Asunto(s)

Edema Encefálico/etiología , Paro Cardíaco Extrahospitalario/complicaciones , Fosfopiruvato Hidratasa/sangre , Apoyo Vital Cardíaco Avanzado , Biomarcadores/sangre , Edema Encefálico/enzimología , Coma/sangre , Femenino , Humanos , Persona de Mediana Edad , Paro Cardíaco Extrahospitalario/terapia

RESUMEN

PURPOSE: Endothelial pathology is considered to play a key role in septic shock. Since endothelial-derived microvesicles (MV) are elevated in various diseases associated with endothelial pathology, they are considered surrogate markers of the endothelial state. By analyzing the signature of circulating MV with high-sensitivity flow cytometry (hsFC), we wanted to test the hypothesis whether endothelial-derived MV are increased in septic shock. METHODS: MV in blood from healthy volunteers and patients with septic shock treated in a medical intensive care unit were quantified by hsFC, which has an improved detection limit of approximately 0.3 µm. RESULTS: Patients with septic shock (n = 30) showed 3-fold higher levels of CD31+/CD41- MV (58.5 (26.4-101.2) [median (25th-75th percentile)] vs. 19.5 (12.8-25.4) MV/µL; P <0.001) compared with healthy volunteers (n = 18). Absolute counts of CD144+, CD62E+, and CD106+ MV, specific for endothelial-derived MV, were low in all groups. The number of CD31+/CD41- MV correlated significantly with leukocyte count (rs = 0.64; P <0.001). Platelet-derived CD41+ MV were significantly elevated in the group dying within 48 h after inclusion (639.1 (321.3-969.7) vs. 221.5 (119.5-456.9) MV/µL; P = 0.037). Patients dying within 48 h had also significantly higher levels of CD31+/CD41-/AnnexinV- MV (51.9 (24.9-259.8) vs. 18.9 (9.7-31) MV/µL; P = 0.028). CONCLUSIONS: Despite an improved detection limit for MV by using hsFC, counts of endothelial-specific MV are unexpectedly low in patients with septic shock. Increased amounts of CD41+ and CD31+/CD41-/AnnexinV- MV indicate release by activated platelets and possibly leukocytes correlating with unfavorable outcome.

Asunto(s)

Micropartículas Derivadas de Células/metabolismo , Citometría de Flujo/métodos , Choque Séptico/metabolismo , Adulto , Anciano , Anexina A5/metabolismo , Antígenos CD/metabolismo , Cadherinas/metabolismo , Selectina E/metabolismo , Células Endoteliales/metabolismo , Femenino , Humanos , Interleucina-10/sangre , Interleucina-6/sangre , Recuento de Leucocitos , Masculino , Persona de Mediana Edad , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Glicoproteína IIb de Membrana Plaquetaria/metabolismo , Choque Séptico/sangre , Molécula 1 de Adhesión Celular Vascular/metabolismo , Adulto Joven

RESUMEN

PURPOSE: The neuropeptide secretoneurin (SN) shows widespread distribution in the brain. We evaluated whether SN is elevated after cardiopulmonary resuscitation (CPR) and could serve as a potential new biomarker for hypoxic brain injury after CPR. METHODS: This was a prospective observational clinical study. All patients admitted to a tertiary medical intensive care unit after successful CPR with expected survival of at least 24 h were consecutively enrolled from September 2008 to April 2013. Serum SN and neuron-specific enolase were determined in 24 h intervals starting with the day of CPR for 7 days. Neurological outcome was assessed with the Cerebral Performance Categories Scale (CPC) at hospital discharge. RESULTS: A total of 134 patients were included with 49 % surviving to good neurological outcome (CPC 1-2). SN serum levels peaked within the first 24 h showing on average a sixfold increase above normal. SN levels were significantly higher in patients with poor (CPC 3-5) than in patients with good neurological outcome [0-24 h: 75 (43-111) vs. 38 (23-68) fmol/ml, p < 0.001; 24-48 h: 45 (24-77) vs. 23 (16-39) fmol/ml, p < 0.001]. SN determined within the first 48 h showed a receiver operating characteristic (ROC) area under the curve (AUC) of 0.753 (0.665-0.841). NSE in the first 72 h had a ROC-AUC of 0.881 (0.815-0.946). When combining the two biomarkers an AUC of 0.925 (0.878-0.972) for outcome prediction could be reached. CONCLUSIONS: SN is a promising early biomarker for hypoxic brain injury. Further studies will be required for confirmation of these results.

