RESUMEN

E. coli associated Hemolytic Uremic Syndrome (epidemic hemolytic uremic syndrome, eHUS) caused by Shiga toxin-producing bacteria is characterized by thrombocytopenia, microangiopathic hemolytic anemia, and acute kidney injury that cause acute renal failure in up to 65% of affected patients. We hypothesized that the mannose-binding lectin (MBL) pathway of complement activation plays an important role in human eHUS, as we previously demonstrated that injection of Shiga Toxin-2 (Stx-2) led to fibrin deposition in mouse glomeruli that was blocked by co-injection of the anti-MBL-2 antibody 3F8. However, the markers of platelet thrombosis in affected mouse glomeruli were not delineated. To investigate the effect of 3F8 on markers of platelet thrombosis, we used kidney sections from our mouse model (MBL-2+/+ Mbl-A/C-/-; MBL2 KI mouse). Mice in the control group received PBS, while mice in a second group received Stx-2, and those in a third group received 3F8 and Stx-2. Using double immunofluorescence (IF) followed by digital image analysis, kidney sections were stained for fibrin(ogen) and CD41 (marker for platelets), von-Willebrand factor (marker for endothelial cells and platelets), and podocin (marker for podocytes). Electron microscopy (EM) was performed on ultrathin sections from mice and human with HUS. Injection of Stx-2 resulted in an increase of both fibrin and platelets in glomeruli, while administration of 3F8 with Stx-2 reduced both platelet and fibrin to control levels. EM studies confirmed that CD41-positive objects observed by IF were platelets. The increases in platelet number and fibrin levels by injection of Stx-2 are consistent with the generation of platelet-fibrin thrombi that were prevented by 3F8.

Asunto(s)

Síndrome Hemolítico-Urémico/metabolismo , Lectina de Unión a Manosa/metabolismo , Trombosis/metabolismo , Lesión Renal Aguda/metabolismo , Animales , Plaquetas/metabolismo , Modelos Animales de Enfermedad , Células Endoteliales/metabolismo , Escherichia coli/metabolismo , Escherichia coli/patogenicidad , Infecciones por Escherichia coli/microbiología , Síndrome Hemolítico-Urémico/microbiología , Humanos , Riñón/metabolismo , Glomérulos Renales/metabolismo , Lectina de Unión a Manosa/inmunología , Ratones , Ratones Noqueados , Ratones Transgénicos , Toxina Shiga/metabolismo , Toxina Shiga II/metabolismo , Tromboembolia/metabolismo

RESUMEN

Mitchell-Riley syndrome/Martinez-Frias syndrome (MRS/MFS) is a rare, autosomal recessive disorder with multisystem involvement and poor prognosis. Most reported cases have been associated with homozygous or compound heterozygous mutations in the RFX6 gene, a transcriptional regulatory factor for pancreatic morphogenesis. Given the limited number of reported cases, the syndrome may be under-recognized. When the particular phenotype of MFS includes a mutation on the RFX6 gene and neonatal diabetes, it has been called Mitchell-Riley syndrome. Because of this, we propose that MFS/MRS is a symptom continuum or an RFX6 malformation complex. We report an infant with all of the key clinical features of MRS/MFS without a definable mutation in RFX6 gene, supporting the consideration of these features as a symptom complex, and raising the question of genetic heterogeneity.

Asunto(s)

Proteínas de Unión al ADN/genética , Diabetes Mellitus/diagnóstico , Diabetes Mellitus/genética , Enfermedades de la Vesícula Biliar/diagnóstico , Enfermedades de la Vesícula Biliar/genética , Atresia Intestinal/diagnóstico , Atresia Intestinal/genética , Fístula Traqueoesofágica/diagnóstico , Fístula Traqueoesofágica/genética , Factores de Transcripción/genética , Hemocromatosis/diagnóstico , Hemosiderosis/diagnóstico , Humanos , Recién Nacido , Imagen por Resonancia Magnética , Factores de Transcripción del Factor Regulador X

RESUMEN

Ischemia/reperfusion injury (IRI) remains a major problem in renal transplantation. Clinical studies have identified that high serum levels of Mannan-binding lectin (MBL), the initiator of the lectin pathway of complement activation, are associated with inferior renal allograft survival. Using a rat model, we identified an entirely novel role for MBL in mediating renal IRI. Therapeutic inhibition of MBL was protective against kidney dysfunction, tubular damage, neutrophil and macrophage accumulation, and expression of proinflammatory cytokines and chemokines. Following reperfusion, exposure of tubular epithelial cells to circulation-derived MBL resulted in internalization of MBL followed by the rapid induction of tubular epithelial cell death. Interestingly, this MBL-mediated tubular injury was completely independent of complement activation since attenuation of complement activation was not protective against renal IRI. Our identification that MBL-mediated cell death precedes complement activation strongly suggests that exposure of epithelial cells to MBL immediately following reperfusion is the primary culprit of tubular injury. In addition, also human tubular epithelial cells in vitro were shown to be susceptible to the cytotoxic effect of human MBL. Taken together, these data reveal a crucial role for MBL in the early pathophysiology of renal IRI and identify MBL as a novel therapeutic target in kidney transplantation.

