RESUMEN
Recently we have demonstrated that recombinant human erythropoietin (EPO) protects neurosensory hair cells in the organotypic culture of the organ of Corti by reducing apoptosis and necrosis. In the present study, we tested the hypothesis that EPO may be involved in reparative angiogenesis. We analyzed in parallel the endogenous erythropoietin (Epo) mRNA expression and that of Epo receptor (Epor) and of genes associated with angiogenesis in the organ of Corti, the modiolus and the stria vascularis using real time reverse transcription polymerase chain reaction and microarray. We compared the expression levels of freshly prepared tissue (control) and tissue cultured for 24 h under normoxia or hypoxia. The basal expression of Epo- and Epor mRNA in controls of all regions was very low. However, after 24 h in culture, a 20-100 fold increase of these two transcripts was measured. In culture, the vascular endothelial growth factor and the Cxcr4 (the receptor for the stromal cell-derived factor-1, Sdf-1) mRNA levels, were found to be increased and the Sdf-1 mRNA level to be decreased. Changes in mRNA expression occurred in all pathways activated in non-erythroid cells by the application of EPO (phosphoinositide 3-kinase/serine-threonine protein kinase B, Janus-type protein tyrosine kinase 2/signal transducer and activator of transcription 3, and the mitogen activated protein kinase). These data suggest that the neuroprotective effect of EPO may include at least two molecular events, the decrease of hair cell death rate and the induction of angiogenic genes.
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Inductores de la Angiogénesis/metabolismo , Oído Interno/metabolismo , Eritropoyetina/metabolismo , Animales , Animales Recién Nacidos , Recuento de Células , Hipoxia de la Célula , Supervivencia Celular , Quimiocina CXCL12/genética , Quimiocina CXCL12/metabolismo , Oído Interno/citología , Oído Interno/lesiones , Células Ciliadas Auditivas , Análisis de Secuencia por Matrices de Oligonucleótidos , Técnicas de Cultivo de Órganos , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Receptores de Eritropoyetina/genética , Receptores de Eritropoyetina/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
OBJECTIVE: Growth differentiation factor-5 (GDF-5), a member of the transforming growth factor (TGF)-beta family, is involved in joint development during embryogenesis and has the potential to regenerate cartilage in adult animals. As progression of chronic joint diseases is influenced by cytokines of the synovial tissue, we examined the expression and effects of GDF-5 in this tissue. METHODS: Microarray experiments were investigated for differential expression of GDF-5 in synovial tissues, synovial fibroblasts, and peripheral blood cells. GDF-5 expression was validated by semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR), immunohistochemistry, double immunofluorescence, and in situ hybridization in synovial tissue of normal donors (ND) and patients with osteoarthritis (OA) and rheumatoid arthritis (RA). Effects of inflammation and therapy were investigated in RA and OA fibroblasts after stimulation with interleukin (IL)-1beta, tumour necrosis factor (TNF)-alpha, methotrexate (MTX), and prednisolone. The influence of GDF-5 on macrophages was studied by chemotaxis assay. RESULTS: Microarray analysis and immunostaining revealed expression predominantly in synovial fibroblasts. Compared to patients without immunomodulating drugs, expression of GDF-5 was decreased significantly in patients receiving glucocorticoids and/or disease-modifying antirheumatic drugs (DMARDs) (p = 0.007), but did not differ between the total group of ND, OA, and RA. Stimulation with prednisolone and TNFalpha reduced GDF-5 expression in OA and RA fibroblasts, whereas MTX and IL-1beta revealed minor or no relevant change. GDF-5 also reduced cell migration of macrophages (p<0.001). CONCLUSION: GDF-5 is expressed in synovial fibroblasts and may counteract macrophage infiltration. Its modulation by inflammation and therapy suggests that glucocorticoids play a conflicting role by suppressing not only inflammation but also putative mechanisms of repair.
