RESUMEN
OBJECTIVE: To provide information on the clinical presentation of sciatic neuropathy and its management in a real-world setting, and to analyze the effects of a multimodal approach based on the association of physical and pharmacological therapy. PATIENTS AND METHODS: A multicentric observational prospective study was conducted in 44 Italian tertiary centers specialized in Physical Medicine and Rehabilitation, Orthopedics, Neurology, Neurosurgery, and Rheumatology. To develop a shared management of LPB with sciatica, a dedicated clinical record was proposed to collect data about diagnosis, treatment, and outcomes. Pain, disability, and quality of life were recorded trough validated questionnaires at baseline and after a two-month follow-up. RESULTS: 394 patients (age, mean ± SD 55.7 ± 14.1 years, 57.1% females) with chronic LBP and sciatica were enrolled in the study. The characteristics of the selected group showed a certain variability in the clinical presentation. At baseline, patients received several different therapeutic options among physical, pharmacological and neurotrophic treatments. A subgroup of 312 patients was treated with a combination of neurotrophic agents containing alpha-lipoic acid (ALA). After a two-month follow-up, a general improvement in both perceived pain and functional disabilities was observed. A significant improvement (p < 0.001) in the Pain Numeric Rating Scale (NRS), Roland e Morris Disability Questionnaire (RMDQ) and Brief Pain Inventory (BPI) Italian short version was observed. CONCLUSIONS: Sciatic neuropathy is a multifaceted condition managed by means of a wide spectrum of therapeutic options. The results of this study suggest that a multimodal approach based on the association of ALA with physical and pharmacological therapies can be beneficial in the treatment of LBP with sciatica.
Asunto(s)
Dolor de la Región Lumbar , Dimensión del Dolor , Adulto , Dolor Crónico , Evaluación de la Discapacidad , Femenino , Humanos , Italia , Dolor de la Región Lumbar/fisiopatología , Dolor de la Región Lumbar/psicología , Dolor de la Región Lumbar/terapia , Persona de Mediana Edad , Estudios Prospectivos , Calidad de Vida , Nervio Ciático , Encuestas y Cuestionarios , Resultado del TratamientoRESUMEN
Increasing evidence suggests that a continuous release of histamine from mast cells occurs in the airways of asthmatic patients and that histamine may modulate functions of other inflammatory cells such as macrophages. In the present study histamine (10(-9)-10(-6) M) increased in a concentration-dependent fashion the basal release of beta-glucuronidase (EC(50) = 8.2 +/- 3.5 x 10(-9) M) and IL-6 (EC(50) = 9.3 +/- 2.9 x 10(-8) M) from human lung macrophages. Enhancement of beta-glucuronidase release induced by histamine was evident after 30 min and peaked at 90 min, whereas that of IL-6 required 2-6 h of incubation. These effects were reproduced by the H(1) agonist (6-[2-(4-imidazolyl)ethylamino]-N-(4-trifluoromethylphenyl)heptane carboxamide but not by the H(2) agonist dimaprit. Furthermore, histamine induced a concentration-dependent increase of intracellular Ca(2+) concentrations ([Ca(2+)](i)) that followed three types of response, one characterized by a rapid increase, a second in which [Ca(2+)](i) displays a slow but progressive increase, and a third characterized by an oscillatory pattern. Histamine-induced beta-glucuronidase and IL-6 release and [Ca(2+)](i) elevation were inhibited by the selective H(1) antagonist fexofenadine (10(-7)-10(-4) M), but not by the H(2) antagonist ranitidine. Inhibition of histamine-induced beta-glucuronidase and IL-6 release by fexofenadine was concentration dependent and displayed the characteristics of a competitive antagonism (K(d) = 89 nM). These data demonstrate that histamine induces exocytosis and IL-6 production from human macrophages by activating H(1) receptor and by increasing [Ca(2+)](i) and they suggest that histamine may play a relevant role in the long-term sustainment of allergic inflammation in the airways.
Asunto(s)
Exocitosis/inmunología , Histamina/análogos & derivados , Histamina/fisiología , Interleucina-6/biosíntesis , Pulmón/inmunología , Pulmón/metabolismo , Macrófagos Alveolares/metabolismo , Receptores Histamínicos H1/metabolismo , Calcio/metabolismo , Calcio/fisiología , Citosol/metabolismo , Dimaprit/farmacología , Relación Dosis-Respuesta Inmunológica , Glucuronidasa/metabolismo , Histamina/farmacología , Agonistas de los Receptores Histamínicos/farmacología , Antagonistas de los Receptores Histamínicos H1/farmacología , Antagonistas de los Receptores H2 de la Histamina/farmacología , Humanos , Interleucina-6/genética , Interleucina-6/metabolismo , Pulmón/citología , Pulmón/enzimología , Macrófagos Alveolares/enzimología , Macrófagos Alveolares/inmunología , ARN Mensajero/biosíntesis , Toluidinas/farmacología , Regulación hacia Arriba/inmunologíaRESUMEN
Fibroblasts are involved in all pathologies characterized by increased ExtraCellularMatrix synthesis, from wound healing to fibrosis. Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF) is a cytokine isolated as an hemopoietic growth factor but recently indicated as a differentiative agent on endothelial cells. In this work we demonstrated the expression of the receptor for GM-CSF (GM-CSFR) on human normal skin fibroblasts from healthy subjects (NFPC) and on a human normal fibroblast cell line (NHDF) and we try to investigate the biological effects of this cytokine. Human normal fibroblasts were cultured with different doses of GM-CSF to study the effects of this factor on GM-CSFR expression, on cell proliferation and adhesion structures. In addition we studied the production of some Extra-Cellular Matrix (ECM) components such as Fibronectin, Tenascin and Collagen I. The growth rate of fibroblasts from healthy donors (NFPC) is not augmented by GM-CSF stimulation in spite of increased expression of the GM-CSFR. On the contrary, the proliferation of normal human dermal fibroblasts (NHDF) cell line seems more influenced by high concentration of GM-CSF in the culture medium. The adhesion structures and the ECM components appear variously influenced by GM-CSF treatment as compared to fibroblasts cultured in basal condition, but newly only NHDF cells are really induced to increase their synthesis activity. We suggest that the in vitro treatment with GM-CSF can shift human normal fibroblasts towards a more differentiated state, due or accompanied by an increased expression of GM-CSFR and that such "differentiation" is an important event induced by such cytokine.
