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1.
Internist (Berl) ; 59(5): 486-493, 2018 May.
Artigo em Alemão | MEDLINE | ID: mdl-28748250

RESUMO

A 28-year-old Syrian refugee presented with right-sided knee pain and progressive deterioration of the general condition over the past months. Laboratory diagnostics revealed severe hypercalcemia due to primary hyperparathyroidism, and computed tomography (CT) scanning demonstrated disseminated osteolytic lesions throughout the skeleton. Histologically, these lesions were characterized by multinuclear giant cells (defining these lesions as so-called brown tumors). Finally, surgical removal of a jugular mass allowed the histopathologic diagnosis of a sporadic parathyroid carcinoma. In the patient, this condition was associated with a mutation in the HPRT2 gene locus.


Assuntos
Hipercalcemia , Hiperparatireoidismo Primário , Osteíte Fibrosa Cística , Neoplasias das Paratireoides , Refugiados , Adulto , Humanos , Hipercalcemia/complicações , Hipercalcemia/diagnóstico , Hiperparatireoidismo Primário/complicações , Hiperparatireoidismo Primário/diagnóstico , Osteíte Fibrosa Cística/diagnóstico , Osteíte Fibrosa Cística/etiologia , Neoplasias das Paratireoides/complicações , Neoplasias das Paratireoides/diagnóstico
2.
Klin Padiatr ; 228(3): 145-8, 2016 Apr.
Artigo em Alemão | MEDLINE | ID: mdl-27135272

RESUMO

BACKGROUND: Since the amendment of the Social Law V in Germany in 2007 the financial basis for a Specialised Home Palliative Care for Children (SHPC) for children was established. In Hesse 3 different SHPC teams entered into collective negotiations with health insurance companies. In 2014, the team of the University Children's Hospital in Giessen started to treat the first patient with a lead time of two months. METHODS: Thus in this paper the development of a SHPC team is described. After the first year anonymized patients data were retrospectively analyzed. RESULTS: Within 12 months 35 patients, 24 females and 11 males, were treated. All of the 6 patients who died, died at home. Calculated 48 weeks survival was 78%. 45% of the patients suffered from malignancies, 34% of malformations and 34% had metabolic disorders. 51% needed crisis intervention and 51% infusion therapy. Only 26% of parents denied cardiopulmonary resuscitation (CPR). Only 10% of the patients or their families received professional psychological care. CONCLUSION: Formation of a SHPC is feasible within a short time period once a financial basis is established. So, empathic guidance of families to help decision making for emergency situations are considered to be important. Analysis of patient's data after one year could help to improve the quality of care. Our data provides information for developing a palliative care team und could motivate colleagues to start the job.


Assuntos
Anormalidades Congênitas/terapia , Serviços de Assistência Domiciliar/organização & administração , Doenças Metabólicas/terapia , Neoplasias/terapia , Cuidados Paliativos/organização & administração , Equipe de Assistência ao Paciente/organização & administração , Adolescente , Causas de Morte , Criança , Pré-Escolar , Anormalidades Congênitas/mortalidade , Feminino , Alemanha , Serviços de Assistência Domiciliar/legislação & jurisprudência , Hospitais Universitários , Humanos , Lactente , Recém-Nascido , Masculino , Doenças Metabólicas/mortalidade , Programas Nacionais de Saúde/legislação & jurisprudência , Neoplasias/mortalidade , Cuidados Paliativos/legislação & jurisprudência , Equipe de Assistência ao Paciente/legislação & jurisprudência , Ordens quanto à Conduta (Ética Médica)/legislação & jurisprudência , Estudos Retrospectivos , Análise de Sobrevida
3.
Eur Respir J ; 36(1): 187-95, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20032014

RESUMO

Anti-neutrophil cytoplasmic antibodies (c-ANCA) targeting proteinase 3 (PR3) are implicated in the pathogenesis of Wegener's granulomatosis (WG). Fulminant disease can present as acute lung injury (ALI). In this study, a model of ALI in WG was developed using isolated rat lungs. Isolated human polymorphonuclear leukocytes (PMNs) were primed with tumour necrosis factor (TNF) to induce surface expression of PR3. Co-perfusion of TNF-primed neutrophils and monoclonal anti-PR3 antibodies induced a massive weight gain in isolated lungs. This effect was not observed when control immunoglobulin G was co-perfused with TNF-primed PMNs. The c-ANCA-induced oedema formation was paralleled by an increase in the capillary filtration coefficient as a marker of increased pulmonary endothelial permeability. In contrast, pulmonary artery pressure was not affected. In the presence of the oxygen radical scavenger superoxide dismutase and a NADPH oxidase inhibitor, c-ANCA-induced lung oedema could be prevented. Inhibition of neutrophil elastase was equally effective in preventing c-ANCA-induced lung injury. In conclusion, anti-PR3 antibodies induced neutrophil mediated, elastase- and oxygen radical-dependent ALI in the isolated lung. This experimental model supports the hypothesis of a pathogenic role for c-ANCA in WG and offers the possibility of the development of therapeutic strategies for the treatment of lung injury in fulminant WG.