Asunto(s)
Antirreumáticos/uso terapéutico , Artritis Reumatoide/metabolismo , Glucocorticoides/uso terapéutico , Factor 5 de Diferenciación de Crecimiento/metabolismo , Membrana Sinovial/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Antirreumáticos/farmacología , Artritis Reumatoide/tratamiento farmacológico , Estudios de Casos y Controles , Ensayos de Migración de Macrófagos , Citocinas/farmacología , Regulación hacia Abajo , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Expresión Génica , Glucocorticoides/farmacología , Humanos , Inmunohistoquímica , Terapia de Inmunosupresión , Hibridación in Situ , Metotrexato/farmacología , Metotrexato/uso terapéutico , Persona de Mediana Edad , Análisis de Secuencia por Matrices de Oligonucleótidos , Prednisolona/farmacología , Prednisolona/uso terapéutico , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Membrana Sinovial/efectos de los fármacos , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
The clinical phenotype of human dilated cardiomyopathy (DCM) encompasses a broad spectrum of etiologically distinct disorders. As targeting of etiology-related pathogenic pathways may be more efficient than current standard heart failure treatment, we obtained the genomic expression profile of a DCM subtype characterized by cardiac inflammation to identify possible new therapeutic targets in humans. In this inflammatory cardiomyopathy (DCMi), a distinctive cardiac expression pattern not described in any previous study of cardiac disorders was observed. Two significantly altered gene networks of particular interest and possible interdependence centered around the cysteine-rich angiogenic inducer 61 (CYR61) and adiponectin (APN) gene. CYR61 overexpression, as in human DCMi hearts in situ, was similarly induced by inflammatory cytokines in vascular endothelial cells in vitro. APN was strongly downregulated in DCMi hearts and completely abolished cytokine-dependent CYR61 induction in vitro. Dysbalance between the CYR61 and APN networks may play a pathogenic role in DCMi and contain novel therapeutic targets. Multiple immune cell-associated genes were also deregulated (e.g., chemokine ligand 14, interleukin-17D, nuclear factors of activated T cells). In contrast to previous investigations in patients with advanced or end-stage DCM where etiology-related pathomechanisms are overwhelmed by unspecific processes, the deregulations detected in this study occurred at a far less severe and most probably fully reversible disease stage.
Asunto(s)
Cardiomiopatía Dilatada/genética , Cardiomiopatía Dilatada/terapia , Perfilación de la Expresión Génica , Genoma Humano/genética , Adiponectina/genética , Adiponectina/metabolismo , Adulto , Anciano , Proteína 61 Rica en Cisteína , Citocinas/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Redes Reguladoras de Genes , Humanos , Proteínas Inmediatas-Precoces/genética , Proteínas Inmediatas-Precoces/metabolismo , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Persona de Mediana Edad , Modelos Biológicos , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismoRESUMEN
HSP90's are overexpressed in different cancer types and they probably are required to sustain aberrant signalling in malignant cells. Recently, pharmacological inhibition of HSP90 was found to suppress growth of myeloma cell lines and in primary myeloma cells. Therefore, we wanted to investigate the role of HSP90alpha and HSP90beta in the pathogenesis of malignant myeloma (MM) in more detail. Immunohistochemistry was employed to examine the expression of HSP90alpha and HSP90beta in MM. The importance of HSP90 for survival of MM -cells was investigated by SiRNA-mediated knockdown of HSP90 and blockade of the IL-6R/STAT3 and the MAPK signaling pathways in vitro. HSP90alpha and HSP90beta were overexpressed in majority of investigated MM cases, but not in MGUS or in normal plasma cells. SiRNA-mediated knockdown of HSP90 or treatment with the novel HSP90 inhibitor 17-DMAG attenuated the levels of STAT3 and phospho-ERK and decreased the viability of MM cells. The knockdown of HSP90alpha was sufficient to induce apoptosis. This effect was strongly increased when both HSP90s were targeted, indicating a cooperation of both. HSP90 critically contributes to myeloma survival in the context of its microenvironment and therefore strengthen the potential value of HSP90 as a therapeutic target.
RESUMEN
Silencing of gene expression by methylation of CpG islands in regulatory elements is frequently observed in cancer. However, an influence of the most common oncogenic signalling pathways onto DNA methylation has not yet been investigated thoroughly. To address this issue, we identified genes suppressed in HRAS-transformed rat fibroblasts but upregulated after treatment with the demethylating agent 5-Aza-2-deoxycytidine and with the MEK1,2 inhibitor U0126. Analysis of gene expression by microarray and Northern blot analysis revealed the MEK/ERK target genes clusterin, matrix metalloproteinase 2 (Mmp2), peptidylpropyl isomerase C-associated protein, syndecan 4, Timp2 and Thbs1 to be repressed in the HRAS-transformed FE-8 cells in a MEK/ERK- and methylation-dependent manner. Hypermethylation of putative regulatory elements in HRAS-transformed cells as compared to immortalized fibroblasts was detected within a CpG island 14.5 kb upstream of clusterin, within the clusterin promoter and within a CpG island of the Mmp2 promoter by bisulphite sequencing. Furthermore, hypermethylation of the clusterin promoter was observed 10 days after induction of HRAS in immortalized rat fibroblasts and a clear correlation between reduced clusterin expression and hypermethlyation could also be observed in distinct rat tissues. These results suggest that silencing of individual genes by DNA methylation is controlled by oncogenic signalling pathways, yet the mechanisms responsible for initial target gene suppression are variable.
