RESUMEN
Loss of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) activity in mammals results in severe combined immuno-deficiency (SCID). This SCID phenotype has been postulated to be due solely to the function of DNA-PKcs in V(D)J recombination, a process critical for lymphocyte maturation. However; we show that DNA-PKcs is required for IL-2 production via regulation of the calcineurin signaling pathway. Reducing DNA-PKcs activity in activated T cells either by shRNA or an inhibitor significantly reduced IL-2 production by blocking calcineurin activity and the translocation of NFAT into the nucleus. Additionally, we show that DNA-PKcs exerts its effect on calcineurin by altering the expression of the endogenous calcineurin inhibitor Cabin1 through activation of the kinase CHK2, a known Cabin1 regulator. The discovery of DNA-PKcs as a potent regulator of IL-2 production will drive continued investigation of small molecule inhibition of this enzyme within the clinic.
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Calcineurina/fisiología , Interleucina-2/biosíntesis , Transporte Activo de Núcleo Celular , Expresión Génica , Humanos , Interleucina-2/genética , Interleucina-2/metabolismo , Células Jurkat , Factores de Transcripción NFATC/metabolismo , Oligopéptidos/genética , Oligopéptidos/metabolismo , Fosforilación , Procesamiento Proteico-Postraduccional , Estabilidad Proteica , Activación TranscripcionalRESUMEN
Constitutive activation of the Rearranged during Transfection (RET) proto-oncogene leads to the development of MEN2A medullary thyroid cancer (MTC). The relatively clear genotype/phenotype relationship seen with RET mutations and the development of MEN2A is unusual in the fact that a single gene activity can drive the progression towards metastatic disease. Despite knowing the oncogene responsible for MEN2A, MTC, like most tumors of neural crest origin, remains largely resistant to chemotherapy. Constitutive activation of RET in a SK-N-MC cell line model reduces cell sensitivity to chemotherapy. In an attempt to identify components of the machinery responsible for the observed RET induced chemoresistance, we performed a proteomic screen of histones and associated proteins in cells with a constitutively active RET signaling pathway. The proteomic approach identified DNA-PKcs, a DNA damage response protein, as a target of the RET signaling pathway. Active DNA-PKcs, which is phosphorylated at site serine 2056 and localized to chromatin, was elevated within our model. Treatment with the RET inhibitor RPI-1 significantly reduced s2056 phosphorylation in RET cells as well as in a human medullary thyroid cancer cell line. Additionally, inhibition of DNA-PKcs activity diminished the chemoresistance observed in both cell lines. Importantly, we show that activated DNA-PKcs is elevated in medullary thyroid tumor samples and that expression correlates with expression of RET in thyroid tumors. These results highlight one mechanism by which RET signaling likely primes cells for rapid response to DNA damage and suggests DNA-PKcs as an additional target in MTC.
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Carcinoma Neuroendocrino/metabolismo , Proteína Quinasa Activada por ADN/metabolismo , Proteínas Nucleares/metabolismo , Proteómica/métodos , Proteínas Proto-Oncogénicas c-ret/metabolismo , Neoplasias de la Tiroides/metabolismo , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Humanos , Indoles/farmacología , Fosforilación/efectos de los fármacos , Proto-Oncogenes Mas , Transducción de SeñalRESUMEN
BACKGROUND: A 2005 survey reported 87% of surgery program directors believed practice management training should occur during residency. However, only 8% of program directors believed residents received adequate training in practice management [1]. In addition to the gap in practice financial management knowledge, we recognized the need for training in personal finance among residents. A literature review and needs assessment led to the development of a novel curriculum for surgery residents combining principles of practice management and personal finance. METHODS: An 18-h curriculum was administered over the 2012 academic year to 28 post graduate year 1-5 surgery residents and faculty. A self-assessment survey was given at the onset and conclusion of the curriculum [2]. Pre-tests and post-tests were given to objectively evaluate each twice monthly session's content. Self-perception of learning, interest, and acquired knowledge were analyzed using the Wilcoxon signed ranks test. RESULTS: Initial self-assessment data revealed high interest in practice management and personal finance principles but a deficiency in knowledge of and exposure to these topics. Throughout the curriculum, interest increased. Residents believed their knowledge of these topics increased after completing the curriculum, and objective data revealed various impacts on knowledge. CONCLUSIONS: Although surgery residents receive less exposure to these topics than residents in other specialties, their need to know is no less. We developed, implemented, and evaluated a curriculum that bridged this gap in surgery education. After the curriculum, residents reported an increase in interest, knowledge, and responsible behavior relating to personal and practice financial management.
Asunto(s)
Educación de Postgrado en Medicina/métodos , Administración Financiera , Cirugía General/educación , Internado y Residencia/métodos , Práctica Privada , Servicios Contratados , Curriculum , Conocimientos, Actitudes y Práctica en Salud , Humanos , Asistentes Médicos , Gestión de RiesgosRESUMEN
Closed claims reviews are a robust source of severe surgical errors for study. Most errors are preventable. Most preventable technical errors and judgment errors occur during care provided by competent surgeons. Failure to operate within a proper scope-of-practice is the most common cause of incompetence. Patient factors and systems failures, including human factors, cause or profoundly contribute to the cause of most technical errors. Regardless of its cause or preventability, a technical error sets the stage for other errors in care that relate to systems' failures and surgeons' judgment failures. Failed judgment and poor decision-making are usually the result of cognitive errors caused by flawed behavioral practices instead of lack of knowledge. Systems of care and good behavioral practices are catalysts that maximize the power of knowledge and skill to achieve good outcomes. The uniform application of knowledge and skill in a favorable environment is as important as the possession of knowledge and skill. Identifying systems and behavioral causes of errors may help to define best practices and lead to safer patient care through improved systems of care and increased diligent attention to ordinary tasks that require more time than knowledge on the part of surgeons.
