RESUMEN
We describe a mechanical method for delivery of adenoviral vector to the adventitial surface of arteries and to other tissues. Our goal was to characterize, principally in intact carotid artery, the morphological, biochemical, and functional effects of mechanical delivery of a recombinant beta-galactosidase-expressing adenoviral vector following its direct application using a small paintbrush. Our ex vivo and in vivo data demonstrate efficient, accurate, and rapid transduction of arteries without compromise of their morphological, biochemical, and functional integrity. We also demonstrate the general applicability of this technique in vivo via transduction of skeletal muscle, fibrotendinous tissue, peritoneum, serosal surface of bowel, and wounded skin. We conclude that direct mechanical delivery of an adenoviral vector to tissues using a suitable paintbrush represents an intuitive, accurate, and effective means of augmenting gene transfer efficiency, and may be a useful adjunct to other delivery methods.
Asunto(s)
Adenoviridae/genética , Arterias Carótidas , Terapia Genética/métodos , Vectores Genéticos/administración & dosificación , Transducción Genética/métodos , Administración Tópica , Animales , Arterias Carótidas/enzimología , Perros , Modelos Animales , beta-Galactosidasa/análisis , beta-Galactosidasa/genéticaRESUMEN
BACKGROUND: In patients with quiescent asthma, macrophages are the most prevalent cells recovered by bronchoalveolar lavage (BAL). Through activation via their FcepsilonRII receptors or by acting as antigen-presenting cells, macrophages could, in theory, promote the late airway response to allergen. OBJECTIVE: In order to investigate the importance of macrophages and other airway luminal cells in inducing the late airway response, a novel washout experiment was designed. METHODS: Five patients with ragweed-allergic asthma underwent bronchoscopy and segmental bronchial challenge with either normal saline or short ragweed extract in two segments of one lung. In a third segment of the opposite lung, 12 successive BALs (25 mL each) were performed, followed by challenge with an identical dose of short ragweed (washed-challenged segment). After 24 h, all three challenged segments underwent BAL. RESULTS: Initially, in the washed-challenged segment, over 80% (mean 80.4%, range 68-88%) of the recoverable airway dwelling cells were removed. Unexpectedly, 24 h later these same washed-challenged segments contained more eosinophils in the BAL than the challenged segments from the opposite lung (P = 0.033). CONCLUSIONS: Removing the majority of airway luminal cells followed by allergen bronchoprovocation increased the number of eosinophils recovered 24 h after challenge. Our results suggest that in quiescent allergic asthma, the airway luminal cells are protective and attenuate the late eosinophilic response to allergen challenge.
Asunto(s)
Antígenos/inmunología , Asma/inmunología , Líquido del Lavado Bronquioalveolar/inmunología , Eosinófilos/inmunología , Adulto , Asma/patología , Pruebas de Provocación Bronquial , Líquido del Lavado Bronquioalveolar/citología , División Celular , Eosinófilos/patología , Femenino , Humanos , Recuento de Leucocitos , Pulmón/inmunología , Macrófagos/fisiología , Masculino , Persona de Mediana Edad , Proteínas de Plantas/inmunología , Polen/inmunología , Factores de TiempoRESUMEN
The current study was designed to determine the effect of recombinant heme oxygenase-1 (HO-1) gene expression on endothelial function in cerebral arteries. Isolated canine basilar arteries were exposed ex vivo (30 minutes at 37 degrees C) to an adenoviral vector (10(10) PFU/mL, total volume 300 microL) encoding either the HO-1 gene (AdCMVHO-1) or the beta-galactosidase (beta-Gal) reporter gene (AdCMVbeta-Gal). Twenty-four hours after transduction, arterial rings were suspended in organ chamber for isometric force recording. Endothelium-dependent relaxations were obtained in response to bradykinin (10(-10) to 10(-6) mol/L) during contraction to uridine-5'-triphosphate (UTP; 3 x 10(-6) to 3 x 10(-5) mol/L). Certain rings were incubated with oxyhemoglobin (OxyHb; 10(-5) mol/L) overnight (16 to 18 hours of 24 hours). Expression and localization of recombinant protein were shown by Western blot analysis and immunohistochemistry. Endothelium-dependent relaxation to bradykinin and endothelium-independent relaxation to forskolin (10(-9) to 10(-5) mol/L) and DEA-NONOate (10(-10) to 10(-5) mol/L) were identical in beta-Gal- and HO-1-transduced arteries. Exposure to OxyHb caused impairment of endothelium-dependent relaxation to bradykinin (P < 0.01). In contrast, OxyHb did not affect endothelium-dependent relaxation in arteries expressing recombinant HO-1 ( P > 0.05). This protective effect of HO-1 was reversed by coincubation with tin protoporphyrin (SnPP9; 10(-5) mol/L), a selective inhibitor of HO-1 (P < 0.01). Basal levels of 3',5'-cyclic monophosphate (cGMP) in HO-1-transduced vessels were not significantly different from those in beta-Gal-transduced vessels. Pretreatment with OxyHb significantly reduced cGMP level in beta-Gal-transduced rings (P < 0.01), whereas it had no effect in HO-1-transduced rings. These results demonstrate that HO-1 gene transfer does not affect endothelial and smooth muscle function of normal arteries, and that expression of recombinant HO-1 in cerebral arteries protects vasomotor function against OxyHb-induced injury.
