RESUMEN
Although intravenous Escherichia coli endotoxin has been used extensively in experimental studies to increase lung endothelial permeability, the effect of E. coli endotoxin on lung epithelial permeability has not been well studied. To examine this issue in sheep, bidirectional movement of protein across the lung epithelial barrier was studied by labeling the vascular space with 131I-albumin and by instilling 3 ml/kg of an isosmolar protein solution with 125I-albumin into the alveoli. E. coli endotoxin was administered according to one of three protocols: intravenous alone (5-500 micrograms/kg), intravenous (5 micrograms/kg) plus low-dose alveolar endotoxin (10 micrograms/kg), and high-dose alveolar endotoxin alone (50-100 micrograms/kg). Alveolar liquid clearance was estimated based on the concentration of the instilled native protein. Sheep were studied for either 4 or 24 h. Although intravenous E. coli endotoxin produced a marked increase in transvascular protein flux and interstitial pulmonary edema, there was no effect on the clearance of either the vascular (131I-albumin) or the alveolar (125I-albumin) protein tracer across the epithelial barrier. High-dose alveolar E. coli endotoxin caused a 10-fold increase in the number of leukocytes, particularly neutrophils, that accumulated in the air spaces. In spite of the marked chemotactic effect of alveolar endotoxin, there was no change in the permeability of the epithelial barrier to the vascular or alveolar protein tracers. Also, alveolar epithelial liquid clearance was normal. Morphologic studies confirmed that the alveolar epithelial barrier was not injured by either intravenous or alveolar E. coli endotoxin. Thus, the alveolar epithelium in sheep is significantly more resistant than the lung endothelium to the injurious effects of E. coli endotoxin.
Asunto(s)
Endotelio/metabolismo , Endotoxinas/toxicidad , Escherichia coli/metabolismo , Pulmón/metabolismo , Albúminas/metabolismo , Animales , Quimiotaxis de Leucocito/efectos de los fármacos , Epitelio/metabolismo , Espacio Extracelular/efectos de los fármacos , Espacio Extracelular/metabolismo , Femenino , Hemodinámica/efectos de los fármacos , Permeabilidad , Alveolos Pulmonares/metabolismo , Edema Pulmonar/etiología , Edema Pulmonar/prevención & control , OvinosRESUMEN
We studied anesthetized sheep to determine the relationship between increased permeability pulmonary edema and the development and mechanism of pleural effusion formation. In 12 sheep with intact, closed thoraces, we studied the time course of pleural liquid formation after 0.12 ml/kg i.v. oleic acid. After 1 h, there were no pleural effusions, even though extravascular lung water increased 50% to 6.0 +/- 0.7 g/g dry lung. By 3 h pleural effusions had formed, they reached a maximum at 5 h (48.5 +/- 16.9 ml/thorax), and at 8 h there was no additional accumulation of pleural liquid (45.5 +/- 16.9 ml). Morphologic studies by light and electron microscopy demonstrated subpleural edema but no detectable injury to the visceral pleura, suggesting that the pleural liquid originated from the lung and not the pleura. In nine sheep, we quantified the rate of formation of pleural liquid by enclosing one lung in a plastic bag. By comparing in the same sheep the volume of pleural liquid collected from the enclosed lung to the volume found in the opposite intact chest, we estimated the rate of liquid absorption from the intact chest to be 0.32 ml/(kg.h); we had previously reported a liquid absorption rate of 0.28 ml/(kg.h) in normal sheep. These studies also supported the conclusion that the majority of the pleural liquid originated from the lung because we could account for all of the pleural liquid that was formed and cleared. The volume of pleural liquid collected from the enclosed lungs was equal to 21% of the excess lung liquid that formed after oleic acid-induced lung injury. Thus, the pleural space and parietal pleural lymphatic pathways are important pathways for the clearance of pulmonary edema liquid after experimentally induced increased permeability pulmonary edema.