Asunto(s)

Reanimación Cardiopulmonar/estadística & datos numéricos , Paro Cardíaco/sangre , Hipoxia Encefálica/diagnóstico , Neuropéptidos/sangre , Fosfopiruvato Hidratasa/sangre , Secretogranina II/sangre , APACHE , Anciano , Área Bajo la Curva , Biomarcadores/sangre , Reanimación Cardiopulmonar/efectos adversos , Técnicas de Diagnóstico Neurológico , Femenino , Paro Cardíaco/complicaciones , Paro Cardíaco/terapia , Humanos , Hipoxia Encefálica/sangre , Hipoxia Encefálica/etiología , Unidades de Cuidados Intensivos/estadística & datos numéricos , Masculino , Persona de Mediana Edad , Análisis Multivariante , Puntuaciones en la Disfunción de Órganos , Paro Cardíaco Extrahospitalario/sangre , Paro Cardíaco Extrahospitalario/complicaciones , Paro Cardíaco Extrahospitalario/terapia , Valor Predictivo de las Pruebas , Estudios Prospectivos , Curva ROC , Centros de Atención Terciaria/estadística & datos numéricos , Tiempo de Tratamiento

RESUMEN

The pharmacokinetics of lipid-bound and liberated amphotericin B (AMB) was assessed in 11 critically ill patients with cholestatic liver disease (CSLD) and in 9 subjects with normal liver function treated with AMB colloidal dispersion (ABCD). Exposure to lipid-bound AMB was higher in patients with CSLD. Levels of liberated AMB were elevated by CSLD only after the first dose, whereas its pharmacokinetics was unaffected at steady state. The standard dosage of ABCD is probably adequate for patients with CSLD.

Asunto(s)

Anfotericina B/farmacocinética , Antifúngicos/farmacocinética , Enfermedad Crítica , Hepatopatías/sangre , Adolescente , Adulto , Anciano , Anfotericina B/sangre , Antifúngicos/sangre , Niño , Coloides , Femenino , Humanos , Hepatopatías/tratamiento farmacológico , Masculino , Persona de Mediana Edad , Adulto Joven

RESUMEN

BACKGROUND: There is growing evidence that TLR2 plays a role in the pathogenesis of atherosclerosis. It is highly expressed in endothelial cells in areas of disturbed blood flow, like plaques or vessel bifurcations, but laminar blood flow suppresses endothelial TLR2 expression and is therefore thought to be atheroprotective. We sought for means to also protect lesion prone sites from TLR2 over-expression and subsequent endothelial activation. METHODS: Human coronary artery endothelial cells (HCAEC) were treated with atorvastatin (ATV) and TLR2 surface expression was determined by FACS analyses. Western blot analyses were used to explore the phosphorylation status of SP1. RESULTS: ATV profoundly inhibited basal and stimulated endothelial TLR2 expression in a time- and dose-dependent manner. It also inhibited HCAEC activation by MALP-2. TLR2 surface expression was inversely correlated to SP1 serine phosphorylation and was casein kinase 2 dependent. CONCLUSION: We demonstrate that ATV can control over-expression of proinflammatory endothelial TLR2 protein and TLR2-mediated endothelial activation. The mechanism involves casein kinase 2 and SP1 phosphorylation. ATV effects on endothelial cell TLR2 are comparable to those of laminar blood flow and might therefore also be atheroprotective.

Asunto(s)

Anticolesterolemiantes/farmacología , Células Endoteliales/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Ácidos Heptanoicos/farmacología , Pirroles/farmacología , Receptor Toll-Like 2/antagonistas & inhibidores , Receptor Toll-Like 2/genética , Atorvastatina , Quinasa de la Caseína II/metabolismo , Línea Celular , Atrios Cardíacos/citología , Humanos , Lipopéptidos/metabolismo , Fosforilación , Factor de Transcripción Sp1/metabolismo , Receptor Toll-Like 2/metabolismo