Assuntos
Lesão Pulmonar Aguda/imunologia , Anticorpos Anticitoplasma de Neutrófilos/imunologia , Granulomatose com Poliangiite/imunologia , Neutrófilos/imunologia , Lesão Pulmonar Aguda/tratamento farmacológico , Lesão Pulmonar Aguda/patologia , Lesão Pulmonar Aguda/prevenção & controle , Animais , Anticorpos Anticitoplasma de Neutrófilos/farmacologia , Anticorpos Monoclonais/farmacologia , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Granulomatose com Poliangiite/tratamento farmacológico , Granulomatose com Poliangiite/prevenção & controle , Humanos , Imunoglobulina G/imunologia , Imunoglobulina G/farmacologia , Elastase de Leucócito/antagonistas & inibidores , Mieloblastina/imunologia , NADPH Oxidases/antagonistas & inibidores , Ativação de Neutrófilo/imunologia , Edema Pulmonar/imunologia , Edema Pulmonar/prevenção & controle , Ratos , Superóxido Dismutase/análise , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/farmacologia
4.
Br J Haematol ; 126(2): 252-4, 2004 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-15238147

RESUMO

The NB1 glycoprotein (CD177, HNA-2a antigen) is exclusively expressed on human neutrophils. As the clinical significance of CD177 expression is unknown, we investigated its expression in healthy individuals before and after stimulation with granulocyte colony-stimulating factor (G-CSF), in patients with rheumatoid arthritis, viral hepatitis, severe bacterial infections and polycythaemia vera. Expression was quantitatively determined by flow cytometry and by real time polymerase chain reaction. Only G-CSF-stimulated individuals and patients with severe bacterial infections and polycythaemia showed a significantly (P < 0.001) increased CD177 expression compared with healthy individuals, indicating that neutrophil CD177 expression can increase significantly in certain clinical conditions.


Assuntos
Infecções Bacterianas/imunologia , Isoantígenos/imunologia , Glicoproteínas de Membrana/imunologia , Neutrófilos/imunologia , Policitemia Vera/imunologia , Adulto , Artrite Reumatoide/imunologia , Estudos de Casos e Controles , Feminino , Citometria de Fluxo , Proteínas Ligadas por GPI , Fator Estimulador de Colônias de Granulócitos/farmacologia , Hepatite Viral Humana/imunologia , Humanos , Masculino , Receptores de Superfície Celular , Reação em Cadeia da Polimerase Via Transcriptase Reversa
5.
J Immunol ; 166(10): 6287-93, 2001 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-11342652

RESUMO

Although cytokine synthesis in polymorphonuclear leukocytes (PMN) was shown to be modulated by soluble mediators, the impact of microenvironmental conditions has not been elucidated. In this study, we investigated the effect of cell density on cytokine release from human neutrophils. PMN were cultured at various cell densities (10 x 10(6) PMN/ml; 60 x 10(6) PMN/ml), and LPS-induced release of cytokines was quantified by ELISA technique. Upon an increase in PMN density, secretion of the CXC chemokine IL-8 was progressively reduced. This effect was paralleled by a decrease in IL-8 mRNA. In contrast, TNF-alpha and IL-1beta rose proportionally with increasing cell density. The inhibition of IL-8 secretion was reproduced by conditioned media of PMN at high cell density, but was not affected by blocking beta(2) integrin-dependent adhesion. When analyzing the supernatant of LPS-challenged neutrophils, large amounts of soluble TNFRs p55 and p75 (sTNFRI, sTNFRII), and IL-1R antagonist (IL-1RA), rising constantly with the cell density, were detected. Interestingly, combined blocking of the bioactivities of these mediators completely restored neutrophil IL-8 secretion at high cell densities, with the anti-IL-1RA Ab being the more potent agent. Moreover, combined application of exogenous IL-1RA and sTNFRs to 10 x 10(6) PMN/ml reproduced the suppression of IL-8 generation. We conclude that neutrophil IL-8 synthesis is autoregulated, being suppressed under conditions of high cell density. IL-1RA and sTNFRs, accumulating under these circumstances, seem to be centrally involved in this regulatory mechanism by interfering with the IL-1beta- and TNF-alpha-dependent IL-8 generation. This feedback mechanism may control further neutrophil recruitment and activation in a neutrophil-rich environment, thereby preventing tissue destruction.


Assuntos
Interleucina-8/biossíntese , Neutrófilos/citologia , Neutrófilos/metabolismo , Receptores de Interleucina-1/antagonistas & inibidores , Receptores do Fator de Necrose Tumoral/fisiologia , Sialoglicoproteínas/fisiologia , Araquidonato 5-Lipoxigenase/metabolismo , Células Cultivadas , Meios de Cultivo Condicionados/farmacologia , Inibidores de Ciclo-Oxigenase/farmacologia , Dinoprostona/antagonistas & inibidores , Dinoprostona/metabolismo , Regulação para Baixo/imunologia , Humanos , Imunossupressores/farmacologia , Indóis/farmacologia , Indometacina/farmacologia , Proteína Antagonista do Receptor de Interleucina 1 , Interleucina-1/metabolismo , Interleucina-8/antagonistas & inibidores , Interleucina-8/metabolismo , Contagem de Leucócitos , Lipopolissacarídeos/farmacologia , Inibidores de Lipoxigenase , Neutrófilos/enzimologia , Fator de Ativação de Plaquetas/antagonistas & inibidores , Compostos de Piridínio/farmacologia , Sialoglicoproteínas/farmacologia , Solubilidade , Fator de Necrose Tumoral alfa/metabolismo , Regulação para Cima/imunologia
6.
Infect Immun ; 69(2): 897-905, 2001 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-11159983