Asunto(s)
Hemo Oxigenasa (Desciclizante)/genética , Oxihemoglobinas/farmacología , Animales , Arteria Basilar/efectos de los fármacos , Arteria Basilar/enzimología , Cloruro de Calcio/farmacología , Bovinos , Línea Celular , Perros , Ácido Edético , Técnicas de Transferencia de Gen , Vectores Genéticos , Hemo-Oxigenasa 1 , Cinética , Sulfato de Magnesio/farmacología , NG-Nitroarginina Metil Éster/farmacología , Cloruro de Potasio/farmacología , Ratas , Proteínas Recombinantes/análisis , Proteínas Recombinantes/biosíntesis , Transfección , Vasodilatación/efectos de los fármacosRESUMEN
The paper describes results of "operationalized psychodynamic diagnostics" (OPD) with 54 adolescent and young adult drug addicts prior to outpatient family therapy. Focus of investigation is the prognostic and factorial validity of the OPD-system. Furthermore, relationships between conflicts and psychic structure are investigated. The use of the OPD-system allows a clinical description of the patient sample. Thus, adolescent drug addicts are characterized by autonomy versus dependence and self-esteem conflicts. A neurotic or sometimes borderline level is found on the structure axis. Low scores on the structure axis are significantly correlated with self-esteem conflicts. There is some evidence for the prognostic validity of the OPD I axis. However, prognostic validity of the structure and conflict axes require further research.
Asunto(s)
Terapia Familiar , Trastornos Relacionados con Sustancias/psicología , Trastornos Relacionados con Sustancias/rehabilitación , Adolescente , Adulto , Femenino , Humanos , Masculino , Reproducibilidad de los Resultados , AutoimagenRESUMEN
Endothelium-dependent relaxations mediated by NO are impaired in a mouse model of human atherosclerosis. Our objective was to characterize the mechanisms underlying endothelial dysfunction in aortas of apolipoprotein E (apoE)-deficient mice, treated for 26 to 29 weeks with a lipid-rich Western-type diet. Aortic rings from apoE-deficient mice showed impaired endothelium-dependent relaxations to acetylcholine (10(-)(9) to 10(-)(5) mol/L) and Ca(2+) ionophore (10(-)(9) to 10(-)(6) mol/L) and endothelium-independent relaxations to diethylammonium (Z)-1-(N,N-diethylamino)diazen-1-ium-1,2-diolate (DEA-NONOate, 10(-)(10) to 10(-)(5) mol/L) compared with aortic rings from C57BL/6J mice (P<0.05). By use of confocal microscopy of an oxidative fluorescent probe (dihydroethidium), increased superoxide anion (O(2)(-)) production was demonstrated throughout the aortic wall but mainly in smooth muscle cells of apoE-deficient mice. CuZn-superoxide dismutase (SOD) and Mn-SOD protein expressions were unaltered in the aorta exposed to hypercholesterolemia. A cell-permeable SOD mimetic, Mn(III) tetra(4-benzoic acid) porphyrin chloride (10(-)(5) mol/L), reduced O(2)(-) production and partially normalized relaxations to acetylcholine and DEA-NONOate in apoE-deficient mice (P<0.05). [(14)C]L-Citrulline assay showed a decrease of Ca(2+)-dependent NOS activity in aortas from apoE-deficient mice compared with C57BL/6J mice (P<0.05), whereas NO synthase protein expression was unchanged. In addition, cGMP levels were significantly reduced in the aortas of apoE-deficient mice (P<0.05). Our results demonstrate that in apoE-deficient mice on a Western-type fat diet, impairment of endothelial function is caused by increased production of O(2)(-) and reduced endothelial NO synthase enzyme activity. Thus, chemical inactivation of NO with O(2)(-) and reduced biosynthesis of NO are key mechanisms responsible for endothelial dysfunction in aortas of atherosclerotic apoE-deficient mice.