Asunto(s)
Dermis/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Adulto , Western Blotting , Adhesión Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Línea Celular , Colágeno Tipo I/biosíntesis , Dermis/metabolismo , Relación Dosis-Respuesta a Droga , Ensayo de Inmunoadsorción Enzimática , Matriz Extracelular/efectos de los fármacos , Matriz Extracelular/metabolismo , Femenino , Fibroblastos/metabolismo , Fibronectinas/biosíntesis , Humanos , Inmunohistoquímica , Masculino , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/biosíntesis , Tenascina/biosíntesisRESUMEN
Interferon-beta (IFN-beta) inhibits mitogen-induced T cell responses, in part through downregulation of interleukin-12 (IL-12) or upregulation of IL-10. We have reexamined these findings using ragweed (RW) stimulated or tetanus toxoid (TT)-stimulated human peripheral blood mononuclear cells (PBMC) and nontransformed, antigen-specific, human Th0, Th1, and Th2 clones. IFN-beta induced concentration-dependent inhibition of phytohemagglutinin (PHA)-stimulated PBMC proliferation and enhancement of RW-stimulated or TTstimulated PBMC proliferation. Monocyte depletion of PBMC isolates resulted in concentration-dependent inhibition of RW-driven or TT-driven proliferation by IFN-beta. This response was unaltered by the addition of either exogenous recombinant human IL-12 (rHuIL-12) or saturating concentrations of anti-IL-10. Moreover, addition of exogenous rHuIL-10 to nondepleted RW-driven or TT-driven PBMC cultures did not alter the concentration-dependent enhancement of antigen-driven proliferation induced by IFN-beta. Th0, Th1, and Th2 clones stimulated in the presence of antigen and autologous, irradiated PBMC displayed concentration-dependent inhibition of proliferation in the presence of IFN-beta that was unaltered by the addition of either exogenous rHuIL-12 or a saturating concentration of anti-IL-10. Finally, whereas IFN-beta inhibited antigen-driven generation of IL-5, IL-12, IL-13, and IFN-gamma, IFN-beta enhanced generation of both IL-4 and IL-10. Thus, IFN-beta, induces a selective, IL-10-independent and IL-12-independent upregulation of antigen-specific T cell responses, supporting the role of IFN-beta as an immunomodulatory rather than an antiproliferative/immunosuppressive cytokine.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Epítopos de Linfocito T/inmunología , Interferón beta/farmacología , División Celular/inmunología , Células Clonales/citología , Células Clonales/inmunología , Células Clonales/metabolismo , Humanos , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Activación de Linfocitos/inmunología , Mitógenos/inmunologíaRESUMEN
Secretory phospholipases A2 (sPLA2s) are a group of extracellular enzymes that release fatty acids at the sn-2 position of phospholipids. Group IIA sPLA2 has been detected in inflammatory fluids, and its plasma level is increased in inflammatory diseases. To investigate a potential mechanism of sPLA2-induced inflammation we studied the effect of group IA (from cobra venom) and group IIA (human synovial) sPLA2s on human macrophages. Both sPLA2s induced a concentration- and Ca2+-dependent, noncytotoxic release of beta-glucuronidase (16.2 +/- 2.4% and 13.1 +/- 1.5% of the total content with groups IA and IIA, respectively). Both sPLA2s also increased the rate of secretion of IL-6 and enhanced the expression of IL-6 mRNA. Preincubation of macrophages with inhibitors of the hydrolytic activity of sPLA2 or cytosolic PLA2 did not influence the release of beta-glucuronidase. Incubation of macrophages with p-aminophenyl-mannopyranoside-BSA (mp-BSA), a ligand of the mannose receptor, also resulted in beta-glucuronidase release. However, while preincubation of macrophages with mp-BSA had no effect on beta-glucuronidase release induced by group IIA sPLA2, it enhanced that induced by group IA sPLA2. A blocking Ab anti-mannose receptor inhibited both mp-BSA- and group IIA-induced beta-glucuronidase release. Taken together, these data indicate that group IA and IIA sPLA2s activate macrophages with a mechanism independent from their enzymatic activities and probably related to the activation of the mannose receptor or sPLA2-specific receptors. The secretion of enzymes and cytokines induced by sPLA2s from human macrophages may play an important role in inflammation and tissue damage associated with the release of sPLA2s.