Asunto(s)
Permeabilidad Capilar , Derrame Pleural/fisiopatología , Edema Pulmonar/fisiopatología , Animales , Agua Corporal/fisiología , Femenino , Hemodinámica , Ácido Oléico , Ácidos Oléicos/administración & dosificación , Pleura/ultraestructura , Derrame Pleural/metabolismo , Derrame Pleural/patología , Proteínas/metabolismo , Alveolos Pulmonares/irrigación sanguínea , Alveolos Pulmonares/fisiopatología , Alveolos Pulmonares/ultraestructura , Edema Pulmonar/metabolismo , Edema Pulmonar/patología , OvinosRESUMEN
The cardioprotective effects of an mAb to P-selectin designated mAb PB1.3 was examined in a feline model of myocardial ischemia (MI) and reperfusion. PB1.3 (1 mg/kg), administered after 80 min of ischemia (i.e., 10 min before reperfusion), significantly attenuated myocardial necrosis compared to a non-blocking mAb (NBP1.6) for P-selectin (15 +/- 3 vs 35 +/- 3% of area at risk, P < 0.01). Moreover, endothelial release of endothelium derived relaxing factor, as assessed by relaxation to acetylcholine, was also significantly preserved in ischemic-reperfused coronary arteries isolated from cats treated with mAb PB1.3 compared to mAb NBP1.6 (67 +/- 6 vs 11 +/- 3, P < 0.01). This endothelial preservation was directly related to reduced endothelial adherence of PMNs in ischemic-reperfused coronary arteries. Immunohistochemical localization of P-selectin was significantly upregulated in the cytoplasm of endothelial cells that lined coronary arteries and veins after 90 min of ischemia and 20 min of reperfusion. The principal site of intracytoplasmic expression was in venous vessels. mAb PB1.3 significantly decreased (P < 0.01) adherence of unstimulated PMNs to thrombin and histamine stimulated endothelial cells in a concentration-dependent manner in vitro. These results demonstrate that PMN adherence to endothelium by P-selectin is an important early consequence of reperfusion injury, and a specific monoclonal antibody to P-selectin exerts significant endothelial preservation and cardioprotection in myocardial ischemia and reperfusion.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Moléculas de Adhesión Celular/metabolismo , Endotelio Vascular/efectos de los fármacos , Corazón/fisiopatología , Daño por Reperfusión Miocárdica/tratamiento farmacológico , Glicoproteínas de Membrana Plaquetaria/metabolismo , Animales , Plaquetas/metabolismo , Gatos , Adhesión Celular/efectos de los fármacos , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/inmunología , Moléculas de Adhesión Celular/aislamiento & purificación , Electrofisiología , Endotelio Vascular/patología , Citometría de Flujo , Regulación de la Expresión Génica , Hemodinámica , Inmunohistoquímica , Masculino , Pruebas de Neutralización , Neutrófilos/metabolismo , Selectina-P , Agregación Plaquetaria/efectos de los fármacos , Glicoproteínas de Membrana Plaquetaria/genética , Glicoproteínas de Membrana Plaquetaria/inmunología , Glicoproteínas de Membrana Plaquetaria/aislamiento & purificaciónRESUMEN
Intrauterine growth retardation (IUGR) increases the risk of neuroendocrine reprogramming. In the rat, IUGR leads to persistent changes in cerebral mRNA levels. This suggests lasting alterations in IUGR cerebral transcriptional regulation, which may result from changes in chromatin structure. Candidate nutritional triggers for these changes include altered cerebral zinc and one-carbon metabolite levels. We hypothesized that IUGR affects cerebral chromatin structure in neonatal and postnatal rat brains. Rats were rendered IUGR by bilateral uterine artery ligation; controls (Con) underwent sham surgery. At day of life 0 (d0), we measured cerebral DNA methylation, histone acetylation, expression of chromatin-affecting enzymes, and cerebral levels of one-carbon metabolites and zinc. At day of life 21 (d21), we measured cerebral DNA methylation and histone acetylation, as well as the caloric content of Con and IUGR rat breast milk. At d0, IUGR significantly decreased genome-wide and CpG island methylation, as well as increased histone 3 lysine 9 (H3/K9) and histone 3 lysine 14 (H3/K14) acetylation in the hippocampus and periventricular white matter, respectively. IUGR also decreased expression of the chromatin-affecting enzymes DNA methyltransferase 1 (DNMT1), methyl-CpG binding protein 2 (MeCP2), and histone deacetylase (HDAC)1 in association with increased cerebral levels of zinc. In d21 female IUGR rats, cerebral CpG DNA methylation remained lower, whereas H3/K9 and H3/K14 hyperacetylation persisted in hippocampus and white matter, respectively. In d21 male rats, IUGR decreased acetylation of H3/K9 and H3/K14 in these respective regions compared with controls. Despite these differences, caloric, fat, and protein content were similar in breast milk from Con and IUGR dams. We conclude that IUGR results in postnatal changes in cerebral chromatin structure and that these changes are sex specific.