Asunto(s)
Clusterina/antagonistas & inhibidores , Clusterina/biosíntesis , Metilación de ADN , Genes Supresores , Genes ras , Regiones Promotoras Genéticas , Animales , Antimetabolitos Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Azacitidina/análogos & derivados , Azacitidina/farmacología , Línea Celular Transformada , Clusterina/genética , Decitabina , Ácidos Hidroxámicos/farmacología , Ratas , Transducción de Señal/genéticaRESUMEN
BACKGROUND: Transcription factor HIF-1 (hypoxia-inducible factor-1) regulates the expression of genes which are involved in glucose supply, growth, metabolism, redox reactions and blood supply. Hypoxia and ischemia play an important role in the pathogenesis of tinnitus and hearing loss. Therefore, HIF-1 activity and the expression of HIF-1 dependent genes in the cochlea were examined under ischemic and hypoxic conditions. MATERIAL AND METHODS: For the HIF-1 analysis, single-cell cultures of the organ of Corti (OC), stria vascularis (SV) and modiolus (MOD) were used. mRNA expression was analyzed in the organotypic culture using a microarray technique (RN U34-chip, Affymetrix). RESULTS: Ischemia (hypoxia without glucose) and pure hypoxia increase the HIF-1 activity identically, with the highest increase found in MOD and OC. The HIF-1 alpha mRNA levels were found to be higher in SV than in the OC and MOD. During culturing, there is a clear increase in HIF-1 alpha mRNA and the expression of a number of HIF-1 dependent genes, such as Gapdh/glyceraldehyde-3-phosphate dehydrogenase, Slc2a1/solute carrier family 2 (facilitated glucose transporter), member 1, Tf/transferrin and Tfrc/transferrin receptor, in all three regions. In SV, MOD and OC, increase in the expression of Hmox1/hemoxygenase 1, Nos2/nitric oxide synthase, inducible and Tfrc is particularly high. Hypoxia (5 h) results in an increased expression of Igf2/Insulin-like growth factor 2. CONCLUSION: The present data underline the contribution of radical forming processes to the pathogenesis of inner ear diseases. For experimental research, it is important to note that organotypic culture may be coupled with hypoxia.
Asunto(s)
Cóclea/metabolismo , Factor 1 Inducible por Hipoxia/metabolismo , Hipoxia/metabolismo , Isquemia/metabolismo , Animales , Animales Recién Nacidos , Hipoxia de la Célula , Células Cultivadas , Regulación de la Expresión Génica , Ratas , Ratas WistarRESUMEN
BACKGROUND: The influence of caspase inhibitors on spontaneous and on CD95-triggered apoptosis was investigated in neutrophils from healthy volunteers and compared with neutrophils from patients with severe sepsis. METHODS: To further elucidate the mechanisms of neutrophil apoptosis, isolated neutrophils from healthy volunteers (n = 9) were either stimulated with the agonistic anti-CD95 antibody (100 ng/mL) or left unstimulated in the presence or absence of the caspase inhibitors zIETD-fmk (10 micromol/L), zDEVD-fmk (10 micromol/L), or zVAD-fmk (20 micromol/L). Apoptosis was determined by measuring DNA fragmentation and Annexin-V binding in FACS, and caspase-3-like activity by DEVD-afc cleavage assay. Results were compared with those from patients with severe sepsis (n = 15). RESULTS: Reduced spontaneous neutrophil apoptosis in patients with sepsis (-48.7%) was completely restored by incubation with agonistic anti-CD95 antibody (p < 0.05). Inhibition of caspases did not influence spontaneous neutrophil apoptosis in both groups. However, zVAD-fmk reduced anti-CD95 antibody-induced apoptosis in neutrophils from controls by -22.6% (p < 0.05) and in patients with sepsis by -43.1% (p < 0.05). CONCLUSION: These results indicate that spontaneous in contrast to CD95-induced neutrophil apoptosis is independent of caspase activity.