RESUMEN

INTRODUCTION: Levosimendan is an extensively investigated inodilator showing also cardioprotective and antiinflammatory effects. The aim of our study was to explore the influence of levosimendan on polymorphonuclear leucocytes (PMN), a main source of reactive oxygen species, in vitro and in patients with acute heart failure or septic myocardial depression. METHODS: PMN isolated from healthy volunteers were incubated with levosimendan in vitro. After stimulation with N-formyl-Met-Leu-Phe (fMLP) or phorbol 12-myristate 13-acetate (PMA) respiratory burst was quantified using a fluorescent dye. Apoptosis and expression of cell adhesion molecules of PMN were measured by flow cytometry. For determination of in vivo effects patients with acute heart failure (n = 16) or septic cardiac failure (n = 9) receiving levosimendan treatment were enrolled consecutively. PMN were isolated to measure respiratory burst activity before treatment as well as one and two hours after initiation of levosimendan administration. Furthermore inflammatory, hemodynamic and renal function parameters were obtained. RESULTS: In vitro, levosimendan suppressed respiratory burst activity in fMLP or PMA stimulated PMN in a dose dependent manner by 30 ± 11% (P < 0.001) at 100 ng/mL and by 27 ± 17% (P < 0.001) at 1000 ng/mL respectively. Markers of apoptosis and PMN cell adhesion molecule expression remained unaffected by levosimendan treatment.In vivo, levosimendan treatment for two hours resulted in a significant reduction of PMA stimulated oxidative burst by 45% (P < 0.01) and fMLP stimulated oxidative burst by 49% (P < 0.05) in patients with acute heart failure. In patients suffering from septic shock levosimendan treatment decreased oxidative burst activity in unstimulated, fMLP and PMA stimulated PMN by 48% (P < 0.05), 46% (P < 0.01) and 43% (P < 0.01) respectively. CONCLUSIONS: Levosimendan appears to exert distinct immunomodulatory effects by decreasing oxidative burst activity of PMN. This property might contribute to the previously described cardioprotective effects of the drug.

Asunto(s)

Cardiotónicos/farmacología , Insuficiencia Cardíaca/fisiopatología , Hidrazonas/farmacología , Neutrófilos/efectos de los fármacos , Piridazinas/farmacología , Especies Reactivas de Oxígeno/antagonistas & inhibidores , Choque Séptico/fisiopatología , Enfermedad Aguda , Anciano , Anciano de 80 o más Años , Austria , Femenino , Citometría de Flujo , Humanos , Técnicas In Vitro , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Simendán

RESUMEN

Albumin dialysis (AD) is a therapeutic option in severe cholestatic liver failure. However, it can significantly enhance drug elimination. Pharmacokinetic data on antimicrobial agents--in particular on antimycotics--administered under this clinical condition are very sparse. Therefore, amphotericin B (AMB) plasma concentrations were measured in two critically ill patients who were treated with AD because of severe cholestatic liver failure and were prescribed lipid formulated AMB--either AMB colloidal dispersion (ABCD) or AMB lipid complex (ABLC)--for suspected invasive fungal infection. AD was performed with the molecular adsorbent recirculating system (MARS). Lipid-associated and liberated AMB were separately quantified on and off AD. The clearance of the liberated AMB fraction was not essentially affected (ABLC) or moderately enhanced during AD by a factor of 2.5 (ABCD). The clearance of the lipid-formulated fraction was increased by a factor of 4 during AD (ABCD) or was similar (ABLC) on and off AD. Despite the fact that there was a four-fold higher clearance of the lipid-formulated fraction of ABCD, the clinically relevant area under the concentration time curve of the liberated AMB fraction was only moderately changed (by 37% in ABCD, 70% in ABLC) during AD. Thus, the effect of AD on lipid formulated AMB appears to be moderate. A daily dose of 5 mg/kg will probably lead to adequate plasma levels in patients on AD.

Asunto(s)

Albúminas/uso terapéutico , Anfotericina B/sangre , Anfotericina B/metabolismo , Antifúngicos/sangre , Antifúngicos/metabolismo , Fallo Hepático/terapia , Diálisis Renal , Anciano , Anfotericina B/uso terapéutico , Antifúngicos/uso terapéutico , Enfermedad Crítica , Femenino , Humanos , Metabolismo de los Lípidos , Masculino , Persona de Mediana Edad , Micosis/prevención & control

RESUMEN

Voriconazole concentrations were determined in autopsy samples of eight patients who had been treated for a median of 7 days (interquartile range [IQR], 5 days). Voriconazole penetrates well into various tissues, with median levels of 6.26 µg/g ((interquartile range [IQR], 7.87 µg/g) in the lung, 3.41 µg/g (IQR, 16.72 µg/g) in the brain, 6.89 µg/g (IQR, 24.16 µg/g) in the liver, 6.47 µg/g (IQR, 6.19 µg/g) in the kidneys, 5.60 µg/g (IQR, 11.49 µg/g) in the spleen, and 7.55 µg/g (IQR, 16.91 µg/g) in the myocardium.