Asunto(s)

Activación de Complemento/inmunología , Lectina de Unión a Manosa/efectos adversos , Insuficiencia Renal/etiología , Insuficiencia Renal/patología , Daño por Reperfusión/etiología , Daño por Reperfusión/patología , Animales , Muerte Celular , Células Cultivadas , Citometría de Flujo , Humanos , Pruebas de Función Renal , Túbulos Renales/efectos de los fármacos , Túbulos Renales/metabolismo , Túbulos Renales/patología , Masculino , Ratas , Ratas Endogámicas Lew

RESUMEN

OBJECTIVE: High-mobility group box-1 (HMGB1) is a potent inflammatory mediator and contributes to acute lung injury in adults. The role of HMGB1 in neonatal lung injury and the development of bronchopulmonary dysplasia (BPD) is unknown. We studied the association between HMGB1 levels in tracheal aspirates (TAs) and adverse outcomes (BPD/death) in ventilated premature infants (VPIs) and modulation of HMGB1 levels with dexamethasone (Dex) use. STUDY DESIGN: Infants born before 32 weeks gestation and requiring mechanical ventilation were enrolled. Serial TA samples were collected on days 1, 3, 5 and 7 and HMGB1 levels were measured. HMGB1 levels in TA samples were compared between infants with no BPD and infants who developed BPD or died. HMGB1 TA levels were also compared before and after using Dex. RESULT: In all, 24 infants (gestational age 26.4+/-1.9 weeks, birth weight 859+/-200 g) had no BPD, 60 infants (gestational age 25.4+/-1.8 weeks, birth weight 749+/-156 g) developed BPD or died before 36 weeks postmenstrual age. Mean HMGB1 level in first week of life was significantly lower in infants with no BPD (27.3+/-16.5 ng mg(-1)) compared with those who developed BPD or died (45.1+/-30.9 ng mg(-1), P=0.004). In total, 29 VPIs received Dex. There was no significant change in HMGB1 levels with steroid therapy (before 47.0+/-43.9, after 60.1.5+/-58.8, P=0.3). CONCLUSION: Our data suggest that higher HMGB1 levels in TA are associated with the development of BPD or death in VPI. Dex use had no effect on HMGB1 levels.

Asunto(s)

Displasia Broncopulmonar/metabolismo , Proteínas de Homeodominio/metabolismo , Esputo/metabolismo , Displasia Broncopulmonar/tratamiento farmacológico , Displasia Broncopulmonar/mortalidad , Dexametasona/uso terapéutico , Femenino , Humanos , Recien Nacido con Peso al Nacer Extremadamente Bajo , Recién Nacido , Recien Nacido Prematuro , Masculino , Respiración Artificial/efectos adversos , Esteroides/uso terapéutico , Tráquea

RESUMEN

The alternative pathway (AP) of complement alone is capable of mediating immune complex-induced arthritis in the collagen antibody-induced arthritis (CAIA) model in mice. Whether the classical pathway (CP) or lectin pathway (LP) alone can mediate CAIA is not known. Using mice genetically deficient in different complement components, our results reported herein establish that the CP and LP alone are each incapable of mediating CAIA. A lower level or absence of C3 and/or C5 activation by the CP may be possible explanations for the importance of the AP in CAIA and in many murine models of disease. In addition, other investigators have reported that CP C5 convertase activity is absent in mouse sera. To address these questions, we employed an in vitro system of adherent immunoglobulin (Ig)G-induced complement activation using plates coated with murine anti-collagen monoclonal antibody (mAb). These experiments used complement-deficient mouse sera and wild-type mouse or normal human sera under conditions inactivating either the CP (Ca(++) deficiency) or the AP (mAb inhibitory to factor B). Robust generation of both C3a and C5a by either the AP or CP alone were observed with both mouse and human sera, although there were some small differences between the species of sera. We conclude that neither the CP nor LP alone is capable of mediating CAIA in vivo and that mouse sera exhibits a high level of IgG-induced C5a generation in vitro through either the CP or AP.