Asunto(s)
Glucuronidasa/metabolismo , Interleucina-6/biosíntesis , Pulmón/enzimología , Pulmón/inmunología , Macrófagos Alveolares/enzimología , Macrófagos Alveolares/inmunología , Fosfolipasas A/fisiología , Compuestos de Anilina/farmacología , Inhibidores Enzimáticos/farmacología , Humanos , Hidrólisis , Indoles/farmacología , Pulmón/citología , Pulmón/metabolismo , Macrófagos Alveolares/efectos de los fármacos , Macrófagos Alveolares/metabolismo , Manósidos/farmacología , Fosfolipasas A/antagonistas & inhibidores , Fosfolipasas A/metabolismo , Fosfolipasas A2 , Albúmina Sérica Bovina/farmacologíaRESUMEN
Interleukin (IL)-13 has been implicated in the pathogenesis of various diseases characterized by fibrosis. We describe the effects of IL-13 on collagen homeostasis from normal (NF) and keloid (KF) fibroblasts and compare these effects with those of IL-4 and transforming growth factor (TGF)-beta(1). Total collagen generation was up-regulated in NF after 48 h of stimulation by IL-13; in KF, IL-13 stimulated a more rapid collagen response. The kinetics and magnitude of collagen generation induced by IL-13 were equivalent to those induced by similar concentrations of IL-4 and TGF-beta(1). Collagen type I production paralleled total collagen generation from both NF and KF; however, IL-4-induced collagen type I and total collagen production from KF was more transient than that induced by either IL-13 or TGF-beta(1). Procollagen 1alpha1 gene expression was induced in KF by stimulation with IL-13 for 24 h. Moreover, IL-13 was unique among these three cytokines in its ability to induce gene expression for procollagen 3alpha1. Finally, IL-13 inhibited IL-1beta-induced matrix metalloproteinase (MMP)-1 and MMP-3 production and enhanced tissue inhibitor of metalloproteinase (TIMP)-1 generation from NF; although similar effects were observed with IL-4, TGF-beta(1) transiently enhanced MMP-1 and MMP-3 generation without effecting TIMP-1. In KF, IL-13 and IL-4 inhibited MMP-3, whereas TGF-beta(1) enhanced MMP-3; TIMP-1 was unaffected by any of the three cytokines. These data demonstrate both the profibrotic effects of IL-13 on collagen homeostasis and the potential differential regulation of collagen homeostasis in fibroblast subtypes by IL-13.
Asunto(s)
Colágeno/metabolismo , Interleucina-13/farmacología , Células Cultivadas , Fibroblastos/metabolismo , Homeostasis/efectos de los fármacos , Humanos , Interleucina-4/farmacología , Metaloproteinasa 3 de la Matriz/biosíntesis , Piel/metabolismo , Inhibidor Tisular de Metaloproteinasa-1/biosíntesis , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
BACKGROUND: Cyclosporin A (CS) and tacrolimus (FK506, FK) are calcineurin antagonists used widely as T-cell immunosuppressants; however, their relative efficacy on antigen-stimulated T-cell subsets remains undefined. OBJECTIVE: We have examined the effects of CS and FK on antigen-driven proliferation and cytokine generation from human PBMCs and T-cell clones. METHODS: Proliferation was assessed by tritiated thymidine incorporation. Cytokine generation was assessed by reverse transcription-PCR and ELISA. RESULTS: Ragweed- and tetanus toxoid-driven proliferation of PBMCs was down-regulated equally by CS or FK. Gene expression for proinflammatory cytokines (IL-4, IL-5, IL-13, and IFN-gamma) assessed by reverse transcription-PCR was down-regulated in a concentration-dependent manner by either drug. Antigen-induced proliferation of ragweed-specific Th0, Th1, or Th2 clones was inhibited by either CS or FK. Cytokine gene expression and protein secretion into culture supernatants (IL-4, IL-5, IL-13, and IFN-gamma) were down-regulated in a concentration-dependent manner by either CS or FK in all relevant T-cell subsets. Interestingly, down-regulation of IL-5 protein generation from Th0 and Th2 clones was consistently less sensitive to either drug than was the effect on either IL-4 or IL-13 protein generation. CONCLUSION: CS and FK promote equivalent down-regulation of Th0, Th1, and Th2 responses; however, IL-5 generation is relatively insensitive to the immunomodulatory effects of calcineurin antagonists.