Asunto(s)
Encéfalo/enzimología , Cromatina/química , Epigénesis Genética , Retardo del Crecimiento Fetal/enzimología , Insuficiencia Placentaria/enzimología , Acetilación , Animales , Animales Recién Nacidos , Encéfalo/ultraestructura , Cromatina/genética , Cromatina/metabolismo , Islas de CpG , ADN/genética , ADN/metabolismo , ADN (Citosina-5-)-Metiltransferasa 1 , ADN (Citosina-5-)-Metiltransferasas/genética , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Metilación de ADN , Femenino , Retardo del Crecimiento Fetal/genética , Histona Desacetilasas/genética , Histona Desacetilasas/metabolismo , Histonas/metabolismo , Inmunohistoquímica , Masculino , Proteína 2 de Unión a Metil-CpG/genética , Proteína 2 de Unión a Metil-CpG/metabolismo , Estructura Molecular , Neuronas/enzimología , Neuronas/ultraestructura , Insuficiencia Placentaria/genética , Embarazo , ARN Mensajero/metabolismo , Ratas , Factores Sexuales , Zinc/metabolismoRESUMEN
Pharmacologic study is needed in the extremely immature newborns who currently survive. Study is needed of both the drug treatment previously established in more mature neonates and of novel drug therapy. Carefully controlled studies are needed to identify accurately both beneficial and harmful drug therapy and the mechanisms of that toxicity. Careful pharmacologic study of drug disposition and its mechanisms might lead to dosing paradigms or patient selection that minimize toxicity and maximize efficacy. In vivo, translational models of neonatal diseases are limited, but can be used to identify novel treatments and study mechanisms of established, successful therapy. Findings from such studies can generate hypotheses for study in humans leading to a continuing scientific interchange from bedside to bench to bedside. Similarly, clinical observations can generate hypotheses for study in translational models where more invasive analyses are possible. Specific areas of drug treatment should focus on neonatal disorders with long-term, adverse outcomes, such as chronic lung disease, that is amenable to translational study with animal models. National data show a progressive decrease in the clinician-scientist pool entering biomedical research. The future of neonatal pharmacology studies requires an increase in training programs for the physician-scientist whose clinical education in neonatology can be complemented by rigorous basic-science training. Success as a clinician-scientist will require collaboration with full-time basic scientists who can continue studies during periods of clinical work and provide critical study methodology to the overall study design. Such a work environment must be supported by academic institutions and may require more flexibility in the promotion and tenure schedule and process, such as the nature of what it rewards. To complement this, the NIH could modify its grant reporting process to identify co-investigators in studies who may provide unique input to the study concepts and design, such as clinical correlations or clinical investigations.
Asunto(s)
Modelos Animales de Enfermedad , Enfermedades del Recién Nacido/tratamiento farmacológico , Neonatología , Farmacología , Animales , Investigación Biomédica , Humanos , Recien Nacido con Peso al Nacer Extremadamente Bajo , Recién Nacido , Neonatología/educación , Farmacología/educación , Apoyo a la Investigación como AsuntoRESUMEN
Non-invasive respiratory support is increasingly used in lieu of intubated ventilator support for the management of neonatal respiratory failure, particularly in very low birth weight infants at risk for bronchopulmonary dysplasia. The optimal approach and mode for non-invasive support remains uncertain. This article reviews the application of high-frequency ventilation for non-invasive respiratory support in neonates, including basic science studies on mechanics of gas exchange, animal model investigations, and a review of current clinical use in human neonates.
Asunto(s)
Displasia Broncopulmonar/terapia , Ventilación de Alta Frecuencia/métodos , Ventilación no Invasiva/métodos , Insuficiencia Respiratoria/terapia , Humanos , Recién NacidoRESUMEN
To study neutrophil circulation time and trafficking in vivo requires labeling the cells so that their movement can be followed temporally and spatially. Labeling procedures used to date, however, have relied on ex vivo separation and labeling methods, an undesired consequence of which may be neutrophil activation. Moreover, the labeled cells preferentially stick in the pulmonary circulation for several hours upon reinjection into the host. Therefore, we devised an alternate labeling procedure that relied on intra-arterial infusion of the fluorescent phagocytic cell linker PKH26. We infused PKH26 (5.5 x 10(-6) M for 1 h) into six awake sheep and measured systemic and pulmonary hemodynamics and lung lymph dynamics continuously for 3-4 h after the infusion. We collected simultaneous arterial and venous blood samples hourly for 4 h, and again 24 h after the infusion ended, for white blood cell and neutrophil differential counts and to identify labeled cells in fresh smears by fluorescence and differential interference contrast (DIC) microscopy. Infusion of PKH26 had minimal and transient physiologic effects on systemic and pulmonary artery pressure, lung lymph flow, and leukocyte counts. Labeled cells were present in venous blood after the infusion for at least 24 h. DIC microscopy observation of the blood smears indicated that the fluorescently labeled cells were exclusively neutrophils. Unstimulated superoxide anion release from neutrophils isolated 2 and 24 h after PKH26 infusion was not different from baseline release. Phorbol myristate acetate-stimulated release was not affected either. The labeled neutrophils responded to chemoattractants by migrating to extravascular sites. Our results indicate that neutrophils can be selectively labeled in vivo, after which trafficking of the labeled neutrophils can be determined.
Asunto(s)
Colorantes Fluorescentes , Neutrófilos/fisiología , Compuestos Orgánicos , Animales , Fluorescencia , Masculino , Ovinos , Superóxidos/metabolismo , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
A short-term bone marrow culture was established to determine the functional and proliferative responses of neutrophils sampled from sheep exposed to endotoxin in vivo. Iliac crest bone marrow was drawn from surgical control animals and from sheep given a 12-h infusion of endotoxin. In these bone marrow aspirates, neutrophils from endotoxin-treated sheep showed primed superoxide anion release compared with controls when stimulated with phorbol myristate acetate. Priming was no longer present in neutrophils tested after 2 days in culture. The neutrophil cell count, however, was higher after 2 days in culture than in controls. Increased neutrophil production/viability was reproduced in naive bone marrow cultures by replacing part of the normal serum medium with plasma taken from sheep several hours after endotoxin infusion. These data suggest that a dissociation exists between priming and increased neutrophil production/viability in response to endotoxin.