Asunto(s)

Artritis Experimental/inmunología , Complemento C3a/metabolismo , Complemento C5a/metabolismo , Vía Alternativa del Complemento/inmunología , Vía Clásica del Complemento/inmunología , Lectina de Unión a Manosa de la Vía del Complemento/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Artritis Experimental/metabolismo , Artritis Experimental/patología , Colágeno Tipo II/inmunología , Complemento C1q/genética , Complemento C1q/metabolismo , Complemento C3/genética , Complemento C3/metabolismo , Complemento C4/metabolismo , Factor B del Complemento/genética , Factor B del Complemento/inmunología , Factor B del Complemento/metabolismo , Factor D del Complemento/genética , Factor D del Complemento/metabolismo , Proteínas del Sistema Complemento/genética , Proteínas del Sistema Complemento/metabolismo , Femenino , Pie/patología , Humanos , Inmunoglobulina G/inmunología , Articulaciones/metabolismo , Articulaciones/patología , Lipopolisacáridos/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Suero/inmunología , Suero/metabolismo

RESUMEN

Though complement (C) deposition within the transplant is associated with allograft rejection, the pathways employed have not been established. In addition, evidence suggests that C-mediated cytolysis may be necessary for the tolerance-inducing activities of mAb therapies. Hence, we assessed the role of the classical C pathway in acute allograft rejection and its requirement for experimental mAb therapies. C1q-deficient (C1q-/-) recipients rejected allografts at a faster rate than wild-type (WT) recipients. This rejection was associated with exacerbated graft pathology but not with enhanced T-cell responses in C1q-/- recipients. However, the humoral response to donor alloantigens was accelerated in C1q-/- mice, as an early IgG response and IgG deposition within the graft were observed. Furthermore, deposition of C3d, but not C4d was observed in grafts isolated from C1q-/- recipients. To assess the role of the classical C pathway in inductive mAb therapies, C1q-/- recipients were treated with anti-CD4 or anti-CD40L mAb. The protective effects of anti-CD4 mAb were reduced in C1q-/- recipients, however, this effect did not correlate with ineffective depletion of CD4+ cells. In contrast, the protective effects of anti-CD40L mAb were less compromised in C1q-/- recipients. Hence, this study reveals unanticipated roles for C1q in the rejection process.

Asunto(s)

Complemento C1q/metabolismo , Vía Clásica del Complemento , Rechazo de Injerto/prevención & control , Inmunoglobulina G/metabolismo , Tolerancia al Trasplante/fisiología , Animales , Anticuerpos Monoclonales , Femenino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Índice de Severidad de la Enfermedad

RESUMEN

AIMS/HYPOTHESIS: Diabetic patients are at increased risk of cardiomyopathy, acute myocardial infarction and loss of cardiac progenitor cells (CPCs), but the aetiology is poorly understood. We hypothesised a significant role for mannose-binding lectin (MBL) in cardiomyopathies associated with hyperglycaemia. METHODS: The role of MBL in myocardial ischaemia and reperfusion (MI/R) injury was investigated in wild-type (WT) and MBL-null mice following 2 weeks of streptozotocin-induced hyperglycaemia. RESULTS: Hyperglycaemic WT mice presented with significantly decreased left ventricular ejection fractions and increased serum troponin I levels and myocardial inflammation compared with non-diabetic WT mice following MI/R. Hyperglycaemic MBL-null mice or insulin-treated diabetic WT mice were significantly protected from MI/R injury compared with diabetic WT mice. In an additional study using diabetic WT mice, echocardiographic measurements demonstrated signs of dilative cardiomyopathy, whereas heart:body weight ratios suggested hypertrophic cardiac remodelling after 2 weeks of hyperglycaemia. Immunohistochemical analysis of CPCs showed significantly lower numbers in diabetic WT hearts compared with non-diabetic hearts. Insulin-treated diabetic WT or untreated diabetic MBL-null mice were protected from dilative cardiomyopathy, hypertrophic remodelling and loss of CPCs. CONCLUSIONS/INTERPRETATION: These data demonstrate that MBL may play a critical role in diabetic MI/R injury. Further, the absence of MBL appears to inhibit hypertrophic remodelling and hyperglycaemia-induced loss of CPCs after just 2 weeks of hyperglycaemia in mice.

Asunto(s)

Diabetes Mellitus Experimental/fisiopatología , Lectina de Unión a Manosa/fisiología , Isquemia Miocárdica/fisiopatología , Daño por Reperfusión/fisiopatología , Animales , Proteínas del Sistema Complemento/fisiología , Cruzamientos Genéticos , Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Tipo 1/complicaciones , Diabetes Mellitus Tipo 1/fisiopatología , Angiopatías Diabéticas/fisiopatología , Modelos Animales de Enfermedad , Femenino , Masculino , Lectina de Unión a Manosa/deficiencia , Lectina de Unión a Manosa/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neutrófilos/fisiología

RESUMEN

The compulsive nature of tobacco use is attributable to nicotine addiction. Nicotine is eliminated by metabolism through the cytochrome P450 2A6 (CYP2A6) enzyme in liver. Inhibition of CYP2A6 by chemical compounds may represent a potential supplement to anti-smoking therapy. The purpose of this study was to rationally design potent inhibitors of CYP2A6. 3D-QSAR models were constructed to find out which structural characteristics are important for inhibition potency. Specifically located hydrophobic and hydrogen donor features were found to affect inhibition potency. These features were used in virtual screening of over 60,000 compounds in the Maybridge chemical database. A total of 22 candidate molecules were selected and tested for inhibition potency. Four of these were potent and selective CYP2A6 inhibitors with IC(50) values lower than 1 muM. They represent novel structures of CYP2A6 inhibitors, especially N1-(4-fluorophenyl)cyclopropane-1-carboxamide. This compound can be used as a lead in the design of CYP2A6 inhibitor drugs to combat nicotine addiction.