Asunto(s)

Riñón/metabolismo , Hígado/metabolismo , Pulmón/metabolismo , Pirimidinas/farmacocinética , Bazo/metabolismo , Triazoles/farmacocinética , Adulto , Anciano , Aspergilosis/tratamiento farmacológico , Aspergilosis/microbiología , Autopsia , Encéfalo/metabolismo , Femenino , Humanos , Masculino , Persona de Mediana Edad , Miocardio/metabolismo , Pirimidinas/administración & dosificación , Distribución Tisular , Triazoles/administración & dosificación , Voriconazol , Adulto Joven

RESUMEN

Nitric oxide (NO) plays a critical role in the regulation of renal hemodynamics and tubular function after post-ischemic damage or sepsis. Diminished NO bioavailability contributes to endothelial dysfunction and may be caused by reduced NO synthesis due to substrate or co-factor deficiency. The aim of this study was to investigate the effects of NOS inhibition and NO depletion in a renal endo-epithelial bilayer model compared to monolayers of proximal tubular epithelial (HK-2) cells and endothelial cells of venous origin (EA.hy 926) with respect to cellular integrity, apoptosis and cytokine release. Two different NOS inhibitors have been used: an arginine-based-inhibitor, L-N(G)monomethyl-arginine (L-NMMA) and a cofactor-based-inhibitor, H4-amino-biopterin (4-ABH(4)) showing iNOS selectivity. We found significantly higher basal NO production by epithelial than by endothelial monolayers, which was significantly reduced by both NOS-inhibitors with a stronger effect demonstrated by 4-ABH(4). Furthermore we detected significant basal iNOS protein expression in unstimulated HK-2 cells. NOS inhibition by 4-ABH(4) was associated with increased LDH release, apoptosis and reduced IL-6 production in epithelial but not in endothelial monolayers. These effects on epithelial cells were abolished under co-culture conditions. In contrast, endothelial cells showed higher IL-6 and IL-8 release under co-culture conditions than in monolayers, with IL-8 production being largely suppressed by L-NMMA but not by 4-ABH(4). In conclusion, inhibition of basal NO production in epithelial monolayers shows detrimental effects on cell integrity and viability. Under co-culture conditions interrelation between epithelial and endothelial cells appears to counteract these potentially harmful effects of epithelial NOS inhibition.

Asunto(s)

Células Endoteliales/metabolismo , Células Epiteliales/metabolismo , Riñón/metabolismo , Óxido Nítrico Sintasa de Tipo II/antagonistas & inhibidores , Óxido Nítrico/metabolismo , Apoptosis , Biopterinas/farmacología , Técnicas de Cocultivo , Citocinas/metabolismo , Células Endoteliales/enzimología , Células Epiteliales/enzimología , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Riñón/citología , Riñón/enzimología , Óxido Nítrico Sintasa de Tipo II/metabolismo , omega-N-Metilarginina/farmacología

RESUMEN

INTRODUCTION: It has been hypothesized that hyperoncotic colloids might contribute to acute kidney injury (AKI). However, the validity of this hypothesis remains unclear. METHODS: A meta-analysis was conducted of randomized controlled trials evaluating AKI after infusion of hyperoncotic albumin and hydroxyethyl starch (HES) solutions. Mortality was a secondary endpoint. Eligible trials were sought by multiple methods, and the pooled odds ratios (OR) for AKI and death and 95% confidence intervals (CI) were computed under a random effects model. RESULTS: Eleven randomized trials with a total of 1220 patients were included: 7 evaluating hyperoncotic albumin and 4 hyperoncotic HES. Clinical indications were ascites, surgery, sepsis and spontaneous bacterial peritonitis. Hyperoncotic albumin decreased the odds of AKI by 76% (OR, 0.24; CI, 0.12-0.48; P < 0.0001), while hyperoncotic HES increased those odds by 92% (OR, 1.92; CI, 1.31-2.81; P = 0.0008). Parallel effects on mortality were observed, with hyperoncotic albumin reducing the odds of death by 48% (OR, 0.52; CI, 0.28-0.95; P = 0.035) and hyperoncotic HES raising those odds by 41% (OR, 1.41; CI, 1.01-1.96; P = 0.043). CONCLUSIONS: This meta-analysis does not support the hypothesis that hyperoncotic colloid solutions per se injure the kidney. Renal effects appear instead to be colloid-specific, with albumin displaying renoprotection and HES showing nephrotoxicity.