Asunto(s)
Apolipoproteínas E/genética , Arteriosclerosis/fisiopatología , Endotelio Vascular/metabolismo , Animales , Aorta/efectos de los fármacos , Aorta/metabolismo , Aorta/fisiopatología , Arteriosclerosis/metabolismo , Western Blotting , Calcio/metabolismo , Técnicas de Cultivo , AMP Cíclico/biosíntesis , GMP Cíclico/biosíntesis , Masculino , Metaloporfirinas/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo II , Óxido Nítrico Sintasa de Tipo III , Superóxido Dismutasa/inmunología , Superóxido Dismutasa/metabolismo , Superóxidos/metabolismo , Vasoconstricción , VasodilataciónRESUMEN
Human eosinophil major basic protein (MBP) is strongly implicated as a mediator of disease, especially bronchial asthma. We recently isolated a highly divergent human homologue of MBP (MBPH). Given human MBP's importance in disease and the restricted expression of it and human MBPH, we isolated the 4.6-kb human MBPH gene (HGMW-approved symbol PRG3). Comparisons among the human MBP (PRG2), human MBPH, and murine MBP-1 (mMBP-1; Prg2) genes suggest that the human MBP and mMBP-1 genes are more closely related than either is to the human MBPH gene. Proximal promoters of these three genes show conservation of potential binding sites for IK2 and STAT and of a known GATA site. However, a known C/EBP site is altered in the human MBPH gene's proximal promoter. The human MBP and MBPH genes localized to chromosome 11 in the centromere to 11q12 region. Thus, the human MBP and MBPH genes have diverged considerably, probably following a gene duplication event. Furthermore, the identified conserved and distinct proximal promoter elements likely contribute to the eosinophil-restricted and relatively reduced transcription of the human MBPH gene.
Asunto(s)
Proteínas Sanguíneas/genética , Proteína Mayor Básica del Eosinófilo , Regiones Promotoras Genéticas , Ribonucleasas , Animales , Secuencia de Bases , Sitios de Unión , Proteínas Sanguíneas/biosíntesis , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Centrómero/ultraestructura , Mapeo Cromosómico , Cromosomas Humanos Par 11 , Secuencia Conservada , ADN Complementario/metabolismo , Proteínas en los Gránulos del Eosinófilo , Evolución Molecular , Exones , Biblioteca de Genes , Humanos , Hibridación Fluorescente in Situ , Intrones , Ratones , Modelos Genéticos , Datos de Secuencia Molecular , Análisis de Secuencia de ADN , Homología de Secuencia de Ácido Nucleico , Transcripción GenéticaRESUMEN
Endothelial nitric oxide synthase (eNOS)-derived nitric oxide (NO) contributes to hepatic vascular homeostasis. The aim of this study was to examine whether delivery of an adenoviral vector encoding eNOS gene to liver affects vasomotor function in vivo and the mechanism of NO production in vitro. Rats were administered adenoviruses encoding beta-galactosidase (AdCMVLacZ) or eNOS (AdCMVeNOS) via tail vein injection and studied 1 wk later. In animals transduced with AdCMVLacZ, beta-galactosidase activity was increased in the liver, most prominently in hepatocytes. In AdCMVeNOS-transduced animals, eNOS protein levels and catalytic activity were significantly increased. Overexpression of eNOS diminished baseline perfusion pressure and constriction in response to the alpha(1)-agonist methoxamine in the perfused liver. Transduction of cultured hepatocytes with AdCMVeNOS resulted in the targeting of recombinant eNOS to a perinuclear distribution and binding with the NOS-activating protein heat shock protein 90. These events were associated with increased ionomycin-stimulated NO release. In summary, this is the first study to demonstrate successful delivery of the recombinant eNOS gene to liver in vivo and in vitro with ensuing NO production.