Asunto(s)
Inhibidores de la Calcineurina , Ciclosporina/farmacología , Activación de Linfocitos/efectos de los fármacos , Linfocitos T Colaboradores-Inductores/efectos de los fármacos , Tacrolimus/farmacología , Relación Dosis-Respuesta a Droga , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Hipersensibilidad/inmunología , Interferón gamma/metabolismo , Interleucinas/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Propionyl-L-carnitine (PrC) has been shown to exert beneficial effects in the treatment of myocardial and peripheral ischemia in man. These conditions are associated with the activation of circulating neutrophils and platelets. To determine whether PrC could affect the synthesis of lipid mediators known to influence neutrophil and platelet functions, we explored the effects of PrC on the synthesis of platelet-activating factor (PAF) and arachidonic acid (AA) metabolites. Preincubation (90 min) of human neutrophils with PrC (0.1-100 microM) inhibited the synthesis of PAF and of a PAF analog (1-alkyl-1'enyl-2-acetyl-sn-glycero-3-phosphoethanolamine: AEGPE) induced in vitro by the calcium ionophore A23187. In contrast, concentrations of PrC up to 100 microM did not influence the uptake of exogenous AA or the A23187-induced release of AA and eicosanoids from neutrophils in vitro. PrC (1 microM) also inhibited PAF synthesis from human platelets stimulated in vitro with thrombin, but had no effect on thrombin-induced aggregation. Oral administration of PrC (2 g/day for two weeks) to five normal volunteers resulted in a significant inhibition of PAF and AEGPE synthesis by neutrophils stimulated with A23187 ex vivo, with no effect on AA or eicosanoid release. These data indicate that PrC selectively inhibits in vitro and ex vivo PAF synthesis from human neutrophils and platelets without influencing AA metabolism or eicosanoid release. This effect of PrC might represent an additional mechanism by which this molecule can exert protective effects in tissue ischemia and in other inflammatory diseases associated with neutrophil and platelet activation.
Asunto(s)
Plaquetas/efectos de los fármacos , Cardiotónicos/farmacología , Carnitina/análogos & derivados , Neutrófilos/efectos de los fármacos , Factor de Activación Plaquetaria/biosíntesis , Adulto , Ácidos Araquidónicos/metabolismo , Plaquetas/metabolismo , Carnitina/farmacología , Eicosanoides/metabolismo , Humanos , Técnicas In Vitro , Isquemia/tratamiento farmacológico , Masculino , Persona de Mediana Edad , Neutrófilos/metabolismo , Factor de Activación Plaquetaria/efectos de los fármacosRESUMEN
BACKGROUND: IL-4 and IL-13 are related cytokines with similar functional properties. Differential regulation of IL-4 and IL-13 has not been described. OBJECTIVE: We have examined the effects of IFN-alpha on antigen-driven proliferation, IL-4 generation, and IL-13 generation from human PBMCs and T-cell clones. METHODS: Proliferation was assessed by 3H-thymidine incorporation. Cytokine generation was assessed by reverse transcription PCR and ELISA. Messenger RNA stability was assessed in the presence of actinomycin D. RESULTS: IFN-alpha induced a concentration-dependent inhibition of antigen-driven proliferation of TH1 and TH2 clones (median effective concentration, 150 to 200 U/mL); the sensitivity of TH1 and TH2 clones to IFN-alpha was not significantly different (P =.6). IFN-alpha induced an analogous concentration-dependent inhibition of antigen-driven IL-13 generation from TH1 and TH2 clones (median effective concentration, 100 U/mL); this effect was evident by 12 hours of culture and persisted beyond 48 hours. However, IL-4 generation from TH2 clones was insensitive to IFN-alpha at all concentrations and times tested (1 to 10,000 U/mL). A similar inhibitory effect of IFN-alpha on mitogen-driven proliferation and IL-13 generation from PBMCs was demonstrated; once again, IL-4 generation from PBMCs was insensitive to IFN-alpha. IL-13 mRNA stability was unaffected by IFN-alpha, suggesting transcriptional regulation. CONCLUSION: IFN-alpha differentially regulates antigen-stimulated IL-4 and IL-13 generation.
Asunto(s)
Antígenos/inmunología , Regulación de la Expresión Génica/efectos de los fármacos , Interferón-alfa/farmacología , Interleucina-13/biosíntesis , Interleucina-4/biosíntesis , Subgrupos de Linfocitos T/efectos de los fármacos , Adulto , Células Clonales/efectos de los fármacos , Células Clonales/metabolismo , Dactinomicina/farmacología , Depresión Química , Ensayo de Inmunoadsorción Enzimática , Humanos , Interleucina-13/genética , Interleucina-4/genética , Células Jurkat/efectos de los fármacos , Células Jurkat/inmunología , Células Jurkat/metabolismo , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Activación de Linfocitos/efectos de los fármacos , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Fitohemaglutininas/farmacología , Lectinas de Plantas , Polen , ARN Mensajero/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Rinitis Alérgica Estacional/sangre , Rinitis Alérgica Estacional/inmunología , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Células TH1/efectos de los fármacos , Células TH1/inmunología , Células TH1/metabolismo , Células Th2/efectos de los fármacos , Células Th2/inmunología , Células Th2/metabolismo , Transcripción Genética/efectos de los fármacosRESUMEN
Twenty-two patients with systemic lupus erythematosus and 13 healthy controls were included in a cerebral blood flow study and underwent brain-dedicated single-photon emission computed tomography using 99m technetium-d, l-hexamethylpropylene amine oxime together with a brain computed tomography scan. Plasma levels of antiphospholipid antibodies (lupus anticoagulant and anticardiolipin IgM and IgG antibodies) were also determined. Brain computed tomography showed signs of focal cerebral ischemia in 4 patients (18%), whereas cerebral blood flow by single-photon emission computed tomography was abnormal in 13 of 22 patients (59%), who showed bilateral or monolateral hypoperfusion in the temporo-parietal regions. Patients with abnormal cerebral blood flow had a longer duration of disease than those with normal blood flow (8.9 +/- 1.9 years vs. 5.3 +/- 1.5 years, P < 0.05). Plasma antiphospholipid antibodies were present in 15 patients (68%), but the prevalence was similar in those with normal (6/9, 66%), or abnormal (9/13, 69%) cerebral blood flow. No statistically significant difference in lupus anticoagulant or anticardiolipin antibodies was observed between patients with and without cerebral blood flow abnormalities. Our study shows that patients with systemic lupus erythematosus frequently have cerebral blood flow abnormalities, which could precede those observed by computed tomography. Plasma lupus anticoagulant and anticardiolipin titers were not correlated with normal cerebral blood flow.