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Actitud del Personal de Salud , Cirugía General/legislación & jurisprudencia , Revisión de Utilización de Seguros/estadística & datos numéricos , Responsabilidad Legal , Mala Praxis/legislación & jurisprudencia , Errores Médicos/legislación & jurisprudencia , Cirugía General/estadística & datos numéricos , Humanos , Revisión de Utilización de Seguros/legislación & jurisprudencia , Mala Praxis/estadística & datos numéricos , Errores Médicos/estadística & datos numéricos , Calidad de la Atención de Salud , Estados UnidosRESUMEN
INTRODUCTION: CXCR4 is a chemokine receptor that has recently been implicated to play a pivotal role in breast cancer growth and metastasis. In animal models, reduction of CXCR4 expression significantly abrogated metastatic disease and prolonged survival. In human breast cancers, CXCR4 overexpression may portend a worse clinical course. Recent data suggest that HER-2 up-regulates CXCR4, but whether this is applicable in the clinical setting is not known. In this study, we evaluated the role of CXCR4 overexpression in breast cancer and determined whether it can serve as a potential marker of tumor recurrence in HER-2 negative tumors. METHODS: One hundred three patients with stages I to III breast cancers and 6 benign breast tissues were prospectively accrued and analyzed. Study homogeneity was maintained by standardized treatment, surveillance, and compliance protocols. CXCR4 levels were detected using Western blots and results were quantified against 1 microg of HeLa cells (positive controls). HER-2 expression was evaluated using the Hercep program, (Dako Corp., Carpinteria, CA) with a positive result defined as > or = 2. CXCR4 expression was defined as low (<6.6-fold) or high (> or = 6.6-fold). Primary endpoints were cancer recurrence and death. Statistical analysis performed included Spearman's correlation, independent samples t-test, Kaplan-Meier survival analysis, and log-rank test. RESULTS: All 103 cancer specimens had CXCR4 overexpression (mean 6.6 +/- 4.7), while none of the 6 benign breast tissues had detectable level of CXCR4. There were 36 HER-2 (+) tumors and 67 HER-2 (-) tumors. There was no statistical significance in mean CXCR4 overexpression between HER-2 (+) [5.6] and HER-2 (-) [6.6] cancers (P = 0.3; independent samples t-test). Recurrences occurred in 18 of 103 patients (17%); 10 occurred in HER-2 (+) tumors, and 8 occurred in HER-2 (-) patients. CXCR4 expression level was not predictive of cancer recurrence (P = 0.80) or overall survival (P = 0.70) in the HER-2 (+) group. However, among HER-2 negative tumors, 7 of 8 recurrences occurred in the high CXCR4 group (P = 0.037). There was no correlation between the degree of CXCR4 overexpression with tumor size (r = 0.13, P = 0.22), nodal status (r = 0.019, P = 0.4), ER/PR status (r = 0.12, P = 0.29), and HER-2 status (r = 0.091, P = 0.36). CONCLUSIONS: CXCR4 overexpression was observed in all 103 breast cancer specimens but was undetectable in benign breast tissues. CXCR4 overexpression does not correlate with tumor size, nodal status, ER/PR status, and HER-2 status. High CXCR4 overexpression had a significant impact on disease-free survival in HER-2 negative breast cancer patients and may help identify a subset of HER-2 negative breast cancers that have a more aggressive biological behavior.
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Neoplasias de la Mama/metabolismo , Recurrencia Local de Neoplasia/diagnóstico , Receptor ErbB-2/metabolismo , Receptores CXCR4/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Progresión de la Enfermedad , Supervivencia sin Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Persona de Mediana Edad , Recurrencia Local de Neoplasia/etiología , Recurrencia Local de Neoplasia/metabolismo , Valor Predictivo de las Pruebas , Pronóstico , Estudios Prospectivos , Receptor ErbB-2/genética , Receptores CXCR4/genética , Factores de RiesgoRESUMEN
BACKGROUND: The role of whole-body fluorine-18-FDG positron emission tomography (FDG-PET) as an adjunct localize recurrence in stages II and III breast cancer patients who present with clinical suspicion for recurrence is not well established. We report our experience in such a patient population. METHODS: A retrospective review of all patients with stages II and III breast cancer who had a whole-body FDG-PET scan was performed. RESULTS: Of the 23 patients who fit the criteria, 9 had stage II and 14 had stage III breast cancer. Overall sensitivity, specificity, and accuracy were 81%, 100%, and 87%, respectively. Positive and negative predictive values for stages II and III were 100% and 83%, respectively, and 100% and 50%, respectively. FDG-PET detected two recurrences that were missed by conventional imagings, but such recurrences were local and amenable for biopsy. CONCLUSIONS: In patients with stages II and III breast cancer who present with a suspicion for recurrent disease, a whole-body FDG-PET scan may be a useful adjunct in the evaluation of recurrence. However, its added benefit over conventional imaging should be questioned.