Asunto(s)
Adenoviridae/genética , Técnicas de Transferencia de Gen , Hepatocitos/enzimología , Hígado/metabolismo , Óxido Nítrico Sintasa/genética , Animales , Calcimicina , Hepatocitos/citología , Técnicas In Vitro , Ionóforos , Operón Lac , Hígado/citología , Masculino , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo III , Procesamiento Postranscripcional del ARN/fisiología , Ratas , Ratas Endogámicas F344 , Proteínas Recombinantes/genética , beta-Galactosidasa/genéticaRESUMEN
Gene therapy is being investigated as a putative treatment option for cardiovascular diseases, including cerebral vasospasm. Because there is presently no information regarding gene transfer to human cerebral arteries, the principal objective of this study was to characterize adenovirus-mediated expression and function of recombinant endothelial nitric oxide synthase (eNOS) gene in human pial arteries. Pial arteries (outer diameter 500 to 1,000 microm) were isolated from 30 patients undergoing temporal lobectomy for intractable seizures and were studied using histologic staining, histochemistry, electron microscopy, and isometric force recording. Gene transfer experiments were performed ex vivo using adenoviral vectors encoding genes for bovine eNOS (AdCMVeNOS) and Escherichia coli beta-galactosidase (AdCMVLacZ). In transduced arteries, studied 24 hours after exposure to vectors, expression of recombinant beta-galactosidase and eNOS was detected by histochemistry, localizing mainly to the adventitia (n = 4). Immunoelectron microscopy localized recombinant eNOS in adventitial fibroblasts. During contractions to U46619, bradykinin-induced relaxations were significantly augmented in AdCMVeNOS-transduced rings compared with control and AdCMVLacZ-transduced rings (P < 0.01; n = 6). The NOS inhibitor L-nitroarginine methylester (L-NAME) caused significantly greater contraction in AdCMVeNOS-transduced rings (P < 0.001; n = 4) and inhibited bradykinin-induced relaxations in control and transduced rings (P < 0.001; n = 6). The current findings suggest that in AdCMVeNOS-transduced human pial arteries, expression of recombinant eNOS occurs mainly in adventitial fibroblasts where it augments relaxations to NO-dependent agonists such as bradykinin. Findings from the current study might be beneficial in future clinical applications of gene therapy for the treatment or prevention of cerebral vasospasm.
Asunto(s)
Arterias Cerebrales/fisiología , Regulación Enzimológica de la Expresión Génica/fisiología , Técnicas de Transferencia de Gen , Óxido Nítrico Sintasa/genética , Adenoviridae , Adolescente , Adulto , Anciano , Animales , Bovinos , Niño , Femenino , Vectores Genéticos , Humanos , Masculino , Persona de Mediana Edad , Óxido Nítrico Sintasa de Tipo III , Proteínas Recombinantes/genéticaRESUMEN
Human eosinophils have been reported to express both the mRNA and protein for the high affinity IgE receptor (FcepsilonRI); it is speculated that this receptor plays a role in eosinophil mediator release in allergic diseases. However, questions still remain. How much of the FcepsilonRI protein is actually expressed on the cell surface of the eosinophil? If they are present, are these IgE receptors associated with effector functions of eosinophils? To address these issues, we studied blood eosinophils from patients with ragweed hay fever. A high level of low affinity IgG receptor (FcgammaRII, CD32), but no expression of FcepsilonRI, was detectable on the eosinophil surface by standard FACS analysis. However, after in vitro sensitization with biotinylated chimeric IgE (cIgE), cell-bound cIgE was detected by PE-conjugated streptavidin. This cIgE binding was partially inhibited by anti-FcepsilonRI mAb, suggesting that eosinophils do express minimal amounts of FcepsilonRI detectable only by a sensitive method. Indeed, FACS analysis of whole blood showed that eosinophils express approximately 0.5% of the FcepsilonRI that basophils express. When stimulated with human IgE or anti-human IgE, these eosinophils did not exert effector functions; there was neither production of leukotriene C4 or superoxide anion nor any detectable degranulation response. In contrast, eosinophils possessed membrane-bound human IgG and showed functional responses when stimulated with human IgG or anti-human IgG. Thus, IgG and/or cytokines, such as IL-5, appear to be more important for eosinophil activation in allergic diseases than IgE.