Asunto(s)

Hidrocarburo de Aril Hidroxilasas/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Nicotina/metabolismo , Hidrocarburo de Aril Hidroxilasas/metabolismo , Citocromo P-450 CYP2A6 , Evaluación Preclínica de Medicamentos , Inhibidores Enzimáticos/química , Humanos , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/enzimología , Estructura Molecular , Relación Estructura-Actividad Cuantitativa

RESUMEN

OBJECTIVES: To study the association between angiopoietin 2 (Ang2) concentrations in tracheal aspirates (TAs) and adverse outcome (bronchopulmonary dysplasia (BPD)/death) in ventilated premature infants (VPIs) and modulation of Ang2 concentrations with dexamethasone (Dex) use. STUDY DESIGN: Serial TA samples were collected on days 1, 3, 5 and 7, and Ang2 concentrations were measured. Ang2 TA concentrations were compared prior to and after 48 to 72 h of using Dex. RESULT: A total of 151 TA samples were collected from 60 VPIs. BPD was defined as the oxygen requirement at 36 weeks postmenstrual age (PMA). Twelve infants (mean+/-s.d.) (gestational age (GA) 26.5+/-2.1 weeks, birth weight (BW) 913+/-230 g) had no BPD, 32 infants (GA 25.8+/-1.4 weeks, BW 768+/-157 g) developed BPD and 16 infants (GA 24.5+/-1.1 weeks, BW 710+/-143 g) died before 36 weeks PMA. Ang2 concentrations were significantly lower in infants with no BPD (median, 25th and 75th percentile) (157, 16 and 218 pg mg(-1)) compared with those who developed BPD (234, 138 and 338 pg mg(-1), P=0.03) or BPD and/or death (234, 157 and 347 pg mg(-1), P=0.017), in the first week of life. Twenty-six VPIs (BW 719+/-136 g, GA 25.1+/-1.3 weeks) received 27 courses of Dex. Ang2 concentrations before starting Dex were 202, 137 and 278 pg mg(-1) and significantly decreased to 144, 0 and 224 pg mg(-1) after therapy (P=0.007). CONCLUSIONS: Higher Ang2 concentrations in TAs are associated with the development of BPD or death in VPIs. Dex use suppressed Ang2 concentrations.

Asunto(s)

Angiopoyetina 2/sangre , Displasia Broncopulmonar/sangre , Dexametasona/farmacología , Glucocorticoides/farmacología , Angiopoyetina 2/fisiología , Líquidos Corporales/química , Displasia Broncopulmonar/fisiopatología , Dexametasona/uso terapéutico , Glucocorticoides/uso terapéutico , Humanos , Recién Nacido , Recien Nacido Prematuro , Tráquea/química

RESUMEN

The role of non-complement-activating alloantibodies in humoral graft rejection is unclear. We hypothesized that the non-complement-activating alloantibodies synergistically activate complement in combination with complement-activating antibodies. B10.A hearts were transplanted into immunoglobulin knock out (Ig-KO) mice reconstituted with monoclonal antibodies to MHC class I antigens. In allografts of unreconstituted Ig-KO recipients, no C4d was detected. Similarly, reconstitution with IgG1 or low dose IgG2b alloantibodies did not induce C4d deposition. However, mice administered with a low dose of IgG2b combined with IgG1 had heavy linear deposits of C4d on vascular endothelium. C4d deposits correlated with decreased graft survival. To replicate this synergy in vitro, mononuclear cells from B10.A mice were incubated with antibodies to MHC class I antigens followed by incubation in normal mouse serum. Flow cytometry revealed that both IgG2a and IgG2b synergized with IgG1 to deposit C4d. This synergy was significantly decreased in mouse serum deficient in mannose binding lectin (MBL) and in serum deficient in C1q. Reconstitution of MBL-A/C knock out (MBL-KO) serum with C1q-knock out (C1q-KO) serum reestablished the synergistic activity. This suggests a novel role for non-complement-activating alloantibodies and MBL in humoral rejection.