Asunto(s)
Médula Ósea/efectos de los fármacos , Médula Ósea/metabolismo , Endotoxinas/farmacología , Neutrófilos/citología , Neutrófilos/efectos de los fármacos , Superóxidos/metabolismo , Animales , Células de la Médula Ósea , Supervivencia Celular , Células Cultivadas , Recuento de Leucocitos/efectos de los fármacos , Modelos Biológicos , Neutrófilos/metabolismo , Ovinos , Estimulación Química , Acetato de Tetradecanoilforbol/farmacología , Factores de TiempoRESUMEN
The time course of endothelial P-selectin, ICAM-1, and E-selectin expression was studied in a feline model of myocardial ischemia and reperfusion. Cats were subjected to 90 min of myocardial ischemia followed by 0, 10, 20, 60, 150, or 270 min of reperfusion. At the end of reperfusion, the coronary vasculature was examined immunohistochemically to localize monoclonal antibodies (mAbs) PB1.3, RR1/1, and Cy1787 directed against P-selectin, ICAM-1, and E-selectin, respectively. Immunohistochemical localization for P-selectin, recognized by mAb PB1.3, was maximally expressed 20 min after reperfusion in 60 +/- 6% of coronary venules (P < 0.05 compared to non-reperfused controls), and covered 59 +/- 3% of the endothelial cell perimeter of immunostained coronary venules. Immunolocalization of mAb PB1.3 gradually declined at 60, 150, and 270 min of reperfusion. Immunohistochemical localization of mAb RR1/1 (anti-ICAM-1) in endothelial cells of coronary venules was observed to a modest extent in non-ischemic myocardium and at 10, 20, and 60 min of reperfusion, but was significantly increased following 150 and 270 min of reperfusion (P < 0.05 compared non-reperfused controls). At 270 min post-reperfusion, mAb RR1/1 was seen in 50 +/- 4% of coronary venules. Endothelial immunolocalization of mAb Cy1787 (anti-E-selectin) was only observed in 13 +/- 1 and 14 +/- 3% of coronary venules after 150 and 270 min of reperfusion, respectively, suggesting that pronounced expression of E-selectin does not occur within 270 min after reperfusion. These results demonstrate sequential expression of three major endothelial cell adherence molecules in situ following myocardial ischemia and reperfusion. The timing of endothelial cell expressed P-selectin and ICAM-1 could coordinate neutrophil trafficking during the early stages of reperfusion.
Asunto(s)
Moléculas de Adhesión Celular/biosíntesis , Molécula 1 de Adhesión Intercelular/biosíntesis , Isquemia Miocárdica/metabolismo , Daño por Reperfusión Miocárdica/metabolismo , Glicoproteínas de Membrana Plaquetaria/biosíntesis , Animales , Anticuerpos Monoclonales/inmunología , Gatos , Moléculas de Adhesión Celular/análisis , Creatina Quinasa/sangre , Selectina E , Endotelio Vascular/química , Endotelio Vascular/metabolismo , Endotelio Vascular/patología , Inmunohistoquímica , Molécula 1 de Adhesión Intercelular/análisis , Masculino , Isquemia Miocárdica/patología , Daño por Reperfusión Miocárdica/patología , Miocardio/química , Miocardio/metabolismo , Miocardio/patología , Neutrófilos/patología , Selectina-P , Glicoproteínas de Membrana Plaquetaria/análisis , Factores de TiempoRESUMEN
Intravenous infusion of zymosan-activated plasma (ZAP) in sheep results in acute lung injury mediated in part by free radical release from stimulated neutrophils that are retained in increased numbers in the pulmonary microcirculation. ZAP infusion is also associated with long-term reduction in elicited superoxide anion generation from segmented neutrophils in the circulating and bone marrow pools for at least 24 h after the infusion. The purpose of our study was to investigate the effect of ZAP infusion on leukocyte counts and neutrophil granule-associated enzyme activity in the circulating and bone marrow pools. Cytochemical methods were used to characterize enzyme activity in primary granules (acid phosphatase and myeloperoxidase) and secondary granules (alkaline phosphatase). During the infusion period, neutrophil differential counts decreased less in venous blood than in matched arterial blood. Release from bone marrow stores was evident as increased numbers of circulating band neutrophils during and after the infusion. Neutrophils in venous and arterial blood smears were degranulated acutely during and 1-3 h after the infusion was stopped. Band and segmented neutrophils in bone marrow also appeared slightly degranulated 1-2 h after the infusion. In vitro experiments showed that band and segmented neutrophils in bone marrow degranulated in response to ZAP incubation. Immature polymorphonuclear leukocytes did not degranulate. Five to 24 h after ZAP infusion, enzyme activity in circulating and bone marrow neutrophils was at baseline levels, suggesting that new cells were being released into the circulating pool because degranulated neutrophils do not synthesize new granules. Another indication of a long-term effect in bone marrow were slight decreases in the percentage of immature polymorphonuclear leukocytes and band neutrophils and a significant decrease in the percentage of eosinophils, all of which coincided with a significant increase in the percentage of segmented neutrophils. Our results demonstrate that circulating complement anaphylatoxins affect neutrophils in bone marrow as well as the vascular space.