Asunto(s)
Sitios de Unión de Anticuerpos , Eosinófilos/metabolismo , Hipersensibilidad/sangre , Hipersensibilidad/inmunología , Inmunoglobulina E/sangre , Adolescente , Adulto , Animales , Antígenos CD/biosíntesis , Antígenos CD/sangre , Basófilos/inmunología , Biomarcadores/sangre , Biotinilación , Degranulación de la Célula/inmunología , Eosinófilos/inmunología , Humanos , Inmunoglobulina E/genética , Leucotrieno C4/biosíntesis , Leucotrieno C4/sangre , Activación de Linfocitos , Ratones , Persona de Mediana Edad , Receptores de IgE/biosíntesis , Receptores de IgE/sangre , Receptores de IgE/fisiología , Receptores de IgG/biosíntesis , Receptores de IgG/sangre , Receptores de IgG/fisiología , Proteínas Recombinantes de Fusión/metabolismo , Rinitis Alérgica Estacional/sangre , Rinitis Alérgica Estacional/inmunología , Superóxidos/sangreRESUMEN
Eosinophils are important effector cells in defense against helminth infection and in allergic diseases. To identify novel eosinophil proteins, large scale sequencing of a cDNA library prepared from interleukin-5-stimulated umbilical cord precursor cells was performed, and the major genes expressed by maturing eosinophils were determined. This resulted in the identification of a cDNA with 64% identity to human prepro-major basic protein (hprepro-MBP). This cDNA was designated hprepro-MBP homolog (hprepro-MBPH). Interestingly, the calculated pI values for hMBPH and hMBP differed by >100-fold, with pI values of 8.7 and 11.4, respectively. Given this pronounced basicity difference, the homolog transcript's abundance (1.1%), and MBP's critical role in eosinophil biological activity, we further characterized the homolog. Reverse transcription-polymerase chain reaction detected transcription of hprepro-MBPH in bone marrow only, and this result was confirmed by analysis of a large cDNA data base (electronic Northern). hMBPH was isolated from human eosinophil granule lysates, and its identity was verified by amino acid sequencing and by mass spectrometry. Analyses of the biological activities showed that hMBPH had effects similar to hMBP in cell killing and neutrophil (superoxide anion production and interleukin-8 release) and basophil (histamine and leukotriene C4 release) stimulation assays, but usually with reduced potency. Overall, this novel homolog's unique physical properties indicated that the high net positive charge of hMBP is important but not essential for biological activity.
Asunto(s)
Proteínas Sanguíneas/química , Eosinófilos/química , Precursores de Proteínas/genética , Precursores de Proteínas/aislamiento & purificación , Ribonucleasas , Secuencia de Aminoácidos , Secuencia de Bases , ADN Complementario/química , Proteínas en los Gránulos del Eosinófilo , Eosinófilos/efectos de los fármacos , Humanos , Interleucina-5/farmacología , Datos de Secuencia Molecular , Precursores de Proteínas/química , ARN Mensajero/metabolismo , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
Eosinophil-derived neurotoxin (EDN) and eosinophil cationic protein (ECP) are generally regarded as eosinophil-specific proteins. We tested whether EDN and ECP are present in mature neutrophils. By indirect immunofluorescence, both eosinophils and neutrophils stained with antibodies to EDN and ECP. Lysates of purified (<0.1% eosinophil contamination) neutrophils contained EDN, 112+/-4 ng/10(6) cells, and ECP, 163+/-2 ng/10(6) cells, whereas eosinophil major basic protein (MBP) was not detectable. Electron microscopic examination of immunogold-labeled buffy coat cells stained with EDN antibody showed that EDN is localized to neutrophil granules. Finally, EDN mRNA was detected in lysates of highly purified neutrophils (0.001% eosinophil contamination) by the reverse transcription-polymerase chain reaction. We conclude that proteins that are either identical to or immunologically cross-reactive with EDN and ECP are present in neutrophils and that EDN is synthesized and localized to neutrophil granules. Thus, caution must be exercised in interpreting the presence of EDN and ECP as specific markers of eosinophil-associated inflammation in human disease.
Asunto(s)
Proteínas Sanguíneas/metabolismo , Neutrófilos/metabolismo , Proteínas/metabolismo , Ribonucleasas , Biopsia , Gránulos Citoplasmáticos/metabolismo , Proteínas en los Gránulos del Eosinófilo , Neurotoxina Derivada del Eosinófilo , Eosinófilos/metabolismo , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Microscopía Inmunoelectrónica , Reacción en Cadena de la Polimerasa , ARN Mensajero/sangre , Piel/metabolismo , Transcripción GenéticaRESUMEN
Cytokines active on eosinophils are important in the pathogenesis of allergic diseases. A study was conducted to determine if nasal eosinophilia in allergic rhinitis is associated with an increase in eosinophil-active cytokines in nasal secretions and to compare the effects of fluticasone propionate aqueous nasal spray with astemizole and placebo on the levels of these cytokines. Forty-five patients with moderately severe ragweed allergic rhinitis were randomly assigned to receive 2 weeks of treatment with fluticasone propionate aqueous nasal spray 200 micrograms once daily, astemizole 10 mg once daily, or placebo. Nasal lavage was performed in July (preseason), August (peak season), September (after 2 weeks of treatment), and October (postseason). The number of eosinophils, the amount of eosinophil-derived neurotoxin (EDN), and the amount of eosinophil survival-enhancing activity were measured. Total mean nasal symptom scores, concentrations of nasal eosinophils and EDN, and eosinophil survival-enhancing cytokine activity in nasal secretions were significantly lower after 2 weeks of treatment with fluticasone propionate compared with astemizole or placebo. Survival-enhancing activity was detected in the nasal secretions of 25 patients. By blocking activity with monoclonal antibodies, specific cytokines were identified (granulocyte macrophage-colony stimulating factor, 3 samples; interleukin-3, 2 samples; interleukin-5, 5 samples). In conclusion, eosinophil-active cytokine concentrations parallel the nasal symptoms of patients with ragweed allergic rhinitis. Unlike astemizole, fluticasone propionate significantly lowers cytokine activity in nasal tissue, which may contribute to the therapeutic efficacy of the drug.