RESUMO

The interaction of Listeria monocytogenes with endothelial cells represents a crucial step in the pathogenesis of listeriosis. Incubation of human umbilical vein endothelial cells (HUVEC) with wild-type L. monocytogenes (EGD) provoked immediate strong NO synthesis, attributable to listerial presentation of listeriolysin O (LLO), as the NO release was missed upon employment of a deletion mutant for LLO (EGD hly mutant) and was reproduced by purified LLO. Studies of conditions lacking extracellular Ca(2+) suggested LLO-elicited Ca(2+) flux as the underlying mechanism. In addition, HUVEC incubation with EGD turned out to be a potent stimulus for sustained (>12-h) upregulation of proinflammatory cytokine generation (interleukin 6 [IL-6], IL-8, and granulocyte-macrophage colony-stimulating factor). Use of deletion mutants for LLO (EGD hly mutant), listerial phosphatidylinositol-specific phospholipase C (EGD plcA mutant), broad-spectrum phospholipase C (EGD plcB mutant) and internalin B (EGD inlB mutant), as well as purified LLO, identified LLO as largely responsible for the cytokine response. Endothelial cells responded with diacylglycerole and ceramide generation as well as nuclear translocation of NF-kappa B to the stimulation with the LLO-producing strains EGD and Listeria innocua. The endothelial PC-phospholipase C inhibitor tricyclodecan-9-yl-xanthogenate as well as two independent inhibitors of NF-kappa B activation, pyrolidine dithiocarbamate and caffeic acid phenethyl ester, suppressed both the NF-kappa B translocation and the upregulation of cytokine synthesis. We conclude that L. monocytogenes is a potent stimulus of NO release and sustained upregulation of proinflammatory cytokine synthesis in human endothelial cells, both events being largely attributable to LLO presentation. LLO-induced transmembrane Ca(2+) flux as well as a sequence of endothelial phospholipase activation and the appearance of diacylglycerole, ceramide, and NF-kappa B are suggested as underlying host signaling events. These endothelial responses to L. monocytogenes may well contribute to the pathogenic sequelae in severe listerial infection and sepsis.


Assuntos
Toxinas Bacterianas , Endotélio Vascular/metabolismo , Proteínas de Choque Térmico/toxicidade , Mediadores da Inflamação/metabolismo , Listeria monocytogenes/patogenicidade , Cálcio/metabolismo , Células Cultivadas , Ceramidas/metabolismo , Diglicerídeos/metabolismo , Endotélio Vascular/citologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Proteínas Hemolisinas , Humanos , Interleucina-6/metabolismo , Interleucina-8/metabolismo , NF-kappa B/metabolismo , Óxido Nítrico/metabolismo , Virulência
7.
Crit Care Med ; 29(1): 1-7, 2001 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-11176149

RESUMO

OBJECTIVE: Myocardial depression, which frequently occurs in the course of septic shock, has been attributed to the cardiodepressant properties of nitric oxide (NO) generated by either the inducible NO synthase (iNOS) or the constitutive isoform (cNOS). We have previously demonstrated that alpha-toxin from Staphylococcus aureus induces thromboxane-mediated vasoconstriction accompanied by severe cardiodepression in isolated rat hearts. In the present study, we investigated the role of NO in the alpha-toxin-induced vascular and contractile abnormalities. DESIGN: Prospective, experimental study. SETTING: Research laboratory at a university hospital. SUBJECTS: Isolated hearts from male Wistar rats. INTERVENTIONS: Isolated hearts were perfused with purified staphylococcal alpha-toxin for 60 mins. MEASUREMENTS AND MAIN RESULTS: At a concentration of 0.25 and 0.5 microg/mL, alpha-toxin induced a rise in coronary perfusion pressure, depressed myocardial contractility, and caused edema formation. Simultaneously, a time- and dose-dependent rapid release of NO into the perfusate was noted as quantified by a chemiluminescence technique. L-NMMA, a nonselective inhibitor of NOS, but not PBITU, an iNOS-selective inhibitor, blocked NO synthesis, markedly increased the rise in coronary perfusion pressure and the loss in contractility, and enhanced edema formation in response to alpha-toxin. In contrast, zaprinast, a selective inhibitor of phosphodiesterase type V that is used for stabilization of cyclic guanosine monophosphate, attenuated the toxin-induced coronary vasoconstrictor response and the myocardial depression. L-arginine, the substrate of NOS, had similar, yet less potent, effects as zaprinast and slightly increased the release of NO caused by alpha-toxin. Immunohistochemical analysis of the myocardium at the end of the perfusion period demonstrated a positive staining for cNOS but not for iNOS. In addition, no up-regulation of iNOS mRNA was detected in the tissue of toxin-exposed hearts. CONCLUSIONS: Staphylococcal alpha-toxin provokes NO biosynthesis via activation of cNOS in rat hearts. NO partly antagonizes the deleterious effects of this pathogenicity factor on coronary vasoregulation and myocardial performance.


Assuntos
Baixo Débito Cardíaco/fisiopatologia , Circulação Coronária/fisiologia , Óxido Nítrico Sintase/fisiologia , Óxido Nítrico/biossíntese , Choque Séptico/fisiopatologia , Animais , Baixo Débito Cardíaco/metabolismo , Imuno-Histoquímica , Técnicas In Vitro , Medições Luminescentes , Masculino , Óxido Nítrico/fisiologia , Reação em Cadeia da Polimerase , Estudos Prospectivos , RNA Mensageiro/análise , Ratos , Ratos Wistar , Choque Séptico/metabolismo , Choque Séptico/microbiologia , Staphylococcus aureus , Estatísticas não Paramétricas , Fosfolipases Tipo C/fisiologia , Vasoconstrição/fisiologia
8.
J Leukoc Biol ; 69(1): 89-97, 2001 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-11200073

RESUMO

Anti-neutrophil cytoplasmic antibodies (ANCA) targeting proteinase 3 (PR3) possess a high sensitivity and specificity for Wegener's granulomatosis. Due to their capacity of directly activating neutrophils, a pathogenetic role for these autoantibodies has been proposed. We investigated the impact of subthreshold concentrations of monoclonal anti-PR3 antibodies (anti-PR3; 0.1 microg/mL) on neutrophil activation elicited by a secondary agent. Preincubation with anti-PR3 resulted in a massive amplification of N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced leukotriene (LT) generation, with a marked increase in the liberation of LTB4, LTA4, and 5-hydroxyeicosatetraenoic acid (5-HETE). This priming commenced within 2.5 min, with a maximum after 5-7.5 min. Moreover, anti-PR3 pretreatment markedly enhanced PMN movement toward fMLP. The priming effect of anti-PR3 toward fMLP challenge was reproduced by c-ANCA, but not by F(ab)2 fragments of the antibodies and isotype-matched control IgG. Generation of superoxide anion and release of elastase were suppressed in anti-PR3-pretreated neutrophils undergoing fMLP challenge. In contrast, neutrophil activation by platelet-activating factor (PAF) or the calcium ionophore A23187 remained unaffected. We conclude that subthreshold concentrations of anti-PR3 antibodies selectively modify neutrophil responses to fMLP, with enhancement of leukotriene generation and chemotaxis, but suppression of respiratory burst and degranulation. Such priming might contribute to localized neutrophil accumulation together with blunted host defense in Wegener's granulomatosis.