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Apoptosis , Caspasas/metabolismo , Neutrófilos/patología , Sepsis/metabolismo , Clorometilcetonas de Aminoácidos/farmacología , Estudios de Casos y Controles , Inhibidores de Cisteína Proteinasa/farmacología , Activación Enzimática , Humanos , Neutrófilos/metabolismo , Sepsis/inmunología , Receptor fas/metabolismoRESUMEN
OBJECTIVE: The ubiquitously expressed intracellular protein formerly designated p68 has been identified as autoantigen at both the antibody and the T cell level in rheumatoid arthritis (RA). METHODS: We used 2 independent approaches, Edman degradation and matrix-assisted laser desorption ionization-time-of-flight mass spectrometry, to characterize p68, and we compared its features with those of the endoplasmic reticulum stress protein BiP. RESULTS: In synovial sections from RA patients, BiP was highly overexpressed as compared with control sections. Under in vitro stress conditions, BiP was found to translocate to the nucleus and the cell surface. BiP-specific autoantibodies were present in 63% of 400 RA patients, in 7% of 200 patients with other rheumatic diseases, and in none of the healthy subjects. Thus, BiP-specific autoantibodies represent a new diagnostic marker in RA. Furthermore, we found that BiP-specific T cell reactivity was altered in RA. In healthy individuals and patients with other rheumatic diseases, BiP-reactive T cells were undetectable. In RA, overt T cell reactivity to BiP was observed or could be induced by specifically blocking antigen presentation to potentially regulatory T cells. CONCLUSION: Since overexpression of BiP has been shown to decrease the sensitivity of cells to killing by cytotoxic T cells, BiP overexpression and BiP-specific autoimmunity may be involved in the pathogenesis of RA.
Asunto(s)
Artritis Reumatoide/metabolismo , Linfocitos B/metabolismo , Proteínas Portadoras/metabolismo , Proteínas de Choque Térmico/metabolismo , Chaperonas Moleculares/metabolismo , Linfocitos T/metabolismo , Artritis Reumatoide/inmunología , Artritis Reumatoide/patología , Proteínas Portadoras/química , Chaperón BiP del Retículo Endoplásmico , Femenino , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Masculino , Chaperonas Moleculares/química , Fragmentos de Péptidos/análisis , Mapeo Peptídico , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Membrana Sinovial/metabolismo , Membrana Sinovial/patologíaRESUMEN
OBJECTIVE: To define gene activation patterns of monocytes (MO) in patients with rheumatoid arthritis (RA). METHODS: A complementary DNA (cDNA) library was constructed from first-leukapheresis MO obtained from an RA patient with active disease; 32P-labeled cDNA from first-leukapheresis MO (activated pool) and third-leukapheresis MO (nonactivated pool) were used as probes for differential hybridization. Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to assess gene activation in MO from an additional 26 RA patients and 6 normal controls. RESULTS: Subtraction of genes from first- and third-leukapheresis MO resulted in 482 differentially expressed clones. In first-leukapheresis MO, these clones included the following: 1) interleukin-1alpha (IL-1alpha), IL-1beta, IL-6, tumor necrosis factor alpha, growth-related oncogene alpha (GROalpha)/melanoma growth-stimulatory activity, macrophage inflammatory protein 2/GRObeta, ferritin, alpha1-antitrypsin, lysozyme, transaldolase, Epstein-Barr virus-encoded RNA 1 (EBER-1)/EBER-2-associated-protein, thrombospondin 1, an angiotensin receptor II (ATRII) C-terminal homolog, and RNA polymerase II elongation factor (elongin); 2) two clones homologous to functionally undefined genes (BSK-67 and BSK-83); and 3) three unknown cDNA sequences (BSK-66, 80, 89). In third-leukapheresis MO, the clones included differentiation genes (HOX-B3, thymosin-beta4, PU.1, glucocerebrosidase, MEL-18, and glucose-6-phosphate dehydrogenase) and 3 unknown/functionally undefined sequences. Differential expression of most genes from the activated pool was confirmed in leukapheresis samples from 2 additional RA patients. In MO from RA patients, not only were IL-1beta and the ATRII homolog significantly overexpressed (maximum 36-fold), but also 4 of the unknown/functionally undefined genes (maximum 102-fold). Notably, messenger RNA levels of BSK-89 correlated positively with the erythrocyte sedimentation rate (ESR), whereas those of BSK-83 correlated negatively with the ESR and C-reactive protein level. CONCLUSION: The combined strategy of gene subtraction and semiquantitative RT-PCR may allow the definition of MO activation patterns during different disease phases (including therapy-induced remission) and the identification of novel MO genes with pathogenetic relevance in RA.