Asunto(s)
Androstadienos/administración & dosificación , Antialérgicos/administración & dosificación , Eosinófilos/efectos de los fármacos , Mucosa Nasal/efectos de los fármacos , Rinitis Alérgica Estacional/tratamiento farmacológico , Ribonucleasas , Adulto , Aerosoles , Astemizol/administración & dosificación , Citocinas/efectos de los fármacos , Citocinas/metabolismo , Neurotoxina Derivada del Eosinófilo , Femenino , Fluticasona , Humanos , Masculino , Mucosa Nasal/metabolismo , Neurotoxinas/aislamiento & purificaciónRESUMEN
Chronic eosinophilic pneumonitis (CEP) is characterized by longstanding respiratory symptoms accompanied by a massive pulmonary eosinophil infiltration. We hypothesized that cytokine(s) produced in the disease sites are implicated in the pathophysiology of CEP. We studied peripheral blood and bronchoalveolar lavage fluids (BALF) obtained from two lung segments of a patient with CEP. Seventy times more eosinophils were found in the BALF from an involved lung segment (showing patchy opacification on a chest roentgenogram) than from an uninvolved segment. The eosinophil-active cytokines interleukin-5 (IL-5), IL-6, and IL-10 were strikingly elevated in the BALF from the involved lung segment, whereas no or minimal levels of these cytokines were detectable in the BALF from the uninvolved segment or serum, respectively. Leukocytes in the involved lung segment, but not those in peripheral blood, expressed messenger ribonucleic acid (mRNA) for IL-5, IL-6, and IL-10. In contrast, IL-2, IL-3, IL-4, interferon-gamma (IFN-gamma), granulocyte-macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor-alpha (TNF-alpha) were not detected in any sample. These findings suggest that increased production of several cytokines, such as IL-5, IL-6, and IL-10, in the involved lung segment, but not in the uninvolved lung segment or peripheral blood, is a critical pathophysiologic feature of CEP.
Asunto(s)
Citocinas/biosíntesis , Eosinofilia Pulmonar/fisiopatología , Adulto , Secuencia de Bases , Líquido del Lavado Bronquioalveolar/química , Femenino , Factor Estimulante de Colonias de Granulocitos y Macrófagos , Humanos , Interleucina-5/análisis , Interleucina-5/biosíntesis , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Eosinofilia Pulmonar/metabolismoRESUMEN
Information and communication technologies are presumed to play a critical role in improving effectiveness and efficiency of clinical care. Although the most promising directions of technological development are microcomputer-generated computerized medical record systems, documenting their value has been a major challenge for health care providers. This paper proposes a 15-item spreadsheet instrument for evaluating computerized medical records, and demonstrates how it was experimentally applied to a 6-year long experience at three sites. In conclusion, preliminary implications and guidelines are drawn with regard to practice and research in this area.
Asunto(s)
Técnicas de Apoyo para la Decisión , Eficiencia Organizacional , Sistemas de Registros Médicos Computarizados/normas , Garantía de la Calidad de Atención de Salud , Análisis Costo-Beneficio , Almacenamiento y Recuperación de la Información , Sistemas de Registros Médicos Computarizados/economía , Programas Informáticos , Interfaz Usuario-Computador , Carga de TrabajoRESUMEN
To test whether eosinophil recruitment after pulmonary allergen challenge is associated with interleukin (IL)-5 in patients with asthma, we performed segmental bronchoprovocation (SBP) with saline, and with low and high dosages of ragweed extract in six patients with allergic asthma. Bronchoalveolar lavage (BAL) of the challenged segments was performed 5 min after challenge (immediate BAL fluid) and repeated 24 h later (late BAL fluid). Allergen challenge resulted in recruitment of eosinophils, and increased levels of eosinophil-active cytokines. A bioassay showed the predominant eosinophil-active cytokine in the late BAL fluids to be IL-5. Analysis of the late BAL fluids revealed that IL-5 levels correlated with the numbers of eosinophils and lymphocytes. This study provides evidence that IL-5 is a critical cytokine associated with eosinophil and lymphocyte recruitment into the airways of patients with asthma following exposure to allergen.