Asunto(s)
Neutrófilos/inmunología , Ovinos/inmunología , Zimosan/administración & dosificación , Anestesia , Animales , Células de la Médula Ósea , Degranulación de la Célula , Gránulos Citoplasmáticos/enzimología , Recuento de Leucocitos , Neutrófilos/enzimologíaRESUMEN
Phorbol myristate acetate (PMA, 10(-7) M) activation of adherent neutrophils (PMNs) led to a markedly attenuated release of superoxide anion (O2-) per cell when PMNs were activated at high density (2.85 fmol O2-/PMN at 2 million in 0.1 ml) in comparison with cells activated at low cell density (12.0 fmol O2-/PMN at 250,000 in 0.1 ml). This "autoregulatory" phenomenon was not due to a defect in the superoxide anion assay employed, to a differential adherence of neutrophils at high vs. low density, or to substrate (cytochrome c) or cell stimulus (PMA) limitation. It was associated with an inhibition of apparent NADPH oxidase activity and a leftward shift (toward a lower level of activation) in the activation profile of PMNs (as determined by FACS analysis using PMNs preloaded with 2'7'-dichlorofluorescin diacetate in which H2O2 production results in the production of the fluorescent product 2'7'-dichlorofluorescein intracellularly). Other aspects of the neutrophil activation response including arachidonic acid mobilization, phospholipid metabolism, and perhaps phosphatidylinositol turnover were also attenuated when PMNs were activated at high cell density. Studies with cells in solution, cells treated with cycloheximide, and cells treated with 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid suggest that PMN contact with a surface, neutrophil protein synthesis, and an increased surface expression of the heterodimer CD11b/CD18 on PMNs all were not required for autoregulation. Finally, morphometric and morphologic examination of PMNs activated at low vs. high density revealed histologic and structural correlates associated with the attenuated PMN activation response of cells triggered at high cell density. We conclude that multiple structural and functional aspects of the PMN activation response are modulated by cell density and suggest that this property is important both in the physiologic control of neutrophil activation and in the design of in vitro assays of the neutrophil activation response.
Asunto(s)
Homeostasis/fisiología , Neutrófilos/fisiología , Ácido Araquidónico , Ácidos Araquidónicos/metabolismo , Recuento de Células , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Citometría de Flujo , Homeostasis/efectos de los fármacos , Humanos , Peróxido de Hidrógeno/metabolismo , Inflamación/patología , Microscopía Electrónica , NADH NADPH Oxidorreductasas/metabolismo , NADPH Oxidasas , Neutrófilos/metabolismo , Neutrófilos/ultraestructura , Superóxidos/metabolismo , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
Endothelial cell dysfunction and cardiac myocyte injury resulting from ischemia and reperfusion have been associated with accumulation of neutrophils in the myocardium. To determine whether the accumulation is related primarily to intravascular sequestration or extravascular infiltration of neutrophils during the early period of reperfusion, we morphometrically quantified the tissue distribution of neutrophils in cats and dogs. At the end of the reperfusion period, the base of the heart was cross-clamped to preserve neutrophil location at the moment of death. Point-counting methods were used to determine the distribution of neutrophils inside and outside coronary arterioles and venules (< or = 100 microns in diameter) as well as coronary capillaries 5-10 microns in diameter in 0.5-microns-thick, plastic-embedded sections. Ischemia-reperfusion resulted in a threefold increase in neutrophil number in the lumen of arterioles and venules at 60 min of reperfusion and up to a sevenfold increase at 270 min of reperfusion (P < .05) compared to time-matched control nonischemic hearts. The ratio of intravascular neutrophils in venules to arterioles was 2:1. Intracapillary neutrophils increased, but not significantly, at 60 min of reperfusion. At 270 min of reperfusion, intracapillary neutrophils increased 11-fold (P < .05). The percentage of total neutrophils that accumulated outside arterioles and venules in cat hearts was 8% at 60 min of reperfusion (not significant, NS) and 28% at 270 min of reperfusion (P < .05). In dog hearts, the percentages were 26% (NS) and 44% (P < .05), respectively. The percentage of total neutrophils that accumulated outside capillaries was < 6% in both cat and dog hearts (NS). The combination of rapid intravascular sequestration, delayed extravascular infiltration, and low incidence of neutrophil-cardiac myocyte contact in situ in these two species suggests that neutrophil-mediated cardiac myocyte injury during early reperfusion may initially depend on diffusion of inflammatory mediators and subsequently require direct contact between neutrophils and cardiac myocytes.