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Neoplasias de la Mama/diagnóstico por imagen , Carcinoma Ductal de Mama/diagnóstico por imagen , Carcinoma Lobular/diagnóstico por imagen , Recurrencia Local de Neoplasia/diagnóstico por imagen , Tomografía de Emisión de Positrones , Femenino , Fluorodesoxiglucosa F18 , Humanos , Estadificación de Neoplasias , Radiofármacos , Estudios RetrospectivosRESUMEN
OBJECTIVE: In a prospective trial, to determine if eIF4E overexpression in breast cancer specimens is correlated with VEGF elevation, increased tumor microvessel density (MVD) counts, and a worse clinical outcome irrespective of nodal status. SUMMARY AND BACKGROUND DATA: In vitro, the overexpression of eukaryotic initiation factor 4E (eIF4E) up-regulates the translation of mRNAs with long 5'-untranslated regions (5'-UTRs). One such gene product is the vascular endothelial growth factor (VEGF). METHODS: A total of 114 stage I to III breast cancer patients were prospectively accrued and followed with a standardized clinical surveillance protocol. Cancer specimens were quantified for eIF4E, VEGF, and MVD. Outcome endpoints were cancer recurrence and cancer-related death. RESULTS: eIF4E overexpression was found in all cancer specimens (mean +/- SD, 12.5 +/- 7.6-fold). Increasing eIF4E overexpression correlated with increasing VEGF elevation (r = 0.24, P = 0.01, Spearman's coefficient), and increasing MVD counts (r = 0.35, P < 0.0002). Patients whose tumor had high eIF4E overexpression had shorter disease-free survival (P = 0.004, log-rank test) and higher cancer-related deaths (P = 0.002) than patients whose tumors had low eIF4E overexpression. Patients with high eIF4E had a hazard ratio for cancer recurrence and cancer-related death of 1.8 and 2.1 times that of patients with low eIF4E (respectively, P = 0.009 and P = 0.002, Cox proportional hazard model). CONCLUSIONS: In breast cancer patients, increasing eIF4E overexpression in the cancer specimens correlates with higher VEGF levels and MVD counts. Patients whose tumors had high eIF4E overexpression had a worse clinical outcome, independent of nodal status. Thus, eIF4E overexpression in breast cancer appears to predict increased tumor vascularity and perhaps cancer dissemination by hematogenous means.
Asunto(s)
Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Factor 4E Eucariótico de Iniciación/biosíntesis , Factor A de Crecimiento Endotelial Vascular/biosíntesis , Neoplasias de la Mama/irrigación sanguínea , Neoplasias de la Mama/química , Factor 4E Eucariótico de Iniciación/análisis , Femenino , Humanos , Persona de Mediana Edad , Estadificación de Neoplasias , Pronóstico , Estudios Prospectivos , Factor A de Crecimiento Endotelial Vascular/análisisRESUMEN
An incidental finding of focal thyroid uptake (thyroid incidentaloma) from an 18F-fluorodeoxyglucose positron emission tomography (PET) positron presents a diagnostic challenge. We evaluated the incidence of thyroid incidentaloma identified by PET scans and the likelihood of malignancy associated with this finding. Records from all patients from January 1, 2000 to November 30, 2003 who had focal thyroid uptake without any history of thyroid disease were culled. Of the 6241 PET scans performed, focal thyroid uptake was observed in 76 patients (1.2%). Only 14 patients (18%) underwent biopsy. Four of 14 patients (28.6%) had papillary thyroid carcinoma, 7 (50%) had hyperplasia, and 1 each had thyroiditis, nodular goiter, and follicular neoplasm. The incidence of PET thyroid incidentalomas was 1.2 per cent and the incidence of malignancy was 28.6 per cent. Given the high likelihood of malignancy, a further diagnostic workup for surgically fit patients is warranted.
Asunto(s)
Tomografía de Emisión de Positrones , Derivación y Consulta/estadística & datos numéricos , Neoplasias de la Tiroides/diagnóstico por imagen , Adulto , Anciano , Anciano de 80 o más Años , Biopsia con Aguja Fina , Diagnóstico Diferencial , Femenino , Humanos , Incidencia , Masculino , Persona de Mediana Edad , Reproducibilidad de los Resultados , Estudios Retrospectivos , Factores de Riesgo , Neoplasias de la Tiroides/epidemiología , Neoplasias de la Tiroides/patologíaRESUMEN
Oxidant-mediated modulation of the intracellular redox state affects the apoptotic cascade by altering the balance between cellular signals for survival and suicide. Apolipoprotein A-IV (Apo A-IV) is known to possess antioxidant-like activity. In the present study, we tested 1) whether Apo A-IV could influence redox-dependent apoptosis and, if so, 2) whether such an effect could be mediated by modulation of intracellular redox balance. Mitotic competent, undifferentiated PC-12 cells were incubated with either tert-butyl hydroperoxide (TBH) or diamide with or without preincubation with human Apo A-IV. Apo A-IV significantly decreased apoptosis produced by both TBH and diamide, and washout of A-IV before incubation with TBH and diamide did not eliminate its protective effect. Apo A-I had no such protective effect. The Apo A-IV effect was not blocked by D,L-buthionine-[S,R]-sulfoximine, but it was reversed by both dehydroisoandrosterone and transfection with an antisense oligodeoxynucleotide to glucose-6-phosphate dehydrogenase (G6PD). Apo A-IV abolished the transient, oxidant-induced rise in glutathione disulfide (GSSG) and cellular redox imbalance previously shown to initiate the apoptotic cascade. Apo A-IV had no effect on GSSG reductase activity, but it stimulated G6PD activity 10-fold. These results suggest a novel role for Apo A-IV in the regulation of intracellular glutathione redox balance and the modulation of redox-dependent apoptosis via stimulation of G6PD activity.