Assuntos
Anticorpos Anticitoplasma de Neutrófilos/imunologia , Quimiotaxia de Leucócito/imunologia , Leucotrienos/imunologia , Neutrófilos/imunologia , Animais , Anticorpos Anticitoplasma de Neutrófilos/farmacologia , Células Cultivadas , Quimiotaxia de Leucócito/efeitos dos fármacos , Humanos , Leucotrienos/biossíntese , Camundongos , Mieloblastina , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Neutrófilos/metabolismo , Serina Endopeptidases/imunologia
9.
Circulation ; 102(22): 2758-64, 2000 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-11094044

RESUMO

BACKGROUND: Although endotoxin (lipopolysaccharides, LPS) is recognized as a mediator of septic cardiodepression, its cardiac effects are still not fully elucidated. METHODS AND RESULTS: Perfusion of isolated rat hearts with LPS for 180 minutes resulted in a decline of left ventricular contractility after 90 minutes, whereas coronary perfusion pressure remained unaffected. This cardiodepression was paralleled by a release of tumor necrosis factor (TNF)-alpha into the perfusate and preceded by myocardial TNF-alpha mRNA upregulation as quantified by real-time polymerase chain reaction. The cardiodepression was abrogated when LPS was perfused with a TNF-alpha antiserum or the ceramidase inhibitor N:-oleoylethanolamine. In contrast, the cardiac release of nitric oxide (NO) was not augmented by LPS. Immunohistochemical studies of LPS-perfused hearts revealed a positive staining for the constitutive (NOSIII) but not for the inducible NO synthase (NOSII). Accordingly, NOSII mRNA levels commenced to increase only at the very end of the LPS perfusion period. Progressive liberation of thromboxane (Tx) A(2) and prostacyclin was induced by LPS together with myocardial cyclooxygenase (Cox)-2 mRNA expression. Both nonselective inhibition of Cox by indomethacin and selective inhibition of the inducible Cox-2 by NS-398 abolished prostanoid release. Interestingly, the generation of TNF-alpha and the associated cardiodepression caused by LPS were reduced by indomethacin, NS-398 and the Tx-receptor antagonist daltroban. CONCLUSIONS: LPS depresses contractility of isolated rat hearts by inducing TNF-alpha synthesis and subsequently activating the sphingomyelinase pathway, whereas no evidence for a role of NOSII- or NOSIII-generated NO was found. Moreover, Cox-2-derived TxA(2) appears to facilitate TNF-alpha synthesis in response to LPS.


Assuntos
Coração/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Contração Miocárdica/efeitos dos fármacos , Miocárdio/metabolismo , Fator de Necrose Tumoral alfa/biossíntese , Animais , Permeabilidade Capilar/efeitos dos fármacos , Creatina Quinase/efeitos dos fármacos , Creatina Quinase/metabolismo , Ciclo-Oxigenase 2 , Relação Dose-Resposta a Droga , Endocanabinoides , Inibidores Enzimáticos/farmacologia , Epoprostenol/metabolismo , Etanolaminas/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Coração/fisiologia , Soros Imunes/farmacologia , Técnicas In Vitro , Indometacina/farmacologia , Isoenzimas/genética , Isoenzimas/fisiologia , L-Lactato Desidrogenase/efeitos dos fármacos , L-Lactato Desidrogenase/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase/efeitos dos fármacos , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo II , Óxido Nítrico Sintase Tipo III , Nitrobenzenos/farmacologia , Ácidos Oleicos , Fenilacetatos/farmacologia , Prostaglandina-Endoperóxido Sintases/genética , Prostaglandina-Endoperóxido Sintases/fisiologia , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Esfingosina/fisiologia , Sulfonamidas/farmacologia , Tromboxano A2/metabolismo , Tromboxanos/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia
10.
Am J Respir Crit Care Med ; 162(2 Pt 1): 566-70, 2000 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-10934088

RESUMO

Obstructive sleep apnea (OSA) is associated with increased cardiovascular morbidity and mortality. Free oxygen radicals have been implicated in the pathogenesis of cardiovascular disorders. Therefore, we aimed to test the hypothesis that increased oxidative stress constitutes one underlying mechanism for the connection between OSA and cardiovascular disease. In 18 patients with OSA the release of superoxide from polymorphonuclear neutrophils was determined after stimulation with the bacterial tripeptide formylmethionylleucylphenylalanine (fMLP) and the calcium ionophore A23. Superoxide production was measured as superoxide dismutase-inhibitable reduction of cytochrome c. Blood samples were obtained before and after two nights of CPAP therapy and after 4.8 +/- 0.6 mo of follow-up. Ten healthy young volunteers and 10 lung cancer patients without OSA but a similar spectrum of comorbidity served as controls. Before CPAP, neutrophil superoxide generation was markedly enhanced in OSA when compared with both control groups. Effective CPAP therapy led to a rapid and long-lasting decrease of superoxide release in OSA. In conclusion, OSA is linked with a "priming" of neutrophils for enhanced respiratory burst. The increased superoxide generation, which might have major impact on the development of cardiovascular disorders, is virtually fully reversed by effective CPAP therapy.