Asunto(s)
Isquemia Miocárdica/patología , Daño por Reperfusión Miocárdica/patología , Neutrófilos/patología , Animales , Recuento de Células Sanguíneas , Gatos , Movimiento Celular/fisiología , Perros , Corazón/fisiología , Masculino , Isquemia Miocárdica/fisiopatología , Reperfusión Miocárdica , Daño por Reperfusión Miocárdica/fisiopatología , Miocardio/patología , Neutrófilos/fisiologíaRESUMEN
Experiments were designed to reexamine the relationship between extracellular calcium and superoxide generation in phorbol myristate acetate (PMA) stimulated neutrophils exploiting a newly adapted method to measure superoxide anion (O2-) generation from adherent cells stimulated at high and low cell density. Human neutrophils were plated in microtiter wells in cell densities of either 0.2 or 2.0 million cells/well. Superoxide release was measured sequentially over 60 min by reduction of ferricytochrome c. Cells were maintained in 1 mM Ca++ or 0 mM Ca++ Hanks' buffer for 60 min prior to activation as well as during measurement of O2-. In 1 mM Ca++, 2.0 million adherent neutrophils released 10.7 +/- 1.2 nmol O2- in 20 min (n = 4). O2- release was not significantly different for high density cells incubated and stimulated in 0 mM Ca++. In the presence of 1 mM Ca++, 0.2 million adherent neutrophils released 6.3 +/- 0.5 nmols O2- in 20 min. With cells stimulated at low density, PMA stimulated O2- release was significantly decreased (3.0 +/- 0.6 nmol O2- in 20 min) as was the initial rate of secretion of O2- in the absence of extracellular calcium. Basal release of superoxide was also greater in the presence of 1 mM Ca++ (0.96 nmol/20 min) compared to basal release in 0 mM Ca++ (0.22 nmol/20 min). Additional experiments with 0.2 million cells/well showed that extracellular Ca++ was required during stimulation with PMA and that prior incubation of cells for up to 60 min in 0 mM Ca++ had no effect on O2- release measured in the presence of calcium. Furthermore, PMA stimulated O2- was independent of verapamil (10(-5)-10(-7) M), suggesting that voltage-dependent calcium channels do not participate in this response. The planar areas for unstimulated neutrophils in 0 mM Ca++ increased after addition of PMA. Unstimulated cells in 1 mM Ca++ tended to be larger and planar areas did not increase after PMA. These studies demonstrate that PMA stimulated O2- secretion is dependent on extracellular calcium particularly when adherent neutrophils are stimulated at low cell density. Furthermore, extracellular calcium at a concentration of 1 mM primes neutrophils by increasing basal secretion of O2- and increasing superoxide release after a maximum stimulating dose of PMA.
Asunto(s)
Calcio/farmacología , Neutrófilos/metabolismo , Superóxidos/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Calcio/fisiología , Recuento de Células , Humanos , Neutrófilos/citología , Neutrófilos/efectos de los fármacosRESUMEN
Plasma levels of tumor necrosis factor-alpha (TNF-alpha) peak between 2 and 4 h during a 12-h continuous infusion of endotoxin in awake sheep. We hypothesized that a source of this TNF-alpha is the pool of leukocytes that accumulate in the pulmonary circulation. To test this hypothesis, we physiologically monitored six anesthetized sheep during baseline and 4-h endotoxin infusion periods (10 ng/kg x min). We obtained open-lung biopsies at baseline and at 20 min and 2 and 4 h during the endotoxin infusion period for immunohistochemical localization of TNF-alpha. The plasma concentration of TNF-alpha increased from an average baseline concentration of 0.06 +/- 0.03 ng/ml (mean +/- SD) to a peak of 1.40 +/- 0.28 ng/ml at 2 h of the endotoxin infusion. We observed increased cytoplasmic TNF-alpha immunoreactivity in situ among neutrophils and intravascular mononuclear phagocytes during the endotoxin infusion compared with baseline. Also, the number of immunopositive leukocytes increased in the pulmonary circulation during the continuous infusion of endotoxin. We conclude that TNF-alpha-producing leukocytes accumulate in the pulmonary circulation during endotoxemia. These cells probably contribute to both the rise in the circulating levels of TNF-alpha and the development of acute lung injury.