Asunto(s)
Anticuerpos Anticardiolipina/sangre , Enfermedades Autoinmunes/fisiopatología , Isquemia Encefálica/diagnóstico por imagen , Circulación Cerebrovascular , Inhibidor de Coagulación del Lupus/sangre , Lupus Eritematoso Sistémico/fisiopatología , Adolescente , Adulto , Enfermedades Autoinmunes/complicaciones , Enfermedades Autoinmunes/inmunología , Isquemia Encefálica/complicaciones , Isquemia Encefálica/etiología , Isquemia Encefálica/inmunología , Trastornos Cerebrovasculares/diagnóstico por imagen , Trastornos Cerebrovasculares/etiología , Niño , Femenino , Humanos , Lupus Eritematoso Sistémico/complicaciones , Lupus Eritematoso Sistémico/inmunología , Masculino , Persona de Mediana Edad , Sensibilidad y Especificidad , Exametazima de Tecnecio Tc 99m , Tomografía Computarizada de Emisión de Fotón Único , Tomografía Computarizada por Rayos X , Vasculitis/diagnóstico por imagen , Vasculitis/etiologíaRESUMEN
Purified human basophils were examined for changes in diacylglycerol levels to determine whether the transient nature of a N-formyl-methionyl-leucyl-phenylalanine (fMLP) -stimulated elevation in membrane protein kinase C (PKC) activity could be explained by the transient production of diacylglycerol (DAG). In preliminary experiments total DAG levels were measured by the DAG kinase assay. Although elevations followed stimulation with 1 microM fMLP (basal levels of 15 pmol/10(6) basophils vs. 45 pmol/10(6) basophils at the 3-min time point), there were no detectable changes in the first 60 s of the reaction. Histamine release is typically complete by 30-45 s. Measurement of inositol trisphosphate indicated a rapid increase by 5 s of 2.5 pmol/10(6) basophils. If DAG were produced at similar levels, the DAG kinase assay would not have detected the elevation. Consequently, fMLP-stimulated basophils were examined for changes in 1-stearoyl, 2-arachidonoyl, 3-sn-glycerol (SA-DAG) and 1-oleoyl, 2-arachidonoyl, 3-sn-glycerol by GC-NICIMS (negative ion chemical ionization mass spectroscopy). A 5-s elevation in these two species averaged 2 pmol/10(6) basophils, consistent with the inositol trisphosphate levels and occurring during the period of histamine release. However, a much more pronounced second phase to the SA-DAG response also occurred, mirroring the total DAG levels. This second phase of the DAG response, either total or SA-DAG, was transient on a time scale temporally coincident with the appearance and resolution of degranulation sacs as measured by fluorescence microscopy. These data suggest that there is selective generation of DAG species in the early reaction and the later appearance of DAG may be related to the formation and resolution of granule structures that follow the secretion of histamine.
Asunto(s)
Basófilos/metabolismo , Basófilos/fisiología , Diglicéridos/biosíntesis , Liberación de Histamina/fisiología , Prueba de Desgranulación de los Basófilos , Basófilos/efectos de los fármacos , Degranulación de la Célula/efectos de los fármacos , Degranulación de la Célula/fisiología , Humanos , Cinética , N-Formilmetionina Leucil-Fenilalanina/farmacología , Proteína Quinasa C/metabolismo , Transducción de Señal/fisiología , Estimulación QuímicaRESUMEN
Investigation of the role of diacylglycerol molecular species in signal transduction in human basophils has been impeded by the lack of an assay method with adequate sensitivity and selectivity. Conversion of 1-stearoyl-2-arachidonoyl-sn-3-glycerol to the pentafluorobenzoyl ester conveys electron-capture properties to the diacylglycerol. The electron-capture derivative of the diacylglycerol is amenable to gas chromatographic analysis and undergoes limited fragmentation under negative ion mass spectrometric conditions with generation of an intense molecular anion at m/z 838. Monitoring m/z 838 for detection of 1-stearoyl-2-arachidonoyl-sn-3-glycerol and m/z 841 for detection of 1-trideuterostearoyl-3-arachidonoyl-sn-2-glycerol employed as the internal standard provides the analytical basis for GC-MS quantitation of the endogenous diacylglycerol in human basophils. The assay displays excellent reproducibility over a wide range of concentrations with variations < or = 10%. The GC-MS assay is highly selective and exquisitely sensitive with a detection limit of < or = 0.20 pg (approximately 30 fmol) for endogenous 1-stearoyl-2-arachidonoyl-sn-3-glycerol per injection. Approximately 400 fmol of the diacylglycerol were extracted from 10(5) stimulated human basophils.