Asunto(s)

Complemento C4b/inmunología , Proteínas del Sistema Complemento/inmunología , Trasplante de Corazón/inmunología , Isoanticuerpos/sangre , Fragmentos de Péptidos/inmunología , Animales , Activación de Complemento/inmunología , Complemento C4b/análisis , Citometría de Flujo , Trasplante de Corazón/patología , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos , Modelos Animales , Fragmentos de Péptidos/análisis , Conejos , Trasplante Homólogo/inmunología

RESUMEN

BACKGROUND: Extremely low birth weight (ELBW) infants are at increased risk for invasive candidiasis and associated morbidity and mortality. The use of fluconazole prophylaxis in this population has raised a benefit versus risk concern among clinicians. OBJECTIVES: To evaluate the effectiveness and safety of fluconazole prophylaxis in ELBW infants. STUDY DESIGN: ELBW infants (BW

Asunto(s)

Profilaxis Antibiótica , Antifúngicos/efectos adversos , Candidiasis/epidemiología , Candidiasis/prevención & control , Colestasis/inducido químicamente , Fluconazol/efectos adversos , Recién Nacido de muy Bajo Peso , Fosfatasa Alcalina/sangre , Antifúngicos/administración & dosificación , Antifúngicos/uso terapéutico , Bilirrubina/sangre , Candidiasis/etiología , Estudios de Casos y Controles , Colestasis/sangre , Femenino , Fluconazol/administración & dosificación , Fluconazol/uso terapéutico , Humanos , Recién Nacido , Enfermedades del Prematuro/epidemiología , Enfermedades del Prematuro/etiología , Enfermedades del Prematuro/prevención & control , Masculino , New Jersey/epidemiología , Estudios Retrospectivos

RESUMEN

Recently we showed that alternative pathway (AP) amplification was responsible for more than 80% of specific classical pathway-induced terminal pathway activation under physiological conditions. The present study aimed to design a system for specific lectin pathway (LP) activation applicable at low serum dilutions with a fully functional AP. Comparison between activation of normal human serum (NHS), a mannose-binding lectin (MBL) homozygous D/D-deficient serum, and sera deficient in C1q and C2, all diluted 1 : 2, was essential to document optimal conditions for LP specificity. Mannan on the solid phase of enzyme-linked immunosorbent assay (ELISA) plates was used for activation, showing 0.5 microg mannan/well to give optimal conditions because at this concentration a good signal was preserved for C4 and TCC deposition in NHS, whereas the C3 deposition observed in C2-deficient serum at higher mannan concentrations reached nadir at 0.5 microg/well, indicating a lack of direct AP activation under these conditions. Pooled NHS and C1q-deficient serum gave the same degree of C4 and terminal complement complex (TCC) deposition, whereas deposition of these products was not obtained with MBL-deficient serum. Reconstitution with purified MBL, however, restored the depositions. A blocking anti-MBL monoclonal antibody (mAb) completely abolished the complement deposition, in contrast to a non-inhibiting anti-MBL mAb. Activation of C2-deficient serum induced C4 deposition similar to NHS, but negligible deposition of C3 and TCC, confirming the lack of direct activation of AP. Thus, this assay is unique in being LP-specific at low serum dilution and thus particularly suitable to study LP activation mechanisms and the role of AP amplification under physiological conditions.

Asunto(s)

Vía Alternativa del Complemento/fisiología , Lectina de Unión a Manosa de la Vía del Complemento/fisiología , Lectina de Unión a Manosa/fisiología , Anticuerpos Monoclonales/inmunología , Complemento C1q/fisiología , Complemento C2/fisiología , Complemento C4/metabolismo , Humanos , Mananos/inmunología , Lectina de Unión a Manosa/inmunología , Suero/inmunología

RESUMEN

OBJECTIVE: To compare the work of breathing (WOB) in premature neonates supported with high-flow nasal cannula (HFNC) and nasal continuous positive airway pressure (NCPAP). STUDY DESIGN: Eighteen preterm neonates <2.0 kg on HFNC or NCPAP support were studied in a random order. A ventilator was used to deliver 6 cm H2O of NCPAP with nasal prongs. High-flow nasal cannula delivered with Vapotherm (VAPO) at 3, 4 and 5 l/min was used. Tidal ventilation was obtained using respiratory inductance plethysmography calibrated with face-mask pneumotachography. An esophageal balloon estimated pleural pressure from which changes in end distending pressure were calculated. Inspiratory, elastic and resistive WOB and respiratory parameters were calculated. RESULTS: No differences were found in the WOB for all settings. Changes in end distending pressure did not vary significantly over all device settings except VAPO at 5 l/min. CONCLUSION: In these preterm infants with mild respiratory illness, HFNC provided support comparable to NCPAP.