Asunto(s)
Alérgenos/efectos adversos , Asma/inmunología , Quimiotaxis de Leucocito/inmunología , Eosinófilos/inmunología , Interleucina-5/inmunología , Linfocitos/inmunología , Ribonucleasas , Adolescente , Adulto , Proteínas Sanguíneas/análisis , Pruebas de Provocación Bronquial , Lavado Broncoalveolar , Líquido del Lavado Bronquioalveolar/química , Líquido del Lavado Bronquioalveolar/citología , Líquido del Lavado Bronquioalveolar/inmunología , Broncoscopía , Proteínas en los Gránulos del Eosinófilo , Humanos , Ensayo Inmunorradiométrico , Mediadores de Inflamación/análisis , Masculino , Elastasa Pancreática/análisis , RadioinmunoensayoRESUMEN
BACKGROUND: The eosinophilia-myalgia syndrome, caused by a contaminant or contaminants in epidemiologically implicated L-tryptophan products, is characterized by eosinophilia and eosinophil degranulation. We hypothesized that immune cells are stimulated by implicated L-tryptophan and produce eosinophil-active cytokines. OBJECTIVES: This study was designed to identify substances in L-tryptophan causing the eosinophilia-myalgia syndrome. METHODS: Peripheral blood mononuclear cells were cultured with L-tryptophan products, and supernatants were tested for their ability to enhance eosinophil degranulation and survival in vitro and for their cytokine content. Subsequently, 46 different L-tryptophan lots were analyzed for their in vitro biologic activities. RESULTS: After peripheral blood mononuclear cells were cultured with implicated L-tryptophan, their supernatants enhanced eosinophil degranulation and survival. These activities were blocked by anti-granulocyte-macrophage colony-stimulating factor (GM-CSF) antibody; immunoreactive GM-CSF was measurable in the supernatants. Monocytes, but not T lymphocytes, were the responding cells. However, no correlation was observed between the in vitro biologic activity and lots of epidemiologically implicated L-tryptophan products. This biologic activity in the L-tryptophan products was characterized as endotoxin. CONCLUSION: Although L-tryptophan products stimulate peripheral blood mononuclear cells to produce GM-CSF, this response is caused by endotoxin contamination of the L-tryptophan products and not by a specific L-tryptophan contaminant. Endotoxin contamination must be considered as a possible cause of eosinophil-active cytokine production by peripheral blood mononuclear cells.
Asunto(s)
Citocinas/análisis , Síndrome de Eosinofilia-Mialgia/sangre , Eosinófilos/inmunología , Leucocitos Mononucleares/inmunología , Triptófano/inmunología , Supervivencia Celular , Células Cultivadas , Endotoxinas/química , Síndrome de Eosinofilia-Mialgia/inmunología , Eosinófilos/patología , Humanos , Triptófano/química , Triptófano/farmacologíaRESUMEN
Segmental bronchoprovocation (SBP) with allergen was used in an attempt to study eosinophils recruited to the airway 24 h after challenge. Unexpectedly, in the first four patients, neutrophils (rather than eosinophils) were recruited in the bronchoalveolar lavage (BAL) fluids, and we hypothesized that the allergen extracts were contaminated with endotoxin. The extracts used for challenge in the first four patients tested positive for bacterial endotoxin in a limulus amebocyte lysate assay. Rechallenge of one patient from the first group with a comparable dose of an endotoxin-free extract and SBP with endotoxin-free extract in five additional patients resulted in preferential recruitment of eosinophils rather than neutrophils. The number of neutrophils recovered from the challenged segments in the patients challenged with endotoxin-free extract was significantly less than that observed in the first four patients. Taken together, these observations suggest that neutrophil recruitment in the 24-h BAL fluids from the first four patients was probably due to endotoxin contamination of the allergen extract. We caution investigators that endotoxin contamination of allergen extract may alter the cellular inflammation during the late airway response following allergen challenge.