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Artritis Reumatoide/sangre , Artritis Reumatoide/genética , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Expresión Génica , Regulación de la Expresión Génica , Biblioteca de Genes , Humanos , Leucaféresis , Leucocitos Mononucleares/metabolismo , Masculino , Persona de Mediana Edad , Monocitos/química , Monocitos/metabolismo , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Activación TranscripcionalRESUMEN
The present study was conducted to elucidate the long-term effects of exposure to hypoxia of dopaminergic neurons during the early developmental period. Primary mesencephalic cell cultures prepared from fetal rats and containing 0.5-2% of dopaminergic neurons were exposed to hypoxia between in vitro days 1 and 6, the putative critical developmental period. Changes in the content, release and uptake of dopamine were found to depend on the degree of hypoxia and on the duration of exposure. Following moderate hypoxia (7 h, 5% O2) on two consecutive days between in vitro days 1 and 3, the cultures showed a small increase in the dopamine levels, by 16%. After severe hypoxia (0% O2/95% N2 for 24 h), during the same time window, the cellular dopamine content was elevated by 100%. Moreover, severe hypoxia produced long-lasting modulations of the dopaminergic system. On in vitro day 14, cells exhibited increased levels of 3,4-dihydroxyphenylacetic acid and homovanillic acid (by 34% and 55%, respectively), and elevations of both the spontaneous and potassium-stimulated dopamine release by 70%. The dopamine transport and metabolism of cells exposed to hypoxia between in vitro days 4 and 6 remained unchanged with regard to long-term effects. The present study provides strong evidence for the induction of long-term changes in dopaminergic cells due to hypoxia during the critical developmental period in mesencephalic culture. The developmental period capable of inducing long-lasting changes in dopamine metabolism is restricted to in vitro days 1-3.
Asunto(s)
Hipoxia de la Célula/fisiología , Dopamina/metabolismo , Hipoxia Fetal/metabolismo , Hipoxia Encefálica/metabolismo , Mesencéfalo/metabolismo , Neuronas/metabolismo , Animales , Transporte Biológico , Células Cultivadas , Femenino , Hipoxia Encefálica/embriología , Mesencéfalo/embriología , Embarazo , Efectos Tardíos de la Exposición Prenatal , Ratas , Ratas Wistar , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
Studies on gene expression during differentiation and maturation processes have to cope with determinations of extremely low steady state levels of specific mRNA. Using the experimental model of dopamine D2 receptor (D2R) expression in a primary mesencephalic cell culture we worked out a quantitative reverse transcription polymerase chain reaction method which allows to analyze and quantify mRNA levels of cells present in a few wells of the culture. The method uses an internal cRNA standard which shares both primer binding sites and PCR product length with the target sequence. The amplicons are quantitated in microplates by hybridization with immobilized capture probes that allow for the distinction of internal standard and target sequences followed by the chemiluminescent detection of hybridized DNA. Applying this method the levels of D2 receptor mRNA of the mesencephalic cell culture on day in vitro 1 amounted to about 250 fg/microgram RNA and increased to about 1200 fg/microgram RNA on day in vitro 13-15.