Asunto(s)
Endotoxinas/toxicidad , Leucocitos/metabolismo , Pulmón/citología , Factor de Necrosis Tumoral alfa/biosíntesis , Anestesia , Animales , Western Blotting , Inmunohistoquímica , Pulmón/metabolismo , Ovinos , Factor de Necrosis Tumoral alfa/análisisRESUMEN
As techniques evolve that allow molecular characterization of disease processes such as cancer, definition of "normal" at a molecular level becomes increasingly important. Increasingly large numbers of mutations are found at the genomic level, but whether all of those mutations contribute to the malignant state of a carcinoma cell is not clear. Without knowledge of what constitutes normality on the proteomic level in an organ or cell, we cannot determine what genomic changes are physiologically important. Traditionally, colon cancer is identified and classified by histological criteria. Margins of the colon are defined as "grossly uninvolved" when the histology is indistinguishable from that of normal (free from disease) colon. By using molecular pathology techniques and working backward from colon adenocarcinoma to hypoplastic polyps to presumably normal mucosa, we defined some of those protein differences. Our results may provide a molecular basis for identifying tumor formation and progression in situ.(J Histochem Cytochem 49:667-668, 2001)
Asunto(s)
Adenocarcinoma/metabolismo , Biomarcadores de Tumor/metabolismo , Colon/metabolismo , Neoplasias del Colon/metabolismo , Pólipos del Colon/metabolismo , ADN-Topoisomerasas de Tipo II , Transactivadores , Antígenos de Neoplasias , Antígenos de Carbohidratos Asociados a Tumores/metabolismo , Biomarcadores de Tumor/clasificación , Proteínas del Citoesqueleto/metabolismo , ADN-Topoisomerasas de Tipo II/metabolismo , Proteínas de Unión al ADN , Mucinas Gástricas/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Humanos , Inmunohistoquímica , Mucosa Intestinal/metabolismo , Isoenzimas/metabolismo , Proteínas de la Membrana/metabolismo , Mucina-1/metabolismo , Fosfoproteínas/metabolismo , Proyectos Piloto , Proteína de la Zonula Occludens-1 , alfa Catenina , beta CateninaRESUMEN
STUDY OBJECTIVES: Bronchioloalveolar carcinoma is a primary lung neoplasm of variable histopathologic, radiologic, and clinical expression. There are three cell types described in bronchioloalveolar carcinoma: Clara cells, mucin-producing cells, and alveolar type II epithelial cells. It is unclear whether these three tumor cell types are associated with a specific radiologic presentation and clinical course. In this study, we investigated whether tumor cell type, identified by transmission electron microscopy, correlated with a specific radiologic pattern, and whether tumor cell type or radiologic presentation correlated with the patient's clinical course and outcome. DESIGN: Transmission electron microscopy was used to restudy tissue blocks from the original surgical histopathologic specimens in 54 patients with primary bronchioloalveolar carcinoma diagnosed over a 10-year period (1980 to 1990). The pretreatment radiographs were reviewed in each case, and the first chest radiograph obtained at the time of the discovery of the tumor in each patient was compared with the results of the ultrastructural study. The medical records of each patient were examined to obtain pertinent radiologic, clinical, and patient outcome information. MEASUREMENT AND RESULTS: There were 32 Clara cell tumors, 10 mucin-producing cell tumors, and 1 alveolar type II epithelial cell tumor in this series. Eleven additional tumors had mixtures of two or more cell types. No statistically significant relationship was detected between tumor cell type and radiologic presentation or patient mortality pattern. There was increased mortality among patients who presented radiologically with segmental, lobar, multifocal, or diffuse disease compared with those patients exhibiting a solitary pulmonary nodule at presentation. CONCLUSION: Radiologic presentation, rather than tumor cell type, provides prognostic information that aids in predicting patient outcome.
Asunto(s)
Adenocarcinoma Bronquioloalveolar/diagnóstico , Neoplasias Pulmonares/diagnóstico , Adenocarcinoma Bronquioloalveolar/diagnóstico por imagen , Adenocarcinoma Bronquioloalveolar/patología , Adenocarcinoma Bronquioloalveolar/ultraestructura , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Pulmón/diagnóstico por imagen , Neoplasias Pulmonares/diagnóstico por imagen , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/ultraestructura , Masculino , Persona de Mediana Edad , Pronóstico , RadiografíaRESUMEN
We investigated the intrathoracic contributions to the caudal mediastinal lymph node (CMN) efferent lymph in 12 anesthetized sheep after removing possible systemic contributions from below the diaphragm. We interrupted various pathways that may send lymph to the CMN (chest wall, esophagus, lung). Because the experiment is destructive, we did the resections in various combinations and waited 1 h between steps. Base-line CMN efferent lymph flow averaged 0.90 +/- 0.52 g/15 min (mean +/- SD). Cutting the pulmonary ligaments bilaterally caused lymph flow to decrease by an average of 58%. In five sheep, cauterizing around the lung hila reduced lymph flow by 16% of base line, cauterizing along the esophagus reduced lymph flow by 18% of base line, and cauterizing along the chest wall increased lymph flow by 6% of base line. After complete isolation of the node, except for the bronchoesophageal artery, dorsal mediastinal vein, and CMN efferent duct, 14% of base-line flow remained. The lymph-to-plasma total protein concentration ratios did not change significantly with any procedure. Under the conditions of our experiments, approximately 74% of base-line intrathoracic CMN efferent flow comes from the lung.