Assuntos
Neutrófilos/metabolismo , Respiração com Pressão Positiva , Apneia Obstrutiva do Sono/terapia , Superóxidos/sangue , Adulto , Feminino , Humanos , Ionóforos/farmacologia , Masculino , Pessoa de Meia-Idade , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Polissonografia , Apneia Obstrutiva do Sono/sangue
11.
J Infect Dis ; 182(1): 191-9, 2000 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-10882597

RESUMO

The nonapeptide of polymyxin B (PMBN) has been reported to sensitize various pathogenic gram-negative bacteria to the direct bactericidal effect of human serum. To investigate the impact of PMBN on human neutrophil-effected killing of the serum- and phagocytosis-resistant Escherichia coli strains C14 and O111, serum was coapplied with PMBN or with neutrophils, but this did not result in decreased numbers of viable bacteria. In contrast, the most potent bacterial killing occurred in the presence of neutrophils plus serum components plus PMBN. The effect of this on E. coli C14 was the appearance of inositol phosphates, diacylglycerol, respiratory burst, elastase liberation, and generation of lipid mediators (leukotriene B(4), 5-HETE, and platelet-activating factor). Strong neutrophil activation required early, but not late, complement components and was blocked by inhibition of phagocytosis with cytochalasin D. PMBN seems to cause dramatic support of natural host defense by complement-dependent sensitization of E. coli to the bactericidal efficacy of human neutrophils.


Assuntos
Antibacterianos/farmacologia , Escherichia coli/efeitos dos fármacos , Lipopolissacarídeos/metabolismo , Neutrófilos/fisiologia , Polimixina B/análogos & derivados , Polimixina B/farmacologia , Respiração Celular , Quimiotaxia , Relação Dose-Resposta a Droga , Resistência Microbiana a Medicamentos , Escherichia coli/metabolismo , Escherichia coli/fisiologia , Humanos , Técnicas In Vitro , Metabolismo dos Lipídeos , Neutrófilos/enzimologia , Neutrófilos/metabolismo , Elastase Pancreática/metabolismo , Fagocitose , Fosfatidilinositóis/metabolismo
12.
Am J Physiol Lung Cell Mol Physiol ; 279(1): L100-9, 2000 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-10893208

RESUMO

Escherichia coli hemolysin (HlyA) is a prototype of a large family of pore-forming proteinaceous exotoxins that have been implicated in the pathogenetic sequelae of severe infection and sepsis, including development of acute lung injury. In the present study in rabbit alveolar macrophages (AMs), subcytolytic concentrations of purified HlyA evoked rapid synthesis of platelet-activating factor, with quantities approaching those in response to maximum calcium ionophore challenge. In parallel, large quantities of leukotriene (LT) B(4) and 5-, 8-, 9-, 12-, and 15-hydroxyeicosatetraenoic acid (HETE) were liberated from HlyA-exposed AMs depending on exogenous arachidonic acid (AA) supply. Coadministration of eicosapentaenoic acid (EPA) dose dependently suppressed generation of the proinflammatory lipoxygenase products LTB(4) and 5-, 8-, 9-, and 12-HETE in parallel with the appearance of the corresponding EPA-derived metabolites LTB(5) and 5-, 8-, 9-, and 12-hydroxyeicosapentaenoic acid (HEPE). At equimolar concentrations, EPA turned out to be the preferred substrate over AA for these AM lipoxygenase pathways, with the sum of LTB(5) and 5-, 8-, 9-, and 12-HEPE surpassing the sum of LTB(4) and 5-, 8-, 9-, and 12-HETE by >80-fold. In contrast, coadminstration of EPA did not significantly reduce HlyA-elicited generation of the anti-inflammatory AA lipoxygenase product 15-HETE. We conclude that AMs are sensitive target cells for HlyA attack, resulting in marked proinflammatory lipid mediator synthesis. In the presence of EPA, lipoxygenase product formation is shifted from a pro- to an anti-inflammatory profile.


Assuntos
Escherichia coli/metabolismo , Proteínas Hemolisinas/farmacologia , Mediadores da Inflamação/metabolismo , Metabolismo dos Lipídeos , Macrófagos Alveolares/metabolismo , Animais , Eicosanoides/biossíntese , Ácido Eicosapentaenoico/farmacologia , Proteínas Hemolisinas/metabolismo , Ácidos Hidroxieicosatetraenoicos/metabolismo , Mediadores da Inflamação/antagonistas & inibidores , Leucotrieno B4/metabolismo , Inibidores de Lipoxigenase/farmacologia , Macrófagos Alveolares/efeitos dos fármacos , Fator de Ativação de Plaquetas/metabolismo , Coelhos
13.
Circulation ; 101(1): 78-85, 2000.
Artigo em Inglês | MEDLINE | ID: mdl-10618308

RESUMO

BACKGROUND: Cardiac performance is severely depressed in septic shock. Endotoxin has been implicated as the causative agent in Gram-negative sepsis, but similar abnormalities are encountered in Gram-positive sepsis. We investigated the influence of the major exotoxin of Staphylococcus aureus, staphylococcal alpha-toxin, in isolated perfused rat hearts. METHODS AND RESULTS: Alpha-toxin 0.25 to 1 microg/mL caused a dose-dependent increase in coronary perfusion pressure that more than doubled. In parallel, we noted a decrease in left ventricular developed pressure and the maximum rate of left ventricular pressure rise (dP/dt(max)), dropping to a minimum of <60% of control. These changes were accompanied by a liberation of thromboxane A(2) and prostacyclin into the coronary effluent. The release of creatine kinase, lactate dehydrogenase, potassium, and lactate did not surpass control heart values, and leukotrienes were also not detected. Indomethacin, acetylsalicylic acid, and the thromboxane receptor antagonist daltroban fully blocked the alpha-toxin-induced coronary vasoconstrictor response and the decrease in left ventricular developed pressure and dP/dt(max), whereas the lipoxygenase inhibitor nordihydroguaiaretic acid, the platelet activating factor antagonist WEB 2086, and the alpha-adrenergic antagonist phentolamine were entirely ineffective. Inhibition of nitric oxide synthase even enhanced the alpha-toxin-induced increase in coronary perfusion pressure and the loss in myocardial performance. CONCLUSIONS: Purified staphylococcal alpha-toxin provokes coronary vasoconstriction and loss in myocardial contractility. The responses appear to be largely attributable to the generation of thromboxane and are even enhanced when the endogenous nitric oxide synthesis is blocked. Bacterial exotoxins, such as staphylococcal alpha-toxin, may thus be implicated in the loss of cardiac performance encountered in Gram-positive septic shock.