Asunto(s)

Lesión Renal Aguda/inducido químicamente , Albúminas/efectos adversos , Coloides/efectos adversos , Derivados de Hidroxietil Almidón/efectos adversos , Ensayos Clínicos Controlados Aleatorios como Asunto/métodos , Lesión Renal Aguda/diagnóstico , Lesión Renal Aguda/epidemiología , Albúminas/administración & dosificación , Coloides/administración & dosificación , Humanos , Derivados de Hidroxietil Almidón/administración & dosificación

RESUMEN

Amphotericin B (AMB) concentrations were determined in pulmonary epithelial lining fluid (ELF) of 44 critically ill patients, who were receiving treatment with liposomal AMB (LAMB) (n = 11), AMB colloidal dispersion (ABCD) (n = 28), or AMB lipid complex (ABLC) (n = 5). Mean AMB levels (+/- standard errors of the means) in ELF amounted to 1.60 +/- 0.58, 0.38 +/- 0.07, and 1.29 +/- 0.71 microg/ml in LAMB-, ABCD-, and ABLC-treated patients, respectively (differences are not significant).

Asunto(s)

Anfotericina B/farmacocinética , Antifúngicos/farmacocinética , Pulmón/metabolismo , Adulto , Anfotericina B/administración & dosificación , Epitelio/metabolismo , Femenino , Humanos , Masculino , Persona de Mediana Edad

Asunto(s)

Anfotericina B/farmacocinética , Líquido Ascítico/química , Adulto , Anciano , Enfermedad Crítica , Humanos , Masculino , Persona de Mediana Edad

RESUMEN

CD14 is a well-known pattern-recognition receptor in the innate immune system. Here, we show that CD14 enhances double-stranded RNA (dsRNA)-mediated Toll-like receptor 3 (TLR3) activation. Bone marrow-derived macrophages (BMDMs) from CD14-/- mice exhibited impaired responses to polyinosine-polycytidylic acid (pIpC) and reduced production of inflammatory cytokines. CD14-/- mice injected with pIpC also showed impaired cytokine production. When tested with [32P] labeled pIpC small fragments (pIpCsf) that maintain the inflammatory activity of crude pIpC, CD14 directly bound pIpCsf and mediated cellular uptake of pIpCsf. Our data show that TLR3 is intracellular and directly interacts with CD14. Internalized pIpCsf was localized in the lysosomes via the endosomes. In unstimulated cells, neither CD14 nor TLR3 was detected in the lysosomes. However, TLR3 was localized in the lysosomes as was CD14 once the cells took up pIpC. We also observed that internalized pIpCsf colocalized with CD14 and TLR3. Consequently, CD14 mediates pIpC uptake and enhances TLR3 signaling.

Asunto(s)

Receptores de Lipopolisacáridos/fisiología , Macrófagos/inmunología , ARN Bicatenario/metabolismo , Receptor Toll-Like 3/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Células CHO , Células Cultivadas , Cricetinae , Cricetulus , Vesículas Citoplasmáticas/metabolismo , Humanos , Receptores de Lipopolisacáridos/genética , Ratones , Ratones Noqueados , FN-kappa B/metabolismo , Fosfatidilcolinas/farmacología , Transfección

RESUMEN

Circulating monocytic cells may mediate neuroinvasion in transmissible spongiform encephalopathies by transporting prion protein (PrP) from sites of entry to the nervous system. Factors regulating monocyte-derived dendritic cell (DC) functions in neuronal tissue include neurogenic mediators, but their interactions with prion-infected DCs are unknown. Here, we report that neuropeptides regulate the interaction of DCs exposed to PrP(106-126). Inhibition of neurokinin-1-receptors activation with neurokinin-1-receptor antagonist or antibody attenuates substance P-induced enhancement of DC migration induced by PrP(106-126) and prion-protein-induced DC maturation. Other neuropeptides arrest chemotaxis. Data support a priming role of the neuropeptide substance P in PrP(106-126)-induced migration of DCs involving neurokinin-1-receptors.