Asunto(s)
Mesencéfalo/metabolismo , Reacción en Cadena de la Polimerasa/métodos , ARN Mensajero/biosíntesis , Receptores de Dopamina D2/biosíntesis , Animales , Secuencia de Bases , Células Cultivadas , Cartilla de ADN , Hibridación in Situ , Cinética , Mesencéfalo/citología , Datos de Secuencia Molecular , ARN Mensajero/genética , Ratas , Ratas Wistar , Receptores de Dopamina D2/genéticaRESUMEN
BACKGROUND: The accumulation of neutrophils at inflammatory sites results in excessive release of toxic metabolites causing tissue injury. Proinflammatory cytokines may cause the breakdown of homeostasis of neutrophil numbers through inhibition of apoptosis. METHODS: Neutrophils were isolated from healthy humans and from patients with multiple injuries on day of admission and during septic complications. Apoptosis was quantitated using propidium iodide fluorescence and the TUNEL method. Tyrosine phosphorylation was measured by flow cytometry. RESULTS: Neutrophil apoptosis was decreased (33.3 +/- 5.5%; p < 0.05) in injured patients with sepsis compared with healthy humans (87.2 +/- 3.0%) and injured patients without sepsis (76.0 +/- 2.0%). Serum from injured patients with sepsis inhibited (p < 0.05) apoptosis of neutrophils from healthy humans in a dose-dependent manner. Serum from healthy humans and from injured patients at admission was ineffective. Neutralization of granulocyte-colony stimulating factor, but not of granulocyte-macrophage-colony stimulating factor, in serum of injured patients with sepsis partially abrogated (+51.2%) serum induced prolongation of neutrophil life span. Reduction of neutrophil apoptosis was concomitant with increased tyrosine phosphorylation. CONCLUSIONS: Septic complications, but not the injury itself, result in inhibition of spontaneous neutrophil apoptosis. Circulating mediators seem to reduce neutrophil apoptosis through up-regulation of tyrosine phosphorylation.
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Apoptosis , Mediadores de Inflamación/sangre , Traumatismo Múltiple/inmunología , Neutrófilos/citología , Sepsis/inmunología , Tirosina/metabolismo , Adulto , Femenino , Humanos , Mediadores de Inflamación/fisiología , Masculino , Traumatismo Múltiple/sangre , Traumatismo Múltiple/complicaciones , Fosforilación , Valores de Referencia , Sepsis/sangre , Sepsis/etiología , Transducción de Señal , Regulación hacia ArribaRESUMEN
The activation of tyrosine phosphorylation plays a pivotal role for endotoxin induced inhibition of PMN-apoptosis. Modulation of tyrosine phosphorylation with specific inhibitors attenuates endotoxin induced prolongation of PMN-lifespan.
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Apoptosis/inmunología , Endotoxemia/inmunología , Neutrófilos/inmunología , Proteínas Tirosina Quinasas/fisiología , Choque Séptico/inmunología , Activación Enzimática/fisiología , Escherichia coli/inmunología , Humanos , Técnicas In Vitro , Lipopolisacáridos/inmunologíaRESUMEN
Neutrophils play a key role in the pathophysiology of septic multiple organ dysfunction syndrome (MODS) through excessive release of toxic granule components and reactive oxygen metabolites with consequent tissue destruction. The increase of senescent neutrophils during sepsis indicates a potential breakdown of autoregulatory mechanisms including apoptotic processes to remove activated neutrophils from inflammatory sites. Therefore, neutrophil apoptosis of patients with severe sepsis and its regulatory mechanisms were investigated. Spontaneous neutrophil apoptosis from patients with severe sepsis was significantly reduced in comparison to healthy individuals. Cytokines detected in the circulation during sepsis (tumor necrosis factor-alpha [TNF-alpha], interferon-gamma [IFN-gamma], granulocyte colony-stimulating factor [G-CSF], granulocyte-macrophage colony-stimulating factor [GM-CSF]) inhibited neutrophil apoptosis in both groups, though the effect was more distinct in neutrophils from healthy humans. Addition of lipopolysaccharide (LPS) to neutrophils from healthy humans markedly (P < .05) reduced apoptosis which was partially restored through addition of anti-TNF-antibody. Interleukin-10 (IL-10) counteracted (P < .05) inhibition of neutrophil apoptosis induced by LPS, recombinant human (rh) TNF-alpha, rhIFN-gamma, rhG-CSF, and rhGM-CSF, whereas rhIL-4 or rhIL-13 were ineffective. Reduced neutrophil apoptosis during sepsis was concomitant with increased tyrosine phosphorylation, while IL-10 markedly inhibited tyrosine phosphorylation in LPS-stimulated neutrophils. These results identify proinflammatory cytokines and IL-10 as strong regulators of spontaneous neutrophil apoptosis during sepsis. Inhibition as well as acceleration of neutrophil apoptosis seems to be associated with alterations of signal transduction pathways.