Asunto(s)

Enfermedades del Prematuro/fisiopatología , Terapia por Inhalación de Oxígeno , Enfermedades Respiratorias/fisiopatología , Trabajo Respiratorio , Presión de las Vías Aéreas Positiva Contínua , Humanos , Recién Nacido , Recien Nacido Prematuro , Enfermedades del Prematuro/terapia , Rendimiento Pulmonar , Respiración , Mecánica Respiratoria , Enfermedades Respiratorias/terapia , Volumen de Ventilación Pulmonar

RESUMEN

It is known that small changes in the amino acid sequence can change the catalytic activity of cytochromes towards substrates dramatically. With the aim to broaden our knowledge about the structural properties of cytochromes and their relation with substrate specificity a model of CYP2A5 was built by homology modelling based on the crystal structure of CYP2C5. Model stability was evaluated by subjection of the model to a free molecular dynamics simulation in a waterbox under almost physiological conditions using the GROMACS program. The protein folding remains stable over 1.5 ns under these conditions. The modelling procedure was repeated for two mutated forms of CYP2A5 with known differing substrate selectivities towards corticosterone and desoxycorticosterone. A detailed analysis of the models and their behaviour in long running molecular dynamics simulations allows an understanding of the requirements for enzyme activity as well as an explanation of respective experimental data on the molecular level.

Asunto(s)

Hidrocarburo de Aril Hidroxilasas/química , Hidrocarburo de Aril Hidroxilasas/genética , Oxigenasas de Función Mixta/química , Oxigenasas de Función Mixta/genética , Secuencia de Aminoácidos , Corticosterona/metabolismo , Citocromo P-450 CYP2A6 , Desoxicorticosterona/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Oxígeno/química , Mutación Puntual , Conformación Proteica , Pliegue de Proteína , Especificidad por Sustrato

RESUMEN

BACKGROUND: Complement consists of a complex cascade of proteins involved in innate and adaptive immunity. The cascade can be activated through 3 distinct mechanisms, designated the classical, alternative, and lectin pathways. Although complement is widely accepted as participating in the pathophysiology of ischemia-reperfusion injury, the specific role of the lectin pathway has not been addressed. METHODS AND RESULTS: Monoclonal antibodies (mAbs; P7E4 and 14C3.74, IgG1kappa isotypes) were raised against rat mannose-binding lectin (rMBL). Both mAbs recognized rMBL-A by Western analysis or surface plasmon resonance. P7E4, but not 14C3.74, exhibited a concentration-dependent inhibition of the lectin pathway, with maximal effect at 10 microg/mL. In vivo, rats were subjected to 30 minutes of left coronary artery occlusion and 4 hours of reperfusion. Complement C3 deposition was greatly attenuated in hearts pretreated with P7E4 compared with 14C3.74-treated hearts. Pretreatment with P7E4 (1 mg/kg) significantly reduced myocardial creatine kinase loss (48%), infarct size (39%), and neutrophil infiltration (47%) compared with 14C3.74-treated animals. In addition, P7E4 pretreatment significantly attenuated the expression of proinflammatory genes (intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and interleukin-6) after ischemia-reperfusion. CONCLUSIONS: The lectin complement pathway is activated after myocardial ischemia-reperfusion and leads to tissue injury. Blockade of the lectin pathway with inhibitory mAbs protects the heart from ischemia-reperfusion by reducing neutrophil infiltration and attenuating proinflammatory gene expression.

Asunto(s)

Proteínas Portadoras/antagonistas & inhibidores , Daño por Reperfusión Miocárdica/prevención & control , Animales , Anticuerpos Monoclonales/metabolismo , Anticuerpos Monoclonales/farmacología , Especificidad de Anticuerpos , Proteínas Portadoras/farmacología , Moléculas de Adhesión Celular/biosíntesis , Moléculas de Adhesión Celular/genética , Colectinas , Activación de Complemento/efectos de los fármacos , Complemento C3/metabolismo , Creatina Quinasa/metabolismo , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Interleucinas/biosíntesis , Interleucinas/genética , Masculino , Manosa/farmacología , Monocinas/biosíntesis , Monocinas/genética , Isquemia Miocárdica/complicaciones , Isquemia Miocárdica/metabolismo , Isquemia Miocárdica/patología , Daño por Reperfusión Miocárdica/etiología , Daño por Reperfusión Miocárdica/patología , Miocardio/metabolismo , Miocardio/patología , Peroxidasa/metabolismo , ARN Mensajero/biosíntesis , Ratas , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacos , Resonancia por Plasmón de Superficie