Asunto(s)

Movimiento Celular/efectos de los fármacos , Senescencia Celular/efectos de los fármacos , Quimiotaxis/efectos de los fármacos , Células Dendríticas/efectos de los fármacos , Fragmentos de Péptidos/farmacología , Priones/farmacología , Receptores de Neuroquinina-1/metabolismo , Anticuerpos/farmacología , Antígeno B7-1/metabolismo , Células Cultivadas , Senescencia Celular/fisiología , Quimiotaxis/fisiología , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Citometría de Flujo/métodos , Antígenos HLA-DR/metabolismo , Humanos , Antagonistas del Receptor de Neuroquinina-1 , Neuropéptidos/farmacología , Receptores de Neuroquinina-1/inmunología , Estadísticas no Paramétricas

RESUMEN

Innate immune system activation is associated with atherosclerotic lesion development. The specific sites of lesion development are believed to be defined by the shear stress of blood flow. Consequently, we investigated the responsiveness of human coronary artery endothelial cells (HCAECs) to Toll-like receptor (TLR) 2 and 4 agonists in an in vitro model of chronic laminar flow. HCAECs under chronic laminar flow were found to be normally responsive to lipopolysaccharide (and tumor necrosis factor) in terms of E-selectin expression but were found to be hyporesponsive to stimulation with the specific TLR2 ligands macrophage activating lipopeptide-2, PAM2-Cys, and Lip19; this was observed to be attributable to downregulation of TLR2 transcription and protein expression. We found that laminar flow induced SP1 serine phosphorylation by protein kinase CK2 and thereby blocked SP1 binding to the TLR2 promoter, which is required for TLR2 expression. This regulatory mechanism also blocked lipopolysaccharide- and tumor necrosis factor-induced TLR2 upregulation in HCAECs and could be important for suppression of other flow-sensitive endothelial proteins. These results extend the role of flow in controlling endothelial responsiveness. Given the current evidence that TLRs are proatherogenic, flow suppression of TLR2 expression may be atheroprotective.

Asunto(s)

Arteriosclerosis/fisiopatología , Vasos Coronarios/metabolismo , Endotelio Vascular/citología , Regulación de la Expresión Génica/fisiología , Hemorreología , Glicoproteínas de Membrana/biosíntesis , Receptores de Superficie Celular/biosíntesis , Factor de Transcripción Sp1/antagonistas & inhibidores , Proteínas Portadoras/farmacología , Quinasa de la Caseína II/antagonistas & inhibidores , Quinasa de la Caseína II/fisiología , Células Cultivadas/metabolismo , Vasos Coronarios/citología , Vasos Coronarios/efectos de los fármacos , Proteínas de Unión al ADN/metabolismo , Células Endoteliales/metabolismo , Endotelio Vascular/metabolismo , Humanos , Lipopéptidos , Lipopolisacáridos/farmacología , Glicoproteínas de Membrana/genética , Oligopéptidos/farmacología , Fosforilación , Procesamiento Proteico-Postraduccional , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Receptores de Superficie Celular/genética , Proteínas Recombinantes/farmacología , Factor de Transcripción Sp1/fisiología , Factor de Transcripción Sp3 , Estrés Mecánico , Receptor Toll-Like 2 , Receptores Toll-Like , Factores de Transcripción/metabolismo , Triazoles/farmacología , Factor de Necrosis Tumoral alfa/farmacología

RESUMEN

TLR4 is the primary recognition molecule for inflammatory responses initiated by bacterial LPS (endotoxin). Internalization of endotoxin by various cell types is an important step for its removal and detoxification. Because of its role as an LPS-signaling receptor, TLR4 has been suggested to be involved in cellular LPS uptake as well. LPS uptake was investigated in primary monocytes and endothelial cells derived from TLR4 and CD14 knockout C57BL/6 mice using tritiated and fluorescein-labeled LPS. Intracellular LPS distribution was investigated by deconvolution confocal microscopy. We could not observe any difference in LPS uptake and intracellular LPS distribution in either monocytes or endothelial cells between TLR4(-/-) and wild-type cells. As expected, CD14(-/-) monocytes showed a highly impaired LPS uptake, confirming CD14-dependent uptake in monocytes. Upon longer incubation periods, the CD14-deficient monocytes mimicked the LPS uptake pattern of endothelial cells. Endothelial cell LPS uptake is slower than monocyte uptake, LBP rather than CD14 dependent, and sensitive to polyanionic polymers, which have been shown to block scavenger receptor-dependent uptake mechanisms. We conclude that TLR4 is not involved in cellular LPS uptake mechanisms. In membrane CD14-positive cells, LPS is predominantly taken up via CD14-mediated pathways, whereas in the CD14-negative endothelial cells, there is a role for scavenger receptor-dependent pathways.