Asunto(s)
Alérgenos , Asma/diagnóstico , Asma/inmunología , Pruebas de Provocación Bronquial/efectos adversos , Líquido del Lavado Bronquioalveolar/citología , Contaminación de Medicamentos , Endotoxinas/efectos adversos , Eosinófilos , Hipersensibilidad/complicaciones , Neutrófilos , Adolescente , Adulto , Asma/etiología , Líquido del Lavado Bronquioalveolar/química , Femenino , Humanos , Hipersensibilidad/diagnóstico , Ensayo Inmunorradiométrico , Inflamación , Recuento de Leucocitos , Elastasa de Leucocito , Prueba de Limulus , Masculino , Elastasa Pancreática/análisis , Pruebas Cutáneas , Factores de TiempoRESUMEN
To study human eosinophils, their efficient purification from peripheral blood is crucial. Although a number of purification procedures, including discontinuous Percoll and metrizamide density gradient centrifugation, have been used, it has been difficult to isolate eosinophils from normal donors with consistently high yields and purities. Recently, a new isolation technique called magnetic cell separation system (MACS) was reported. To evaluate this procedure, we isolated eosinophils from human peripheral blood using either MACS or the standard discontinuous Percoll density methods, and compared cellular viability, morphology, and response to degranulation stimuli. MACS gave a higher yield of eosinophils than Percoll density centrifugation; for example, 6.6 +/- 1.1 x 10(6) eosinophils were isolated from 20 ml of blood by MACS compared to 6.4 +/- 2.4 x 10(6) from 120 ml by Percoll density gradient. Further, the purity of eosinophils isolated by MACS was 97.1 +/- 0.5% (X +/- SEM) compared to 77.8 +/- 2.9% with Percoll. As part of the MACS protocol, erythrocytes are lysed with either 155 mM ammonium chloride or hypotonic lysis. With 155 mM ammonium chloride treatment, the eosinophils showed a striking reduction in cytokine mediated survival due to interleukin (IL)-3, IL-5 and granulocyte-macrophage colony-stimulating factor (GM-CSF), marked morphologic abnormalities and a reduced degranulation response. With hypotonic lysis, no differences were observed in survival and morphology between eosinophils purified by MACS and Percoll methods; the degranulation responses to stimuli were essentially the same between the two methods. Taken together, these observations suggest that the exposure of eosinophils to 155 mM ammonium chloride results in cellular damage. Therefore, MACS with hypotonic lysis is a useful technique to isolate eosinophils for biological study.
Asunto(s)
Cloruro de Amonio/farmacología , Degranulación de la Célula/fisiología , Citocinas/antagonistas & inhibidores , Eosinófilos/efectos de los fármacos , Separación Celular/métodos , Supervivencia Celular/efectos de los fármacos , Centrifugación por Gradiente de Densidad , Eosinófilos/fisiología , Eosinófilos/ultraestructura , Humanos , Separación Inmunomagnética , Recuento de Leucocitos , Povidona , Dióxido de SilicioRESUMEN
The mechanism of airway eosinophilia during antigen-induced inflammation was investigated by measurement of eosinophil-active cytokines utilizing an eosinophil survival assay. In the first study, 4 patients with allergic rhinitis underwent segmental bronchoprovocation (SBP) with low, medium, and high doses of ragweed extract instilled into different bronchial subsegments; bronchoalveolar lavage (BAL) fluids were collected from each segment 12 min and 48 h after challenge. Eosinophil granule proteins and eosinophil survival activity were significantly elevated in the 48-h (late-phase) BAL fluids from these segments. Correlations were observed between the concentrations of eosinophil granule proteins and eosinophil survival activity (rs = 0.717 to 0.880, p < 0.001) in BAL fluids. Eosinophil survival activity was completely neutralized by anti-IL-5 monoclonal antibody in five of the seven 48-h samples tested representing three of the 4 patients. In the two remaining samples, eosinophil survival activity was only partially neutralized by either anti-IL-5 antibody or anti-granulocyte-macrophage colony-stimulating factor (GM-CSF) but was completely neutralized by anti-IL-5 and anti-GM-CSF in combination. Subsequently, in the second study, 10 patients with allergic rhinitis were challenged by SBP with ragweed extract. Eosinophil survival activity was significantly elevated in the 48-h BAL fluids; this activity was partially neutralized by anti-IL-5 antibody about (48%) and completely neutralized by the combination of anti-IL-5 and anti-GM-CSF antibodies. These findings suggest that the eosinophil survival activity in the late inflammatory lesions following SBP with allergen is mainly associated with IL-5, with small contributions from GM-CSF.(ABSTRACT TRUNCATED AT 250 WORDS)