RESUMEN

Oxidative stress increases endothelial mannose-binding lectin (MBL) binding and activates the lectin complement pathway (LCP). However, the molecular mechanism of MBL binding to the endothelium after oxidative stress is unknown. Intermediate filaments have been previously reported to activate the classical complement pathway in an antibody-independent manner. We investigated whether oxidative stress increases human umbilical vein endothelial cell (HUVEC) cytokeratin 1 (CK1) expression and activates the LCP via MBL binding to CK1. Reoxygenation (3 hours, 21% O(2)) of hypoxic HUVECs (24 hours, 1% O(2)) significantly increased CK1 mRNA (in situ hybridization) and membrane protein expression [enzyme-linked immunosorbent assay (ELISA)/confocal microscopy]. Incubating human serum (HS) with N-acetyl-D-glucosamine or anti-human MBL monoclonal antibody attenuated MBL and C3 deposition on purified CK1 (ELISA). CK1 and MBL were co-immunoprecipitated from hypoxic HUVECs reoxygenated in HS. Treatment with anti-human cytokeratin Fab fragments attenuated endothelial MBL and C3 deposition after oxidative stress (ELISA/confocal microscopy). We conclude that: 1) endothelial oxidative stress increases CK1 expression, MBL binding, and C3 deposition; 2) inhibition of MBL attenuates purified CK1-induced complement activation; and 3) anti-human cytokeratin Fab fragments attenuate endothelial MBL and C3 deposition after oxidative stress. These results suggest that MBL binding to endothelial cytokeratins may mediate LCP activation after oxidative stress.

Asunto(s)

Proteínas del Sistema Complemento/metabolismo , Endotelio Vascular/metabolismo , Queratinas/fisiología , Lectinas/metabolismo , Estrés Oxidativo , Anticuerpos/farmacología , Western Blotting , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/metabolismo , Células Cultivadas , Colectinas , Complemento C3/antagonistas & inhibidores , Complemento C3/metabolismo , Humanos , Queratinas/genética , Queratinas/inmunología , Queratinas/metabolismo , Estrés Oxidativo/efectos de los fármacos , Pruebas de Precipitina , ARN Mensajero/metabolismo

RESUMEN

The recently identified lectin pathway of the complement system, initiated by binding of mannan-binding lectin (MBL) to its ligands, is a key component of innate immunity. MBL-deficient individuals show an increased susceptibility for infections, especially of the mucosal system. We examined whether IgA, an important mediator of mucosal immunity, activates the complement system via the lectin pathway. Our results indicate a dose-dependent binding of MBL to polymeric, but not monomeric IgA coated in microtiter plates. This interaction involves the carbohydrate recognition domain of MBL, because it was calcium dependent and inhibited by mannose and by mAb against this domain of MBL. Binding of MBL to IgA induces complement activation, as demonstrated by a dose-dependent deposition of C4 and C3 upon addition of a complement source. The MBL concentrations required for IgA-induced C4 and C3 activation are well below the normal MBL plasma concentrations. In line with these experiments, serum from individuals having mutations in the MBL gene showed significantly less activation of C4 by IgA and mannan than serum from wild-type individuals. We conclude that MBL binding to IgA results in complement activation, which is proposed to lead to a synergistic action of MBL and IgA in antimicrobial defense. Furthermore, our results may explain glomerular complement deposition in IgA nephropathy.

Asunto(s)

Proteínas Portadoras/inmunología , Activación de Complemento , Inmunoglobulina A/metabolismo , Anticuerpos Monoclonales , Sitios de Unión , Proteínas Portadoras/química , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Colectinas , Complemento C3/metabolismo , Complemento C4/metabolismo , Glomerulonefritis por IGA/genética , Glomerulonefritis por IGA/inmunología , Humanos , Técnicas In Vitro , Lectinas/metabolismo , Mananos/metabolismo , Serina Proteasas Asociadas a la Proteína de Unión a la Manosa , Mutación , Unión Proteica , Estructura Terciaria de Proteína , Serina Endopeptidasas/inmunología , Serina Endopeptidasas/metabolismo

RESUMEN

Binding of mannose-binding lectin (MBL), a C-type lectin, and its associated serine proteases, MASP-1 and MASP-2, to cell surface carbohydrates activates the lectin complement pathway. As MBL plays an important role in innate immunity, it has been cloned and characterized in several species. While the pig may be used as a source of organs/tissues for xenotransplantation, little is known about its MBL, thus, we report the isolation of three monomeric forms of MBL from porcine serum. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Coomassie staining of reduced porcine MBL revealed the presence of three monomeric forms with approximate molecular masses of 30 000, 32 000 and 34 000. Protein sequencing identified these monomeric forms as one single protein, suggesting post-translational modification. Western blot analysis demonstrated the cross-reactivity of anti-human MBL polyclonal antibody with porcine MBL. A full-length porcine liver MBL cDNA was isolated and the predicted amino acid sequence exhibited 64.9% identity with human MBL and 50.2% and 56.7% identity with rat A and C MBL, respectively. Furthermore, Northern blot analysis demonstrated the presence of a single ( approximately 1.4-1.6 kilobase pair) transcript in porcine liver. Addition of purified porcine MBL to MBL-deficient human sera augmented N-acetylglucosamine inhibitable C3 deposition to mannan-coated plates in a dose-dependent manner. Taken together, these data demonstrate that porcine and human MBL are highly conserved, sharing structural and functional characteristics.