Asunto(s)
Apolipoproteínas A/metabolismo , Apoptosis/fisiología , Glutatión/metabolismo , Estrés Oxidativo/fisiología , Animales , Apolipoproteína A-I/farmacología , Apolipoproteínas A/farmacología , Apoptosis/efectos de los fármacos , Diferenciación Celular , Glucosafosfato Deshidrogenasa/metabolismo , Disulfuro de Glutatión/metabolismo , Humanos , Mitosis , Oxidantes/farmacología , Oxidación-Reducción/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Células PC12 , Ratas , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , terc-Butilhidroperóxido/farmacologíaRESUMEN
BACKGROUND: The rigorous maintenance of normoglycemia by the administration of insulin is beneficial to critically ill patients. Because insulin induces endothelial nitric oxide (NO) release, and the constitutive release of NO maintains normal microvascular permeability, the authors postulated that insulin would prevent peroxide (H(2)O(2))-induced endothelial barrier dysfunction, an effect dependent on endothelial NO synthase (eNOS) activity. METHODS: Murine lung microvascular endothelial cells (LMEC) grown to confluence on 8 micro pore polyethylene filters were exposed to media (control), H(2)O(2) (20 to 500 micromol/L), insulin (1 to 1,000 nmol/L) or insulin (100 nmol/L) + H(2)O(2) (10(-4)mol/L). Endothelial monolayer permeability was quantitated by measuring the transendothelial electrical resistance at 15-minute intervals for 120 minutes. Other cells were exposed to H(2)O(2) and insulin after pretreatment with a NO scavenger (PTIO), an eNOS inhibitor (L-NIO), or a phosphoinositol-3-kinase inhibitor (LY-294002). RESULTS: H(2)O(2) caused a concentration- and time-dependent reduction in electrical resistance consistent with an increase in monolayer permeability. This effect was prevented by insulin. Inhibiting NO release (L-NIO, LY-294002) or scavenging NO (PTIO) abolished this protective effect. CONCLUSIONS: These data suggest that insulin may modulate endothelial barrier function during oxidant stress by inducing the release of NO.
Asunto(s)
Permeabilidad de la Membrana Celular/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Insulina/farmacología , Óxido Nítrico/metabolismo , Oxidantes/farmacología , Animales , Células Cultivadas , Cromonas/farmacología , Medios de Cultivo , Óxidos N-Cíclicos/farmacología , Relación Dosis-Respuesta a Droga , Impedancia Eléctrica , Células Endoteliales/fisiología , Inhibidores Enzimáticos/farmacología , Depuradores de Radicales Libres/farmacología , Glucosa/administración & dosificación , Glucosa/farmacología , Peróxido de Hidrógeno/administración & dosificación , Peróxido de Hidrógeno/farmacología , Imidazoles/farmacología , Ratones , Microcirculación/efectos de los fármacos , Morfolinas/farmacología , Óxido Nítrico/antagonistas & inhibidores , Óxido Nítrico Sintasa/antagonistas & inhibidores , Ornitina/análogos & derivados , Ornitina/farmacología , Concentración Osmolar , Oxidantes/administración & dosificación , Conejos , Factores de TiempoRESUMEN
The antiatherogenic properties of apoA-IV suggest that this protein may act as an anti-inflammatory agent. We examined this possibility in a mouse model of acute colitis. Mice consumed 3% dextran sulfate sodium (DSS) in their drinking water for 7 days, with or without daily intraperitoneal injections of recombinant human apoA-IV. apoA-IV significantly and specifically delayed the onset, and reduced the severity and extent of, DSS-induced inflammation, as assessed by clinical disease activity score, macroscopic appearance and histology of the colon, and tissue myeloperoxidase activity. Intravital fluorescence microscopy of colonic microvasculature revealed that apoA-IV significantly inhibited DSS-induced leukocyte and platelet adhesive interactions. Furthermore, apoA-IV dramatically reduced the upregulation of P-selectin on colonic endothelium during DSS-colitis. apoA-IV knockout mice exhibited a significantly greater inflammatory response to DSS than did their WT littermates; this greater susceptibility to DSS-induced inflammation was reversed upon exogenous administration of apoA-IV to knockout mice. These results provide the first direct support for the hypothesis that apoA-IV is an endogenous anti-inflammatory protein. This anti-inflammatory effect likely involves the inhibition of P-selectin-mediated leukocyte and platelet adhesive interactions.