Asunto(s)
Basófilos/química , Diglicéridos/análisis , Cromatografía de Gases y Espectrometría de Masas/métodos , Benzoatos , Calibración , Catálisis , Esterificación , Humanos , Microquímica , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
Increasing evidence suggests that the metabolism of arachidonic acid (AA) may be different in inflammatory cells isolated from blood or migrating into tissues. To explore the possibility that changes in AA metabolism between blood and tissue inflammatory cells could be due in part to a different content or distribution of AA in glycerolipid classes, we studied these parameters in six human inflammatory cells isolated from blood (eosinophils, monocytes, neutrophils, and platelets) or from the lung tissue (mast cells and macrophages). Lung cells generally had a higher total cellular content of AA than that found in the blood cells. In addition, both mast cells and macrophages had a large endogenous pool of AA associated with triglycerides (TG), containing 45 and 22% of their total cellular AA, respectively. To address the hypothesis that cells migrating into the lung had a higher cellular level of AA and a larger AA pool in TG, we studied neutrophils isolated from the bronchoalveolar lavage (BAL) of patients with adult respiratory distress syndrome. BAL neutrophils had a fourfold increase in cellular AA as compared with blood neutrophils and contained 25% of their AA in TG versus 3% in blood neutrophils. BAL neutrophils also had a higher number of cytoplasmic lipid bodies (8 +/- 3/cell) relative to blood neutrophils (2 +/- 1/cell). High concentrations of free AA were also found in the cell-free BAL fluid of adult respiratory distress syndrome patients. To explore whether changes in BAL neutrophils may be due to the exposure of the cells to high concentrations of exogenous AA found in BAL, we incubated blood neutrophils in culture with AA (10-100 microM) for 24 h. Neutrophils supplemented with AA had a 10-fold increase in the amount of AA associated with TG and a sixfold increase in the number of lipid bodies. In addition, supplementation with AA induced a dose-dependent formation of hypodense cells. Taken together, these data indicate that human inflammatory cells undergo a fundamental and consistent remodeling of AA pools as they mature or enter the lung from the blood. These biochemical and morphological changes can be mimicked in vitro by exposing the cells to high levels of AA. This mechanism may be responsible for the changes in AA mobilization and eicosanoid metabolism observed in tissue inflammatory cells.
Asunto(s)
Ácido Araquidónico/metabolismo , Células Sanguíneas/fisiología , Pulmón/patología , Mastocitos/fisiología , Fagocitos/fisiología , Triglicéridos/metabolismo , Adulto , Ácido Araquidónico/farmacología , Plaquetas/fisiología , Líquido del Lavado Bronquioalveolar/citología , Movimiento Celular , Quimiotaxis de Leucocito , Eosinófilos/fisiología , Humanos , Inflamación , Metabolismo de los Lípidos , Pulmón/metabolismo , Macrófagos/fisiología , Monocitos/fisiología , Neutrófilos/fisiología , Edema Pulmonar/patologíaRESUMEN
The distribution of arachidonic acid (AA) within intracellular lipid pools of inflammatory cells is thought to be an important factor regulating the production of eicosanoids. We have recently identified a pool of AA associated with triglycerides (TGs) in human lung macrophages. This pool is also present in other human inflammatory cells (mast cells, eosinophils, monocytes and platelets) and it contains a percentage of total cellular AA ranging from 10 to 45%. Kinetic studies of AA incorporation have shown that TG are the first acceptor pool for exogenous AA that is subsequently transferred to phospholipid pools. During cell activation, AA is released from phospholipids; however, a large amount of AA is rapidly reincorporated into TGs. The TG pool also supplies AA to the phospholipid pools once these have been depleted during cell activation. These data suggest that TGs are not only a storage site for AA but may also be important as regulators of AA metabolism and eicosanoid biosynthesis in human inflammatory cells.
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Ácido Araquidónico/metabolismo , Triglicéridos/metabolismo , Compartimento Celular , Humanos , Líquido Intracelular/metabolismo , Leucocitos/inmunología , Leucocitos/metabolismo , Mastocitos/inmunología , Mastocitos/metabolismo , Fagocitos/inmunología , Fagocitos/metabolismo , Fosfolípidos/metabolismo , Transducción de SeñalRESUMEN
Platelet-activating factor (PAF) can exert profound inflammatory effects at very low concentrations. In plasma, PAF is hydrolyzed to lyso-PAF by acetylhydrolase, an enzyme that circulates bound to LDL. Previous studies suggest that oxygen radicals may act synergistically with PAF to potentiate tissue injury. However, mechanisms underlying this interaction have not been elucidated. In this study we investigated whether oxygen radicals may inactivate PAF acetylhydrolase. PAF acetylhydrolase activity was measured in human plasma and purified LDL before and after exposure to radicals (10-20 nmol/min per ml) generated by xanthine/xanthine oxidase. Oxygen radicals induced > 50% loss of PAF acetylhydrolase activity within 60 s and almost complete inactivation by 10 min. This phenomenon was irreversible and independent of oxidative modification of LDL. Inactivation occurred without changes in the affinity constant of the enzyme (Km was 17.9 microM under control conditions and 15.1 microM after exposure to oxygen radicals). Inactivation was prevented by the scavengers superoxide dismutase or dimethylthiourea or by the iron chelator deferoxamine. Thus, superoxide-mediated, iron-catalyzed formation of hydroxyl radicals can rapidly and irreversibly inactivate PAF acetylhydrolase. Since concomitant production of PAF and oxygen radicals can occur in various forms of tissue injury, inactivation of acetylhydrolase might represent one mechanism by which oxygen radicals may potentiate and prolong the proinflammatory effects of PAF.