Assuntos
Toxinas Bacterianas/farmacologia , Epoprostenol/metabolismo , Coração/fisiologia , Proteínas Hemolisinas/farmacologia , Contração Miocárdica/efeitos dos fármacos , Tromboxano A2/metabolismo , Animais , Aspirina/farmacologia , Azepinas/farmacologia , Edema , Exotoxinas/farmacologia , Coração/efeitos dos fármacos , Técnicas In Vitro , Indometacina/farmacologia , L-Lactato Desidrogenase/análise , Lactatos/metabolismo , Masculino , Masoprocol/farmacologia , Perfusão , Fenilacetatos/farmacologia , Fator de Ativação de Plaquetas/antagonistas & inibidores , Inibidores da Agregação Plaquetária/farmacologia , Potássio/análise , Ratos , Ratos Wistar , Staphylococcus aureus , Sulfonamidas/farmacologia , Triazóis/farmacologia , Vasoconstrição/efeitos dos fármacos , Função Ventricular Esquerda/efeitos dos fármacos , Função Ventricular Esquerda/fisiologia
14.
Am J Respir Crit Care Med ; 160(3): 846-51, 1999 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-10471607

RESUMO

Inhalative vasodilator therapy, employing gaseous nitric oxide (NO) or aerosolized prostaglandin PGI(2), is of interest for regional pulmonary vasodilation in ARDS and pulmonary hypertension. We investigated the impact of the NO donor spermine NONOate as well as PGI(2) and its stable chemical analog iloprost on cultured rat alveolar epithelial type II cell (ATII) surfactant secretion. The NO donor provoked a significant increase in the ATII cGMP content, further enhanced by type V phosphodiesterase (PDE) inhibition, but affected neither baseline nor mechanical stretch-induced surfactant secretion. The prostanoids caused a marked increase in the epithelial cAMP content, further amplified by coadministration of type III/IV PDE inhibitors. Baseline surfactant secretion was not altered by this approach, but mechanical stretch-induced liberation of surfactant was significantly increased, most prominently in the ATII with the highest cAMP levels due to the presence of both iloprost and PDE III/IV inhibitors. In contrast, epithelial phosphoinositide metabolism, well responsive to purinergic stimulation as positive control, was unchanged in prostanoid-exposed cells. We conclude that the PGI(2)-cAMP axis, but not the NO-cGMP axis, forwards a markedly enhanced secretory response to the physiological stimulus of cell surface stretching, which may be relevant for therapeutic use of these agents.


Assuntos
Anti-Hipertensivos/farmacologia , Epoprostenol/farmacologia , Inibidores de Fosfodiesterase/farmacologia , Alvéolos Pulmonares/efeitos dos fármacos , Alvéolos Pulmonares/metabolismo , Surfactantes Pulmonares/metabolismo , Vasodilatadores/farmacologia , Análise de Variância , Animais , Células Cultivadas , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Iloprosta/farmacologia , Masculino , Estimulação Física , Alvéolos Pulmonares/citologia , Ratos , Ratos Sprague-Dawley , Transdução de Sinais
15.
Infect Immun ; 67(3): 1125-30, 1999 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-10024552

RESUMO

Polymorphonuclear leukocytes (PMN) are essential for resolution of infections with Listeria monocytogenes. The present study investigated the role of the listerial exotoxins listeriolysin (LLO) and phosphatidylinositol-specific phospholipase C (PlcA) in human neutrophil activation. Different Listeria strains, mutated in individual virulence genes, as well as purified LLO were used. Coincubation of human neutrophils with wild-type L. monocytogenes provoked PMN activation, occurring independently of phagocytosis events, with concomitant elastase secretion, leukotriene generation, platelet-activating factor (PAF) synthesis, respiratory burst, and enhanced phosphoinositide hydrolysis. Degranulation and leukotriene formation were noted to be solely dependent on LLO expression, as these features were absent when the LLO-defective mutant EGD- and the avirulent strain L. innocua were used. These effects were fully reproduced by a recombinant L. innocua strain expressing LLO (INN+) and by the purified LLO molecule. LLO secretion was also required for PAF synthesis. However, wild-type L. monocytogenes was more potent in eliciting PAF formation than mutants expressing LLO, suggesting the involvement of additional virulence factors. This was even more obvious for phosphoinositide hydrolysis and respiratory burst: these events were provoked not only by INN+ but also by the LLO-defective mutant EGD- and by a recombinant L. innocua strain producing listerial PlcA. We conclude that human neutrophils react to extracellularly provided listerial exotoxins by rapid cell activation. Listeriolysin is centrally involved in triggering degranulation and lipid mediator generation, and further virulence factors such as PlcA apparently contribute to trigger neutrophil phosphoinositide hydrolysis and respiratory burst. In this way, listerial exotoxins may influence the host defense against infections with L. monocytogenes.