Asunto(s)
Antiinflamatorios/metabolismo , Apolipoproteínas A/metabolismo , Colitis/metabolismo , Animales , Antiinflamatorios/inmunología , Apolipoproteínas A/genética , Apolipoproteínas A/inmunología , Colitis/inducido químicamente , Colitis/inmunología , Colitis/patología , Colon/anatomía & histología , Colon/patología , Sulfato de Dextran/administración & dosificación , Sulfato de Dextran/toxicidad , Modelos Animales de Enfermedad , Células Epiteliales/citología , Células Epiteliales/metabolismo , Humanos , Indicadores y Reactivos/administración & dosificación , Indicadores y Reactivos/toxicidad , Inflamación/inmunología , Inflamación/metabolismo , Mucosa Intestinal/citología , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Leucocitos/inmunología , Leucocitos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Selectina-P/metabolismo , Adhesividad Plaquetaria/fisiología , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismoRESUMEN
Reduced plasma concentrations of the extracellular actin-binding proteins gelsolin and Gc-globulin correlate with pulmonary failure and death in humans after injury. The purpose of this study was to investigate the role of plasma gelsolin in the pathophysiology of inflammation-induced lung injury. We postulated that plasma gelsolin levels decrease at an early time point after burn injury and that the intravenous infusion of gelsolin prevents burn-induced pulmonary microvascular dysfunction. Adult Sprague-Dawley rats were randomized to undergo a 40% body surface area thermal injury (Burn) or manipulation without burn (Sham). Plasma gelsolin and Gc-globulin concentrations were determined at various times during the first 6 days of injury by Western blotting. Other animals were randomized to receive either recombinant human gelsolin (0.078, 0.78, or 7.8 mg) or albumin (7.8 mg) before and 8 h after Burn or Sham. Twenty-four hours later, pulmonary microvascular permeability was assessed by measuring the capillary filtration by use of an isolated, perfused lung model. We found that plasma gelsolin levels of burn-injured rats decreased to 10% of normal levels within 12 h and remained below normal levels for up to 6 days postinjury. Gc-globulin values also fall, but to a lesser extent and only transiently. Treatment of burned animals with intravenous infusions of recombinant human gelsolin prevented the increase in pulmonary microvascular permeability that accompanies this injury. Our findings are consistent with the hypothesis that plasma gelsolin depletion contributes to the pathophysiology of pulmonary microvascular dysfunction during inflammation.
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Quemaduras/tratamiento farmacológico , Quemaduras/fisiopatología , Gelsolina/farmacología , Neumonía/prevención & control , Animales , Proteínas Sanguíneas/metabolismo , Quemaduras/complicaciones , Gelsolina/sangre , Hematócrito , Infusiones Intravenosas , Microcirculación/efectos de los fármacos , Neumonía/etiología , Circulación Pulmonar/efectos de los fármacos , Distribución Aleatoria , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/sangre , Proteínas Recombinantes/farmacología , Piel/irrigación sanguínea , Piel/lesiones , Piel/fisiopatología , Proteína de Unión a Vitamina D/sangreRESUMEN
Previous studies indicate that deficiency of endothelial nitric oxide (NO) synthase (eNOS)-derived NO exacerbates myocardial reperfusion injury. We hypothesized that overexpression of eNOS would reduce the extent of myocardial ischemia-reperfusion (MI/R) injury. We investigated two distinct strains of transgenic (TG) mice overexpressing the eNOS gene (eNOS TG). Bovine eNOS was overexpressed in one strain (eNOS TG-Kobe), whereas the human eNOS gene was overexpressed in the other strain (eNOS TG-RT). Non-TG (NTG) and eNOS TG mice were subjected to 30 min of coronary artery occlusion followed by 24 h of reperfusion, and the extent of myocardial infarction was determined. Myocardial infarct size was reduced by 33% in the eNOS TG-Kobe strain (P < 0.05 vs. NTG) and by 32% in the eNOS TG-RT strain (P < 0.05 vs. NTG). However, postischemic cardiac function (cardiac output, fractional shortening) was not improved in the eNOS TG-Kobe mouse at 24 h of reperfusion [P = not significant (NS) vs. NTG]. In additional studies, eNOS TG-Kobe mice were subjected to 30 min of myocardial infarction and 7 days of reperfusion. Fractional shortening and the first derivative of left ventricular pressure were measured in eNOS TG-Kobe and NTG mice, and no significant differences in contractility were observed (P = NS) between the eNOS TG mice and NTG controls. Left ventricular end-diastolic pressure was significantly (P < 0.05 vs. NTG) reduced in the eNOS TG-Kobe strain at 7 days of reperfusion. The cardioprotective effects of eNOS overexpression on myocardial infarct size were ablated by Nomega-nitro-l-arginine methyl ester (300 mg/kg) pretreatment. Thus genetic overexpression of eNOS in mice attenuates myocardial infarction after MI/R but fails to significantly protect against postischemic myocardial contractile dysfunction in mice.