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Oxígeno/farmacología , Fosfolipasas A/antagonistas & inhibidores , Factor de Activación Plaquetaria/metabolismo , 1-Alquil-2-acetilglicerofosfocolina Esterasa , Radicales Libres , Humanos , Lipoproteínas LDL/metabolismo , Fosfolipasas A/sangreRESUMEN
Arachidonic acid (AA) incorporation into and release from cellular glycerolipids are thought to be crucial events for the regulation of eicosanoid biosynthesis in inflammatory cells. The goal of our study was to determine the distribution of AA in the lipid pools of human lung macrophages (HLM) isolated from human lung parenchyma and to define the changes in AA pools occurring during cell activation. Mass spectrometry analysis indicated that the major pools of AA in HLM were located in phosphatidylethanolamine (PE), phosphatidylcholine (PC), and triglycerides (TG), and, to a lesser extent, in phosphatidylinositol/phosphatidylserine (PI/PS) and in a phospholipid similar to bis-(monoacylglyceryl)-phosphate (BMP). Exogenous AA was initially incorporated into TG and subsequently distributed within phospholipids. After 24 h of labeling, the distribution of exogenous AA in glycerolipid classes closely mimicked that of endogenous AA. Under these equilibrium conditions, HLM released 18.7 and 20.2% of cellular AA when stimulated with TPA or A23187, respectively. Free AA was the major product released by TPA- or A23187-stimulated HLM. However, A23187 but not TPA also induced the formation of leukotriene B4 and 5-HETE. AA was released from PC > PI/PS > or = PE > BMP. In contrast to phospholipids, the amount of AA in TG increased 15 to 90 min after cell activation and returned to prestimulation levels after 180 min. These data indicate that AA is stored in several glycerolipid pools of HLM. Among these pools, BMP is unique of macrophages and TG are particularly enriched in AA in HLM. During the early stage of cell activation, phospholipids act as a source of AA, whereas TG function as a reacylation pool for AA released from phospholipids. These data indicate that TG and phospholipid pools have a differential role in the control of free AA levels generated during HLM activation.
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Ácido Araquidónico/metabolismo , Activación de Macrófagos , Macrófagos Alveolares/metabolismo , Fosfolípidos/metabolismo , Triglicéridos/metabolismo , Calcimicina/farmacología , Eicosanoides/metabolismo , Humanos , Técnicas In Vitro , Fosfolipasas/metabolismo , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
BACKGROUND: Platelet activating factor (PAF) is a phospholipid released upon stimulation by a variety of cells and has been implicated in several pathophysiological events such as asthma and inflammatory diseases. However, although the ability to aggregate platelets in vitro was the first biological activity ascribed to PAF, its role in contributing to the in vivo formation of arterial thrombi has not been thoroughly clarified. METHODS AND RESULTS: Intravascular platelet aggregation was initiated in two different animal models of arterial stenosis and endothelial injury. An external constrictor was positioned around rabbit carotid arteries and canine coronary arteries. After placement of the constrictor, a typical pattern of flow developed in the stenotic vessels. This pattern of flow, characterized by progressive reductions of carotid or coronary blood flow followed by spontaneous or induced restorations of flow (cyclic flow variations, CFVs), is related to recurrent platelet aggregation at the site of the stenosis followed by dislodgment of the thrombus. After observing CFVs for 30 minutes, BN52021 (up to 1.2 mg/kg), a potent and selective PAF antagonist, was given intravenously to rabbits (n = 12) and dogs (n = 10). BN52021 completely inhibited CFVs in 10 of 12 rabbits, whereas it was relatively ineffective in abolishing CFVs in dogs (only 2 of 10 animals inhibited). This different effect of BN52021 was not explained by too small a dose of the drug to achieve a complete blockade of PAF receptors in dogs, since ex vivo platelet aggregation was completely inhibited in both rabbits and dogs in response to exogenous PAF at concentrations up to 10(-5) mol/L. In a second group of 10 dogs, the hypothesis that PAF may become an important mediator of CFVs in dogs only several hours after endothelial injury was tested. After 30 minutes of baseline CFVs, these animals received a bolus of BN52021 up to 1.2 mg/kg. After this treatment, CFVs were completely abolished in 2 of 10 animals. The remaining 8 dogs were followed for an additional 8-hour period, at the end of which a second bolus of BN52021 was given. At this time, BN52021 was effective, as CFVs were abolished in 6 of 8 animals. These effects of BN52021 at 8 hours were not the consequence of a cumulative dose of the compound, since ex vivo platelet aggregation in response to PAF returned to baseline values immediately before administering the second dose. To identify possible sources of PAF other than aggregating platelets at the site of arterial stenosis, dogs in a third group were killed after 30 minutes (n = 7) and after 8 hours (n = 8) of CFVs. Histological sections of the stenotic coronary artery showed a marked leukocyte infiltration in these arterial segments after 8 hours of CFVs, whereas sections from dogs killed after 30 minutes showed only moderate or no infiltration. CONCLUSIONS: These data demonstrate that PAF plays an important role as a mediator of platelet aggregation in vivo in rabbits and dogs. In the canine model, PAF appears to become more important after leukocyte infiltration of the arterial wall, as it may contribute to initiating enough platelet activation to lead to cyclic flow variations at sites of arterial stenosis and endothelial injury. Data from the present study suggest that PAF antagonists may be used as antiplatelet agents.