Asunto(s)

Lipopolisacáridos/metabolismo , Glicoproteínas de Membrana/metabolismo , Receptores de Superficie Celular/metabolismo , Transducción de Señal/fisiología , Animales , Células Endoteliales/metabolismo , Receptores de Lipopolisacáridos/metabolismo , Glicoproteínas de Membrana/genética , Ratones , Ratones Noqueados , Monocitos/metabolismo , Receptores de Superficie Celular/genética , Receptores Inmunológicos/metabolismo , Receptores Depuradores , Receptor Toll-Like 4 , Receptores Toll-Like

RESUMEN

Endothelial cells are activated by microbial agonists through Toll-like receptors (TLRs) to express inflammatory mediators; this is of significance in acute as well as chronic inflammatory states such as septic shock and atherosclerosis, respectively. We investigated mechanisms of lipopolysaccharide (LPS)-induced cell activation in human coronary artery endothelial cells (HCAEC) using a combination of FACS, confocal microscopy, RT-PCR, and functional assays. We found that TLR4, in contrast to TLR2, is not only located intracellularly but also functions intracellularly. That being the case, internalization of LPS is required for activation. We further characterized the HCAEC LPS uptake system and found that HCAEC exhibit an effective LPS uptake only in the presence of LPS binding protein (LBP). In addition to its function as a catalyst for LPS-CD14 complex formation, LBP enables HCAEC activation at low LPS concentrations by facilitating the uptake, and therefore delivery, of LPS-CD14 complexes to intracellular TLR4-MD-2. LBP-dependent uptake involves a scavenger receptor pathway. Our findings may be of pathophysiological relevance in the initial response of the organism to infection. Results further suggest that LBP levels, which vary as LBP is an acute phase reactant, could be relevant to initiating inflammatory responses in the vasculature in response to chronic or recurring low LPS.

Asunto(s)

Vasos Coronarios/citología , Células Endoteliales/efectos de los fármacos , Endotelio Vascular/citología , Líquido Intracelular/metabolismo , Lípido A/análogos & derivados , Lipopolisacáridos/farmacología , Glicoproteínas de Membrana/fisiología , Receptores de Superficie Celular/fisiología , Proteínas de Fase Aguda/fisiología , Reacción de Fase Aguda , Adulto , Animales , Antígenos de Neoplasias/biosíntesis , Antígenos de Neoplasias/genética , Antígenos de Superficie/biosíntesis , Antígenos de Superficie/genética , Proteínas Portadoras/fisiología , Compartimento Celular , Células Cultivadas/efectos de los fármacos , Células Cultivadas/metabolismo , Células Endoteliales/metabolismo , Células Endoteliales/ultraestructura , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Citometría de Flujo , Glucolípidos/farmacología , Humanos , Lípido A/farmacología , Antígeno 96 de los Linfocitos , Sustancias Macromoleculares , Glicoproteínas de Membrana/análisis , Glicoproteínas de Membrana/biosíntesis , Glicoproteínas de Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Monocitos/metabolismo , Monocitos/ultraestructura , ARN Mensajero/biosíntesis , Receptores de Superficie Celular/análisis , Receptores de Superficie Celular/biosíntesis , Receptores de Superficie Celular/genética , Receptores Inmunológicos/fisiología , Receptores Depuradores , Receptor Toll-Like 2 , Receptor Toll-Like 4 , Receptores Toll-Like , Venas Umbilicales/citología

RESUMEN

Inflammatory bowel disease (IBD) constituting Crohn's disease (CD) and ulcerative colitis (UC) is related to a dysregulated T cell response. CCL20 attracts memory T lymphocytes and dendritic cells. We asked whether CCL20 expression is altered in IBD. Colonic biopsies were obtained from 114 subjects with IBD, non-IBD colitis, irritable bowel syndrome, and healthy controls. CCL20 and CCR6 mRNA expression was measured by Taqman-PCR, and protein secretion from colonic explant cultures (CEC) and its regulation by TNF-alpha by ELISA. CCL20, CCR6, and Langerin were identified by immunohistochemistry and immunofluorescence. CCL20 mRNA and protein were severalfold increased in involved CD and UC but not in non-IBD colitis. TNF-alpha increased and anti-TNF-alpha decreased CCL20 release in healthy control CEC but not in involved IBD colonic specimens. CCL20 localized to follicle-associated epithelium, and CCR6 to the adjacent mantle zone of lymphoid follicles. Furthermore, abundant numbers of Langerin(+) immature dendritic cells were identified in the subepithelial space of IBD specimens. CCL20 might regulate the attraction of T lymphocytes and dendritic cells in IBD.