Asunto(s)

Proteínas Portadoras/aislamiento & purificación , Porcinos/metabolismo , Secuencia de Aminoácidos , Animales , Northern Blotting , Western Blotting , Proteínas Portadoras/química , Proteínas Portadoras/genética , Colectinas , Complemento C3/metabolismo , Secuencia Conservada , ADN Complementario/genética , Ensayo de Inmunoadsorción Enzimática , Humanos , Lectinas/química , Lectinas/genética , Lectinas/aislamiento & purificación , Mananos/metabolismo , Datos de Secuencia Molecular , Especificidad de la Especie

RESUMEN

Like most retroviruses and retrotransposons, the retrotransposon Ty3 expresses its pol gene analog (POL3) as a translational fusion to the upstream gag analog (GAG3). The Gag3-Pol3 fusion occurs by frameshifting during translation of the mRNA that encodes the two separate but overlapping ORFs. We showed previously that the shift occurs by out-of-frame binding of a normal aminoacyl-tRNA in the ribosomal A site caused by an aberrant codonoanticodon interaction in the P site. This event is unlike all previously described programmed translational frameshifts because it does not require tRNA slippage between cognate or near-cognate codons in the mRNA. A sequence of 15 nt distal to the frameshift site stimulates frameshifting 7.5-fold. Here we show that the Ty3 stimulator acts as an unstructured region to stimulate frameshifting. Its function depends on strict spacing from the site of frameshifting. Finally, the stimulator increases frameshifting dependent on sense codon-induced pausing, but has no effect on frameshifting dependent on pauses induced by nonsense codons. Complementarity between the stimulator and a portion of the accuracy center of the ribosome, Helix 18, implies that the stimulator may directly disrupt error correction by the ribosome.

Asunto(s)

Sistema de Lectura Ribosómico , ARN Mensajero/genética , ARN Ribosómico/genética , Ribosomas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Proteínas de Fusión gag-pol/genética , Proteínas de Fusión gag-pol/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Mutación Missense , Conformación de Ácido Nucleico , Plásmidos , Biosíntesis de Proteínas , Virus ARN/fisiología , ARN Mensajero/química , ARN Mensajero/metabolismo , ARN Ribosómico/química , ARN Ribosómico/metabolismo , Retroelementos/genética , Ribosomas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo

RESUMEN

Complement plays a significant role in mediating endothelial injury following oxidative stress. We have previously demonstrated that the lectin complement pathway (LCP), which is initiated by deposition of the mannose-binding lectin (MBL), is largely responsible for activating complement on endothelial cells following periods of oxidative stress. Identifying functional inhibitors that block MBL binding will be useful in characterizing the role of the LCP in disease models. The human cytokeratin peptide SFGSGFGGGY has been identified as a molecular mimic of N-acetyl-D-glucosamine (GlcNAc), a known ligand of MBL. Thus, we hypothesized that this peptide would specifically bind to MBL and functionally inhibit the LCP on endothelial cells following oxidative stress. Using a BIAcore 3000 optical biosensor, competition experiments were performed to demonstrate that the peptide SFGSGFGGGY inhibits binding of purified recombinant human MBL to GlcNAc in a concentration-dependent manner. Solution affinity data generated by BIAcore indicate this peptide binds to MBL with an affinity (K(D)) of 5 x 10(-5) mol/L. Pretreatment of human serum (30%) with the GlcNAc-mimicking peptide (10-50 microg/ml) significantly attenuated MBL and C3 deposition on human endothelial cells subjected to oxidative stress in a dose-dependent manner, as demonstrated by cell surface ELISA and confocal microscopy. Additionally, this decapeptide sequence attenuated complement-dependent VCAM-1 expression following oxidative stress. These data indicate that a short peptide sequence that mimics GlcNAc can specifically bind to MBL and functionally inhibit the proinflammatory action of the LCP on oxidatively stressed endothelial cells.

Asunto(s)

Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/metabolismo , Queratinas/inmunología , Queratinas/metabolismo , Lectinas/metabolismo , Mananos/metabolismo , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/metabolismo , Acetilglucosamina/metabolismo , Unión Competitiva/inmunología , Línea Celular , Células Cultivadas , Colectinas , Activación de Complemento/inmunología , Complemento C3/antagonistas & inhibidores , Complemento C3/metabolismo , Endotelio Vascular/citología , Endotelio Vascular/inmunología , Endotelio Vascular/metabolismo , Humanos , Inmunosupresores/metabolismo , Imitación Molecular , Estrés Oxidativo/inmunología , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/metabolismo , Resonancia por Plasmón de Superficie , Transfección