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Isquemia Miocárdica/patología , Isquemia Miocárdica/fisiopatología , Daño por Reperfusión Miocárdica/patología , Daño por Reperfusión Miocárdica/fisiopatología , Miocardio/enzimología , Óxido Nítrico Sintasa/metabolismo , Alquenos/farmacología , Animales , Bovinos , Ácidos Decanoicos/farmacología , Ecocardiografía , Corazón/fisiopatología , Hemodinámica , Humanos , Hidroxiácidos/farmacología , Ratones , Ratones Endogámicos , Ratones Transgénicos , Infarto del Miocardio/patología , Isquemia Miocárdica/diagnóstico por imagen , Daño por Reperfusión Miocárdica/diagnóstico por imagen , Donantes de Óxido Nítrico/farmacología , Óxido Nítrico Sintasa de Tipo II , Óxido Nítrico Sintasa de Tipo III , Factores de TiempoRESUMEN
Our laboratory previously demonstrated that MAPK activation is an important signal during cytokine-induced endothelial permeability (Nwariaku FE, Liu Z, Terada L, Duffy S, Sarosi G, and Turnage R. Shock 18: 82-85, 2002). Because GTP-binding proteins have been implicated in MAPK activation, we now hypothesize that the GTP-binding protein Rho is a mediator of TNF-induced MAPK activation and increased endothelial permeability. Transmonolayer permeability was assessed in human lung microvascular cells by measuring transmonolayer electrical resistance. MAPK activity was assessed by using a phospho-specific immunoprecipitation kinase assay and by comparing Western blots for phospho-MAPK with total MAPK. MAPK inhibitors used were SB-202190 and PD-098059, whereas Clostridium botulinum C3 transferase was used as a Rho inactivator. Rho-associated coiled-coil kinase was inhibited with Y-27632. TNF increased pulmonary endothelial permeability in vitro and caused a rapid, sustained increase in endothelial p38 and extracellular signal-regulated kinase MAPK activity. Inhibition of p38 and extracellular signal-regulated kinase MAPK with SB-202190 and PD-098059, respectively, decreased TNF-induced endothelial permeability. C3 transferase attenuated TNF-induced MAPK activation and blocked TNF-induced endothelial permeability. Finally, inhibition of Rho-associated coiled-coil kinase with Y-27632 prevented both MAPK activation and TNF-induced decreases in transmonolayer resistance. Rho acts upstream of mitogen-activated protein kinases in mediating TNF-induced pulmonary endothelial leak.
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Antineoplásicos/farmacología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Proteínas de Unión al GTP rho/antagonistas & inhibidores , ADP Ribosa Transferasas/farmacología , Amidas/farmacología , Toxinas Botulínicas/farmacología , Permeabilidad Capilar/efectos de los fármacos , Permeabilidad Capilar/fisiología , Línea Celular , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/enzimología , Inhibidores Enzimáticos/farmacología , Humanos , Pulmón/irrigación sanguínea , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Piridinas/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos , Proteínas de Unión al GTP rho/metabolismoRESUMEN
This systematic review examines the evidence for commonly employed strategies of managing patients with recurrent ulcer disease after acid-reducing operations. Particular attention is given to recent evidence relating Helicobacter pylori (H. pylori ) and nonsteroidal anti-inflammatory drugs (NSAIDs) to ulcer recurrence after operative therapy. MEDLINE word searches of the literature from 1966 to 2001 identified 895 articles that cross-reference the terms "peptic ulcer disease (PUD)," "surgery," and "recurrence." Articles were selected for systematic review of evidence relating incomplete vagotomy, NSAIDs, and H. pylori to postoperative ulcer recurrence and evidence supporting common medical and surgical strategies. The relationship between incomplete vagotomy and recurrent ulcer disease is suggested by randomized controlled trials and well-designed prospective case series. The evidence that NSAID use is an important pathogenic factor in recurrent ulcer disease includes the relationship between NSAIDs and primary PUD, the occurrence of NSAID-induced ulcers in patients taking proton pump inhibitors, and case series demonstrating virulent ulcer disease in patients taking aspirin despite prior acid-reducing operations. The relationship between H. pylori infection and postoperative ulcer recurrence remains uncertain despite multiple controlled trials and well-designed case series that have documented high rates of H. pylori infection in postoperative patients. The initial management of patients with recurrent ulcer disease after acid-reducing operations consists of a protein pump inhibitor or a histamine-2 receptor antagonist and antibiotics directed at H. pylori, if present. Evidence for this regimen includes prospective randomized trials demonstrating the efficacy of cimetidine in healing ulcers after acid-reducing operations and prospective, randomized studies documenting the efficacy of histamine-2 receptor antagonists and protein pump inhibitors in the management of patients with primary PUD. The critical role that H. pylori infection plays in primary PUD and the minimal risks associated with H. pylori eradication strongly support the initiation of antibiotic therapy when H. pylori is present. The principal indication for operative management of recurrent PUD is the occurrence of ulcer complications that cannot be managed by medical or endoscopic means. The operative management of patients with failed acid-reducing operations is based on ulcer recurrence rates and morbidity and mortality rates in randomized and nonrandomized prospective trials of patients with primary PUD and retrospective case series of patients undergoing remedial operative procedures after various failed acid-reducing operations.