Asunto(s)

Quimiocinas CC/inmunología , Colitis Ulcerosa/inmunología , Enfermedad de Crohn/inmunología , Proteínas Inflamatorias de Macrófagos/inmunología , Adulto , Anticuerpos/inmunología , Anticuerpos/metabolismo , Antígenos CD , Antígenos de Superficie/inmunología , Antígenos de Superficie/metabolismo , Biopsia , Quimiocina CCL20 , Quimiocinas CC/genética , Quimiocinas CC/metabolismo , Colitis Ulcerosa/genética , Colitis Ulcerosa/metabolismo , Colitis Ulcerosa/patología , Enfermedad de Crohn/genética , Enfermedad de Crohn/metabolismo , Enfermedad de Crohn/patología , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Epitelio/inmunología , Epitelio/metabolismo , Epitelio/patología , Femenino , Humanos , Lectinas Tipo C/inmunología , Lectinas Tipo C/metabolismo , Proteínas Inflamatorias de Macrófagos/genética , Proteínas Inflamatorias de Macrófagos/metabolismo , Masculino , Lectinas de Unión a Manosa/inmunología , Lectinas de Unión a Manosa/metabolismo , Persona de Mediana Edad , ARN Mensajero/metabolismo , Receptores CCR6 , Receptores de Quimiocina/inmunología , Receptores de Quimiocina/metabolismo , Factor de Necrosis Tumoral alfa/inmunología , Factor de Necrosis Tumoral alfa/metabolismo

RESUMEN

Opioid receptors are expressed in cells of the immune system, and potent immunomodulatory effects of their natural and synthetic ligands have been reported. In some studies, the opiate receptor antagonist naloxone itself displayed immunomodulatory actions. We investigated effects of naloxone on leukocyte chemotaxis. Cell migration was tested in micropore filter assays using modified Boyden chambers, and receptor expression was investigated using radiolabel binding assays. Naloxone induced peripheral blood nonadherent mononuclear cell and neutrophil chemotaxis at nanomolar concentrations and deactivated their migration toward beta-endorphin, angiotensin II, somatostatin, or interleukin-8 but not toward RANTES, vasoactive intestinal peptide, or substance P. Ligand binding studies showed no alteration in the binding of interleukin-8 to neutrophils by naloxone. Cleavage of heparan sulfate from proteoglycans on the cells' surface completely inhibited chemotactic and deactivating properties of naloxone but not other attractants. Chemotactic properties were abolished by pretreating cells with heparinase, chondroitinase, sodium chlorate, and anti-syndecan-4 antibodies, indicating the involvement of syndecan-4. The extent of migration toward naloxone was diminished by pretreatment with dimethylsphingosine, a specific sphingosine kinase inhibitor. As syndecan-4 signaling in leukocyte chemotaxis involves activation of sphingosine kinase, results indicate that naloxone interacts with syndecan-4 function in cell migration and suggest a role for heparan sulfate proteoglycans as coreceptors to members of the delta-opiate receptor family.

Asunto(s)

Quimiotaxis de Leucocito/fisiología , Proteoglicanos de Heparán Sulfato/fisiología , Receptores Opioides/fisiología , Esfingosina/análogos & derivados , Anticuerpos Monoclonales/farmacología , Inhibición de Migración Celular , Separación Celular , Quimiotaxis de Leucocito/efectos de los fármacos , Quimiotaxis de Leucocito/inmunología , Cloratos/farmacología , Condroitinasas y Condroitín Liasas/farmacología , Liasa de Heparina/farmacología , Humanos , Interleucina-8/metabolismo , Linfocitos/citología , Linfocitos/efectos de los fármacos , Glicoproteínas de Membrana/inmunología , Glicoproteínas de Membrana/fisiología , Naloxona/antagonistas & inhibidores , Naloxona/metabolismo , Naloxona/farmacología , Neutrófilos/citología , Neutrófilos/efectos de los fármacos , Neutrófilos/enzimología , Neutrófilos/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Fosfotransferasas (Aceptor de Grupo Alcohol)/fisiología , Unión Proteica/efectos de los fármacos , Proteoglicanos/inmunología , Proteoglicanos/fisiología , Esfingosina/farmacología , Sindecano-4