 expression may help to stabilize the lipoprotein status in the blood compartment important for host defense.