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Úlcera Duodenal/diagnóstico , Úlcera Duodenal/terapia , Antiinflamatorios no Esteroideos/efectos adversos , Antiinflamatorios no Esteroideos/uso terapéutico , Úlcera Duodenal/epidemiología , Úlcera Duodenal/etiología , Infecciones por Helicobacter/epidemiología , Helicobacter pylori/aislamiento & purificación , Humanos , Incidencia , Complicaciones Posoperatorias/epidemiología , Ensayos Clínicos Controlados Aleatorios como Asunto , Recurrencia , VagotomíaRESUMEN
BACKGROUND: Adherens junctions (AJ), by their association with the endothelial cytoskeleton, maintain microvascular barrier integrity. Phosphorylation states of AJ proteins, such as vascular endothelial (VE) cadherin, can potentially alter the interactions between component AJ proteins. Furthermore, AJ protein phosphorylation is susceptible to regulation by inflammatory mediators. We previously demonstrated the importance of VE cadherin in tumor necrosis factor (TNF)-mediated endothelial permeability. We now postulate that TNF-induced endothelial permeability is associated with tyrosine phosphorylation of VE cadherin. METHODS: Confluent monolayers of human umbilical vein endothelial cells were exposed to saline solution, TNF-alpha (100 U/mL) or TNF and the Src-tyrosine kinase inhibitor, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine. Permeability was assessed by fluorescein isothiocyanate-dextran flux. VE-cadherin phosphorylation was determined by immunoprecipitation and immunoblotting with antiphosphotyrosine antibody. Data are expressed as mean +/- SEM and analyzed by analysis of variance. RESULTS: TNF-alpha-induced tyrosine phosphorylation of VE cadherin and increased intercellular gap formation. These changes were associated with increased endothelial-cell monolayer permeability, all of which were prevented by 4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine. Exposure to an inactive tyrphostin, AG9 (negative control), did not prevent TNF-induced endothelial permeability. CONCLUSIONS: We conclude that tyrosine phosphorylation of VE cadherin is an important regulatory pathway associated with TNF-induced endothelial barrier dysfunction. Modulating AJ protein phosphorylation may provide targets for therapy during inflammation.
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Cadherinas/metabolismo , Permeabilidad Capilar/fisiología , Dextranos/farmacocinética , Endotelio Vascular/metabolismo , Fluoresceína-5-Isotiocianato/farmacocinética , Tirosina/metabolismo , Antígenos CD , Antineoplásicos/farmacología , Permeabilidad Capilar/efectos de los fármacos , Células Cultivadas , Endotelio Vascular/citología , Fluoresceína-5-Isotiocianato/análogos & derivados , Humanos , Fosforilación , Pirimidinas/farmacología , Factor de Necrosis Tumoral alfa/farmacología , Venas Umbilicales/citología , Familia-src Quinasas/antagonistas & inhibidoresRESUMEN
INTRODUCTION: The mechanism by which hypertension is maintained in renovascular hypertension remains poorly defined. Because plasma angiotensin II does not correlate with blood pressure in RVH, we postulated that activation of tissue-specific autocrine-paracrine renin-angiotensin systems may upregulate local production of angiotensin II and maintain hypertension in chronic RVH. METHODS: RVH was induced with a two-kidney one-clip (2K1C) rat model. Animals were killed at 1 or 12 weeks after surgery (acute or chronic RVH). Angiotensin II was quantitated with radioimmunoassay. Angiotensin II-type 1 (AT(1)) receptor density was determined with immunoblotting and immunohistochemistry. RESULTS: Blood pressure was significantly elevated in 2K1C animals compared with sham animals at 1 week (141 +/- 5 mm Hg versus 98 +/- 3 mm Hg; P <.0005) and at 12 weeks (164 +/- 14 mm Hg versus 110 +/- 7 mm Hg; P <.0005) after surgery. No significant difference was seen in plasma angiotensin II levels between 2K1C and control animals during acute (38.2 +/- 6.5 fmol/mL versus 27.6 +/- 6.8 fmol/mL; P = not significant) or chronic (40.1 +/- 17.4 fmol/mL versus 27.1 +/- 6.5 fmol/mL; P = not significant) RVH. During acute RVH, intrarenal angiotensin II was significantly increased in both the clipped (126.0 +/- 16.2 fmol/g versus 62.0 +/- 6.2 fmol/g; P <.005) and unclipped (78.9 +/- 6.3 fmol/g versus 39.9 +/- 2.5 fmol/g; P <.05) kidneys of 2K1C animals compared with control animals. Increased intrarenal angiotensin II levels persisted in chronic RVH in the clipped (147.4 +/- 37.7 fmol/g versus 59.2 +/- 8.7 fmol/g; P <.05) and unclipped (130.8 +/- 31.8 fmol/g versus 63.0 +/- 11.0 fmol/g; P <.05) kidneys of 2K1C animals compared with controls. Adrenal angiotensin II content of 2K1C animals was unchanged in acute RVH (493.7 +/- 51.4 fmol/g versus 522.6 +/- 80.5 fmol/g; P = not significant) but increased nearly three-fold over control animals during chronic RVH (1129.0 +/- 149.3 fmol/g versus 400.6 +/- 59.1 fmol/g; P <.0005). No significant difference in AT(1) receptor density was noted in renal tubules of clipped and unclipped kidneys or in the adrenal glands of 2K1C animals during acute or chronic RVH compared with control animals. CONCLUSION: Tissue angiotensin II production is upregulated in the kidneys and adrenal glands in chronic RVH, and AT(1) receptor density is maintained in these tissues, providing a potential mechanism for maintenance of hypertension in RVH.