Assuntos
Toxinas Bacterianas , Proteínas de Choque Térmico/fisiologia , Proteínas Hemolisinas/fisiologia , Listeria monocytogenes/patogenicidade , Ativação de Neutrófilo , Fosfolipases Tipo C/fisiologia , Humanos , Elastase de Leucócito/metabolismo , Leucotrienos/biossíntese , Fosfatidilinositol Diacilglicerol-Liase , Fosfatidilinositóis/metabolismo , Fosfoinositídeo Fosfolipase C , Explosão Respiratória
16.
Am J Respir Crit Care Med ; 159(1): 206-12, 1999 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-9872840

RESUMO

Alveolar epithelial type II cells (AET2) respond with exocytosis of surfactant containing lamellar bodies to stimulation with mechanical stretch and secretagogues, a process that is fundamental for maintaining alveolar stability and lung gas exchange. In the present study in cultured rat AET2, we employed botulinum C2 toxin, a binary toxin which ADP ribosylates nonmuscle G-actin, as a specific tool to probe the role of the actin microfilament system in the surfactant secretory process. Incubation of AET2 with C2 toxin caused a dose-dependent decay of the cellular F-actin content to a minimum of 20% of baseline, concomitant with an increase in monomeric actin. In parallel, a significant augmentation of baseline surfactant secretion up to twofold elevated levels above control was noted, as assessed by the release of prelabeled phosphatidylcholine. Pretreatment with phalloidin, which stabilized F-actin and reduced the level of G-actin, prevented the C2 toxin-elicited enhancement of baseline surfactant secretion. Even low C2 toxin concentrations, resulting in a reduction of total cellular F-actin content of approximately 10%, sufficed to augment secretagogue (ATP) and, more impressively, mechanical stress elicited an increase in surfactant secretion; the response to the biophysical challenge more than doubled. When investigated in the absence of toxin, different secretagogues (ATP, phorbol ester, betamimetics) caused a rapid-onset, transient reduction of F-actin in the range between 15 and 25% as a consistent part of their secretory response pattern. These data suggest that the state of actin polymerization is intimately linked to the exocytosis process underlying surfactant secretion in AET2. Microfilament system-related compartmentalization effects and/or or the impact of the state of actin assembly on signaling events may be considered as underlying events.


Assuntos
Actinas/metabolismo , Polímeros/metabolismo , Alvéolos Pulmonares/metabolismo , Surfactantes Pulmonares/metabolismo , Adulto , Animais , Toxinas Botulínicas/farmacologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Humanos , Isomerismo , Masculino , Faloidina/farmacologia , Estimulação Física , Alvéolos Pulmonares/citologia , Alvéolos Pulmonares/efeitos dos fármacos , Ratos , Ratos Endogâmicos , Estimulação Química
17.
Anal Biochem ; 261(1): 16-28, 1998 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-9683507

RESUMO

Quantification of lipoxygenase and cytochrome P450 products of both arachidonic acid (AA) and eicosapentaenoic acid (EPA) is of broad interest due to the multiple biological activities of these compounds. We developed a method combining (i) solid-phase extraction, (ii) isocratic reversed-phase high-performance liquid chromatographic separation, and (iii) online photodiode array detection with spectrum analysis for identification and measurement of all main 4- and 5-series eicosanoids (leukotrienes, hydroxyeicosatetraenoic acids/hydroxyeicosapentaenoic acids, epoxyeicosatrienoic acids) within one run. With these procedures, standard mixtures of AA- and EPA-derived lipid mediators were recovered from different biological liquids, like lung perfusate, human bronchoalveolar lavage fluid, and cell supernatant with linear characteristics for each compound. Recoveries of the different lipid mediators exceeded 80% showing excellent reproducibility. Application of the method to isolated, perfused, and ventilated human lungs challenged with the calcium ionophore A23187 and to human neutrophils stimulated in the presence of arachidonic acid and eicosapentaenoic acid with N-formyl-methionyl-leucyl-phenylalanine demonstrated the generation of a large array of lipoxygenase and cytochrome P450 products. Thus, convenient quantification of 4- and 5-series eicosanoids in fluids of biological interest is achieved by a technique comprising solid-phase extraction, isocratic reversed-phase high-performance liquid chromatography, and photodiode array-based online spectrum analysis of eluting compounds.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Eicosanoides/análise , Araquidonato 5-Lipoxigenase/metabolismo , Ácido Araquidônico/metabolismo , Líquido da Lavagem Broncoalveolar/química , Técnicas de Química Analítica , Sistema Enzimático do Citocromo P-450/metabolismo , Eicosanoides/química , Eicosanoides/metabolismo , Ácido Eicosapentaenoico/metabolismo , Granulócitos/metabolismo , Humanos , Técnicas In Vitro , Lipoxigenase/metabolismo , Pulmão/metabolismo , Perfusão , Espectrofotometria Ultravioleta
18.
J Exp Med ; 187(4): 497-503, 1998 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-9463400

RESUMO

Anti-neutrophil cytoplasmic antibodies (ANCAs) targeting proteinase 3 (PR3) have a high specifity for Wegener's granulomatosis (WG), and their role in activating leukocytes is well appreciated. In this study, we investigated the influence of PR3-ANCA and murine monoclonal antibodies on human umbilical vascular endothelial cells (HUVECs). Priming of HUVECs with tumor necrosis factor alpha induced endothelial upregulation of PR3 message and surface expression of this antigen, as measured by Cyto-ELISA, with a maximum occurrence after 2 h. Primed cells responded to low concentrations of both antibodies (25 ng-2.5 microg/ml), but not to control immunoglobulins, with pronounced, dose-dependent phosphoinositide hydrolysis, as assessed by accumulation of inositol phosphates. The signaling response peaked after 20 min, in parallel with the appearance of marked prostacyclin and platelet-activating factor synthesis. The F(ab)2 fragment of ANCA was equally potent as ANCA itself. Disrupture of the endothelial F-actin content by botulinum C2 toxin to avoid antigen-antibody internalization did not affect the response. In addition to the metabolic events, anti-PR3 challenge, in the absence of plasma components, provoked delayed, dose-dependent increase in transendothelial protein leakage. We conclude that anti-PR3 antibodies are potent inductors of the preformed phosphoinositide hydrolysis-related signal tranduction pathway in human endothelial cells. Associated metabolic events and the loss of endothelial barrier properties suggest that anti-PR3-induced activation of endothelial cells may contribute to the pathogenetic sequelae of autoimmune vasculitis characterizing WG.