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Diterpenos , Factor de Activación Plaquetaria/fisiología , Agregación Plaquetaria/fisiología , Animales , Velocidad del Flujo Sanguíneo/fisiología , Trombosis de las Arterias Carótidas/sangre , Trombosis de las Arterias Carótidas/fisiopatología , Trombosis Coronaria/sangre , Trombosis Coronaria/fisiopatología , Perros , Femenino , Ginkgólidos , Lactonas/farmacología , Leucocitos/metabolismo , Masculino , Extractos Vegetales/farmacología , Factor de Activación Plaquetaria/antagonistas & inhibidores , Conejos , Flujo Sanguíneo Regional/fisiología , Factores de TiempoRESUMEN
FK-506 and the structurally related macrolide rapamycin are high-affinity ligands for a specific binding protein (FK-506 binding protein). We examined the effects of FK-506 and rapamycin on the release of pre-formed (histamine) and de novo synthesized inflammatory mediators (prostaglandin D2) from mast cells isolated from human skin tissue. FK-506 (0.1 to 100 nM) concentration-dependently inhibited (5 to 65%) histamine release from skin mast cells activated by anti-IgE. FK-506 was more potent in skin mast cells than in basophils (IC40 = 2.15 +/- 0.78 nM versus 5.12 +/- 1.34 nM; p < 0.001), whereas the maximal inhibitory effect was higher in basophils than in skin mast cells (88.77 +/- 2.44% versus 67.30 +/- 3.98%; p < 0.01). FK-506 had little or no inhibitory effect on histamine release from skin mast cells challenged with compound A23187 and substance P, respectively, whereas it completely suppressed A23187-induced histamine release from basophils. FK-506 (0.1 to 100 nM) also inhibited (up to 65%) the de novo synthesis of prostaglandin D2 from skin mast cells challenged with anti-IgE. Despite its structural similarity to FK-506, rapamycin (10 to 300 nM) had little or no effect on the release of histamine from skin mast cells induced by anti-IgE, A23187, and substance P. However, rapamycin competitively antagonized the inhibitory effect of FK-506 on anti-IgE-induced histamine release from skin mast cells with a dissociation constant of about 14 nM. These data indicate that FK-506, but not rapamycin, is a potent anti-inflammatory agent acting on skin mast cells presumably by binding to the FK-506 binding protein. It thus appears that binding to the FK-506 binding protein is necessary, but not sufficient, to deliver an inhibitory signal to skin mast cells.
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Antiinflamatorios no Esteroideos/farmacología , Mastocitos/efectos de los fármacos , Piel/citología , Tacrolimus/farmacología , Interacciones Farmacológicas , Femenino , Liberación de Histamina/efectos de los fármacos , Humanos , Inmunoglobulina E/farmacología , Inmunosupresores/farmacología , Mastocitos/metabolismo , Polienos/farmacología , Prostaglandina D2/biosíntesis , Sirolimus , Tacrolimus/farmacocinéticaRESUMEN
Epidemiological and clinical evidence have indicated that apolipoprotein A1 and B determination can better define the lipoprotein pattern in normal subjects and in subjects with coronary heart disease. In this paper, a recent immunoturbidimetric method for routine apolipoprotein A1 and B measurement (using the Turbitimer system and commercially available antisera) has been evaluated. The precision and the accuracy of the method have been previously considered. Within-run and between-run coefficients of variation (ranging from 1.67% to 5.04%) for both assays indicate good precision of the method. Accuracy was evaluated on 2 consecutive days (n = 10 each run) using a standard serum for apolipoprotein A1 and B. The bias obtained was 3.79% for apolipoprotein A1 and 2.30% for B. Apolipoproteins A1 and B were then measured in 100 normal and hyperlipemic sera with the immunoturbidimetric assay and radial immunodiffusion (using the monoclonal antibodies). The data obtained were evaluated by linear regression analysis (Al, r = 0.893; B, r = 0.862). The good correlation between the two methods suggests that the immunoturbidimetric assay can be usefully performed for routine apolipoprotein A1 and B determination because of its lower cost, rapidity, and simplicity.