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Glándulas Suprarrenales/metabolismo , Hipertensión Renovascular/fisiopatología , Riñón/metabolismo , Receptores de Angiotensina/metabolismo , Sistema Renina-Angiotensina/fisiología , Regulación hacia Arriba , Angiotensina II/metabolismo , Animales , Comunicación Autocrina , Enfermedad Crónica , Modelos Animales de Enfermedad , Inmunohistoquímica , Túbulos Renales Proximales/metabolismo , Radioinmunoensayo , Distribución Aleatoria , Ratas , Ratas Sprague-DawleyRESUMEN
This study examines the hypotheses that TNF-alpha causes a dose-dependent increase in the microvascular permeability of ex vivo buffer perfused lungs that is quantitatively similar to that caused by lipopolysaccharide (LPS) or thromboxane A2 (TxA2). We also postulated that TNF-alpha potentiates the effect of interleukin-1beta (IL-1beta) or TxA2 receptor activation on pulmonary microvascular permeability. Lungs harvested from Wistar rats were perfused ex vivo with Krebs-Henseleit buffer containing 0, 10, 100, or 1000 ng/mL recombinant rat TNF-alpha. Twenty minutes later pulmonary microvascular permeability was determined by measuring the capillary filtration coefficient (Kf) using a gravimetric technique. The effect of TNF-alpha (100 ng/mL) on pulmonary Kf was compared with that of lungs exposed to LPS (400 microg/mL; E. coli 0111:B4) or a TxA2 receptor agonist (U-46619; 7 x 10(-8)). In other experiments, perfused lungs were exposed to TNF-alpha plus IL-1beta (1 ng/mL) or TNF-alpha plus U-46619 after which Kf was measured. Exposure of ex vivo buffer perfused lungs to 10-1000 ng/mL TNF-alpha had no effect on Kf whereas LPS and U-46619 was associated with a two- and six-fold increase in Kf, respectively (P < 0.05). The Kf of lungs exposed to TNF-alpha plus IL-1 was similar to that of lungs exposed to TNF-alpha alone. Lastly, the Kf of lungs exposed to TNF-alpha plus U-46619 was not different than that of lungs exposed to U-46619 alone. In conclusion, TNF-alpha at least when administered for a relatively brief period of time does not affect microvascular permeability in an isolated, buffer-perfused lung model.
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Permeabilidad Capilar/efectos de los fármacos , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacología , Animales , Bronquios/metabolismo , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Técnicas In Vitro , Interleucina-1/farmacología , Pulmón/irrigación sanguínea , Perfusión , Ratas , Ratas Wistar , Receptores de Tromboxanos/efectos de los fármacos , Receptores de Tromboxanos/metabolismo , Tromboxano A2/metabolismo , Tromboxano A2/farmacología , Factor de Necrosis Tumoral alfa/metabolismo , Factor de Necrosis Tumoral alfa/farmacocinéticaRESUMEN
The mechanism by which tumor necrosis factor alpha (TNFalpha) increases endothelial permeability is unclear. Vascular endothelial (VE) cadherin (cadherin 5) is an important contributor to endothelial monolayer integrity. The purpose of our study was to determine the effect of TNFalpha on VE-cadherin cell-surface expression and to identify the signaling pathways involved in TNF-induced changes in cadherin expression. Human umbilical vein endothelial cell monolayer permeability was measured by enzyme-linked immunosorbent assay for biotin-labeled albumin. Immunofluorescence, laser confocal microscopy, and Western immunobloting were used to assess VE cadherin distribution. Mitogen-activated protein kinase (MAPK) activity was determined using functional kinase assays and was inhibited with the compounds SB202190 and PD98059. TNFalpha significantly increased permeability and induced p38 and ERK MAPK activation compared with controls (P < 0.05). These changes were associated with a loss of membrane-associated VE cadherin. Inhibition of p38 but not ERK MAPK significantly reduced the effect of TNFalpha on endothelial permeability and cell-surface VE cadherin expression. p38 MAP kinase activation appears to be an important upstream signaling event associated with increased endothelial permeability and vascular endothelial cadherin redistribution.
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Cadherinas/metabolismo , Permeabilidad Capilar/fisiología , Endotelio Vascular/fisiología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Antígenos CD , Cadherinas/efectos de los fármacos , Permeabilidad Capilar/efectos de los fármacos , Membrana Celular , Células Cultivadas , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Flavonoides/farmacología , Humanos , Imidazoles/farmacología , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas Activadas por Mitógenos/efectos de los fármacos , Piridinas/farmacología , Transducción de Señal , Factor de Necrosis Tumoral alfa/metabolismo , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
Even in the absence of inhalational injury, acute lung injury is a common cause of morbidity and mortality for patients sustaining severe burns. Other than general supportive measures, there are few therapeutic options for improving survival in these critically ill patients. Numerous clinical and laboratory studies have implicated tumor necrosis factor (TNF)-a and neutrophils as important participants in the pathogenesis of burn-induced lung injury. There is, however, little information regarding the mechanism by which these and other pro-inflammatory mediators affect the movement of fluid and protein across the microvascular barrier into the interstitium of the lung. In addition to reviewing the evidence implicating TNF-a and neutrophils in the pathophysiology of burn-induced lung injury, this report summarizes current theories regarding potential mechanisms by which these mediators may alter microvascular barrier function to lead ultimately to the development of pulmonary edema.