Assuntos
Anticorpos Anticitoplasma de Neutrófilos/imunologia , Anticorpos Monoclonais/imunologia , Endotélio Vascular/imunologia , Granulomatose com Poliangiite/imunologia , Serina Endopeptidases/imunologia , Transdução de Sinais , Comunicação Celular , Células Cultivadas , Selectina E/metabolismo , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/patologia , Ensaio de Imunoadsorção Enzimática , Granulomatose com Poliangiite/patologia , Humanos , Mieloblastina , Fosfatidilinositóis/metabolismo , Fator de Ativação de Plaquetas/metabolismo , Reação em Cadeia da Polimerase , Fator de Necrose Tumoral alfa/farmacologia , Molécula 1 de Adesão de Célula Vascular/metabolismo
19.
Eur J Clin Invest ; 27(11): 893-9, 1997 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-9395784

RESUMO

At present, the exact mechanism of the pathogenic effect of anti-PR-3 antibodies remains unknown. Interaction of anti-neutrophil cytoplasmic antibodies (ANCAs) with human umbilical vein endothelial cells (HUVECs) may play a key role. Recently we were able to show that ANCAs recognize their target antigen, PR-3, translocated into the membrane of HUVECs. The objective of this study was to investigate regulation, i.e. signal transduction pathways, of PR-3 expression in endothelial cells. HUVECs were isolated according to the method of Jaffe et al. and cultured under standard conditions. A cyto-enzyme-linked immunosorbent assay (ELISA) with unfixed cells was performed. Membrane-expressed PR-3 was detected by affinity-purified and monoclonal anti-PR-3 Ab. Tumour necrosis factor alpha (TNF-alpha)-induced membrane expression of PR-3 could be blocked with the RNA synthesis inhibitor actinomycin D, the protein kinase C (PKC) and proteinase A (PKA) inhibitor staurosporine, the specific PKA inhibitor calphostin C, the c-AMP-dependent PKA inhibitor KT5720 and the tyrosine kinase inhibitor genistein in a dose-dependent manner. The effect of calphostin C was the most significant. In addition, the effect of phorbol 12-myristate 13-acetate (PMA), a mediator of intracellular second messengers, was investigated. In our study, pretreatment of cells with PMA for 48 h led to a down-regulation of PR-3 expression. This effect, however, could be overridden by TNF-alpha stimulation, i.e. TNF-alpha-induced membrane expression of PR-3 was resistant to down-regulation of PKC. In conclusion, our data suggest that translocation of PR-3 in HUVECs is an active process depending on protein synthesis. PR-3 expression by HUVECs may involve a PKC reactive to cytokines such as TNF-alpha which induces PR-3 expression at a transcriptional level.


Assuntos
Endotélio Vascular/enzimologia , Serina Endopeptidases/metabolismo , Transdução de Sinais , Membrana Celular/enzimologia , Dactinomicina/farmacologia , Humanos , Mieloblastina , Proteína Quinase C/fisiologia , Acetato de Tetradecanoilforbol/farmacologia , Fator de Necrose Tumoral alfa/farmacologia
20.
J Immunol ; 159(4): 1909-16, 1997 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-9257856

RESUMO

Escherichia coli hemolysin (HlyA) and Staphylococcus aureus alpha-toxin are membrane-perturbating bacterial exotoxins that have been implicated as significant virulence factors in human diseases. We investigated the capacity of these toxins to cause cell activation and mediator release in human endothelial cells, compared with the efficacies of thrombin and the Ca2+ ionophore A23187. Concentration ranges tested were 1 to 1000 ng/ml (HlyA), 0.01 to 10 micro/ml (alpha-toxin), 0.01 to 10 U/ml (thrombin), and 0.01 to 10 microM (A23187). All stimuli caused dose-dependent generation of platelet-activating factor, nitric oxide, and prostaglandin I2. HlyA and thrombin effected time- and dose-dependent accumulation of large quantities of inositol phosphates, with maximum effects at 100 ng/ml and 1 U/ml, respectively. Corresponding time course and dose dependency were noted for HlyA-elicited diacylglycerol formation. In contrast, only the highest concentrations of alpha-toxin (10 microg/ml) and A23187 (10 microM) effected some moderate inositol phosphate accumulation, and this was suppressed in the presence of the platelet-activating factor antagonist WEB 2086. Metabolic and secretory responses elicited by alpha-toxin were dependent on the presence of extracellular Ca2+. We conclude that both HlyA and alpha-toxin are potent inductors of inflammatory and vasodilatory mediators in human endothelial cells. HlyA-elicited effects may proceed predominantly via activation of the phosphatidylinositol hydrolysis-related signal transduction pathway, whereas transmembrane Ca2+ flux appears to be the major event underlying the release of mediators in response to alpha-toxin. These toxin properties may contribute to vasoregulatory and inflammatory disturbances encountered in states of severe infection and sepsis.


Assuntos
Proteínas de Bactérias/toxicidade , Toxinas Bacterianas/toxicidade , Endotélio Vascular/efeitos dos fármacos , Proteínas de Escherichia coli , Proteínas Hemolisinas/toxicidade , Animais , Cálcio/metabolismo , Endotélio Vascular/citologia , Endotélio Vascular/fisiologia , Epoprostenol/biossíntese , Humanos , Óxido Nítrico/biossíntese , Fosfatidilinositóis/metabolismo , Fator de Ativação de Plaquetas/biossíntese , Ratos , Trombina/farmacologia
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