Asunto(s)
Ganglios Linfáticos/fisiología , Linfa/fisiología , Mediastino/fisiología , Músculos Abdominales/fisiología , Animales , Esófago/fisiología , Pulmón/fisiología , Ovinos , Tórax , Factores de TiempoRESUMEN
The purpose of this study was twofold: one concerns carotid blood flow and tissue PO2 and the other the effect of chronic hypoxic hypoxia on enhanced catecholamine content. The rationale was that chronic CO inhalation would not mimic the effect of hypoxia on the carotid body if its tissue blood flow is sufficiently high to counteract the effect of CO on O2 delivery and, hence, on tissue PO2. The differential effects of CO on the carotid body and erythropoietin-producing tissue would also indicate that the effect of hypoxic hypoxia on the carotid body is the result of a direct action of a local low O2 stimulus rather than secondary to a systemic effect initiated by other O2-sensing tissues. To test these alternatives we studied the effects of chronic CO inhalation on carotid body catecholamine content and hematocrit in the rats, which were exposed to an inspired PCO of 0.4-0.5 Torr at an inspired PO2 of approximately 150 Torr for 22 days. The hematocrit of CO-exposed rats was 75 +/- 1.1% compared with 48 +/- 0.7% in controls. Dopamine and norepinephrine content of the carotid bodies (per pair) was 5.88 +/- 0.91 and 3.02 +/- 0.19 ng, respectively, in the CO-exposed rats compared with 6.20 +/- 1.0 and 3.29 +/- 0.6 ng, respectively, in the controls. Protein content of the carotid bodies (per pair) was 18.4 +/- 1.6 and 20.5 +/- 0.9 micrograms, respectively. Thus, despite a vigorous erythropoietic response, the CO-exposed rats failed to show any significant stimulation of carotid body in terms of the content of either catecholamine or protein. The results suggest that carotid body tissue PO2 is not compromised by moderate carboxyhemoglobinemia because of its high tissue blood flow and that the chronic effect of hypoxic hypoxia on carotid body is direct.
Asunto(s)
Monóxido de Carbono/farmacología , Cuerpo Carotídeo/efectos de los fármacos , Dopamina/análisis , Norepinefrina/análisis , Administración por Inhalación , Animales , Monóxido de Carbono/administración & dosificación , Cuerpo Carotídeo/análisis , Hipoxia/fisiopatología , Masculino , Ratas , Ratas EndogámicasRESUMEN
We studied the effects of liposomes on the pulmonary circulation of sheep and found a close correlation between liposome retention in the lung and the intravascular macrophages. A test dose of liposomes (5.5 mumol of total lipids) injected intravenously transiently increased pulmonary arterial pressure from 24 +/- 2 to 55 +/- 16 (SD) cmH2O. The pulmonary arterial pressure responses were dose dependent and reproducible. The rise in pulmonary arterial pressure was blocked completely by indomethacin and 75% by a thromboxane synthase inhibitor. Systemic arterial thromboxane B2 concentration increased from a base-line level of less than 50 pg/ml to 250 +/- 130 pg/ml at the peak of the pressor response. Larger doses of liposomes (220 mumol of total lipids) infused intravenously over 1 h increased pulmonary arterial pressure maximally within the first 15 min. Lymph flow increased and lymph protein concentration decreased, suggesting venoconstriction. Over half (62.4 +/- 15.7%) of 111In-labeled liposomes remained in the lung after 2 h. Fluorescence and transmission electron microscopy showed that greater than 90% of the liposomes were associated with mononuclear cells in the lumen of the alveolar wall microvessels. We conclude that liposomes affect pulmonary arterial pressure transiently by a mechanism involving the arachidonate cascade, principally thromboxane. Our observations suggest that a population of pulmonary intravascular macrophages is likely to be the source of the thromboxane and the pulmonary hemodynamic and lymph dynamic changes that occur in a dose-dependent fashion, although interactions between liposomes, leukocytes, or endothelial cells, in addition to the macrophages, have not been completely ruled out. We believe this is the first demonstration that pulmonary intravascular macrophages may be the source of the arachidonate metabolites rather than endothelial cells, neutrophils, or perivascular interstitial cells.
Asunto(s)
Liposomas/farmacología , Pulmón/citología , Macrófagos/fisiología , Circulación Pulmonar , Animales , Presión Sanguínea , Gasto Cardíaco , Adhesión Celular , Relación Dosis-Respuesta a Droga , Indometacina/farmacología , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Macrófagos/ultraestructura , Microcirculación , Microscopía Electrónica , Microscopía Fluorescente , Arteria Pulmonar/fisiología , Ovinos , Tromboxano-A Sintasa/antagonistas & inhibidoresRESUMEN
The effect of chronic CO exposure, which stimulates erythropoietin production and erythropoiesis, was studied on carotid body cells in the rat. The hypothesis to be tested was that chronic CO inhalation would stimulate cellular hypertrophy and hyperplasia of carotid body if it caused local tissue hypoxia as in chronic hypoxia. The failure of an appropriate response would indicate a lack of a specific local effect on carotid body tissue PO2 presumably because of its unusually high tissue blood flow. Six young male rats were exposed to 0.4-0.5 Torr (0.05-0.07%) inspired PCO in air for 22 days. Control rats (n = 6) were maintained under similar conditions except for CO exposure. After the exposure period the rats were anesthetized, blood was collected for hematocrit, and the carotid bodies were surgically exposed and fixed for electron microscopy and morphometry of type I and type II cells and capillary endothelium. Hematocrit was significantly greater in the CO-exposed group (75 vs. 48%), whereas no significant difference was found in the carotid body parenchyma between the control and CO-exposed groups. We conclude that the lack of an effect of chronic CO exposure on the carotid bodies in contrast to the strong erythropoietic response indicates a relatively high tissue blood flow rate in the carotid body and that CO did not exert a direct cellular effect. The results also suggest that the hypertrophic response of carotid body glomus cells to chronic hypoxic hypoxia is the result of a local direct effect of low PO2 rather than secondary to systemic effects.