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Apoptosis , Citocinas/fisiología , Interleucina-10/fisiología , Neutrófilos/patología , Sepsis/sangre , Apoptosis/efectos de los fármacos , Células Cultivadas , Humanos , Interleucina-10/farmacología , Neutrófilos/fisiología , Sepsis/patologíaRESUMEN
The present study was undertaken in order to study the effects of perinatal asphyxia on tyrosine hydroxylase (TH) activity, dopamine levels and turnover, and dopamine metabolites (3,4-dihydroxyphenylacetic acid, DOPAC, homovanillic acid, HVA, and 3-methoxytyramine, 3-MT, analyzed by high-performance liquid chromatography, HPLC) measured in the basal ganglia of the 20- to 40-min-old newborn and 4-week-old male rat. Asphyxia was induced in pups by placing the fetuses, still in their uterus horns removed by hysterectomy from pregnant rats at full term, in a 37 degrees C water bath for 15-16 min or 19-20 min. Following asphyxia, the uterus horns were opened, and the pups were removed and stimulated to breathe. A 100% and 50-80% pup survival was obtained following 15-16 min and 19-20 min of asphyxia, respectively. Acute changes were studied in brains from newborn pups 20-40 min after delivery, and long-term changes were studied in brains from 4-week-old rats. No changes in TH-activity could be observed in the substantia nigra/ventral tegmental area (SN/VTA), the striatum, or the accumbens nucleus/olfactory tubercle (ACC/TUB), in the newborn or the 4-week-old rat. In the newborn rat, 19-20 min of asphyxia increased (as compared to controls) dopamine levels in the SN/VTA to 136 +/- 14% and in the ACC/TUB to 160 +/- 10%, indicating an increased synthesis and/or release of dopamine. DO-PAC levels were increased in the SN/VTA to 150 +/- 14% and in the ACC/TUB to 151 +/- 10%, and HVA levels were increased to 152 +/- 16% in the striatum and to 117 +/- 4% in the ACC/TUB. Following 15-16 min of asphyxia, dopamine levels were increased to 130 +/- 12% in the ACC/TUB, and DOPAC levels were increased to 135 +/- 6% and 130 +/- 12% in the SN/VTA and the ACC/TUB, respectively. This suggests that the increased dopamine levels may preferably reflect an increased release of dopamine following perinatal asphyxia. In the 4-week-old rat, dopamine levels were decreased in the SN/VTA to 71 +/- 4%, in the striatum to 52 +/- 8%, and in the ACC/TUB to 53 +/- 7%, following 19-20 min of perinatal asphyxia as compared to controls. No changes were observed in DOPAC, HVA, or 3-MT levels, indicating that the reduced dopamine levels reflect a reduced dopamine synthesis following perinatal asphyxia. A decrease in dopamine utilization was observed in the striatum to 15 +/- 8% and in the ACC/TUB to 9 +/- 13% following 19-20 min of perinatal asphyxia as compared to controls. This indicates that perinatal asphyxia produced long-lasting reductions in activity in the mesostriatal/mesolimbic dopamine systems in the 4-week-old rat.
Asunto(s)
Animales Recién Nacidos/metabolismo , Asfixia/metabolismo , Ganglios Basales/metabolismo , Dopamina/metabolismo , Ácido 3,4-Dihidroxifenilacético/metabolismo , Animales , Femenino , Masculino , Embarazo , Ratas , Ratas Sprague-DawleyRESUMEN
OBJECTIVE: To study the responsiveness of peripheral blood mononuclear cells to lipopolysaccharide (LPS) after severe trauma and its regulatory mechanisms. MATERIALS AND METHODS: The release of proinflammatory reacting cytokines (tumor necrosis factor-alpha, interleukin (IL)-1 beta, IL-6, IL-8, interferon (IFN)-gamma) into whole blood from 12 patients on day 1, 5, 10, and 14 after severe trauma (Injury Severity Score, 39.3 +/- 2.8 points) and 10 healthy volunteers was studied after stimulation with LPS, concanavalin A, phorbol myristate acetate (PMA), and the addition of recombinant IFN-gamma. MAIN RESULTS: Trauma caused a significant reduction of LPS and concanavalin A induced release of inflammation activating cytokines into whole blood, including IFN-gamma. However, the diminished release of proinflammatory cytokines could be increased with recombinant IFN-gamma or even attenuated after stimulation of peripheral blood mononuclear cells with the protein kinase C activator PMA. CONCLUSIONS: Trauma leads to reduced responsiveness of blood monocytes to LPS and a decreased secretion of proinflammatory reacting lymphokines. Because activation of the protein kinase C pathway with PMA or the addition of IFN-gamma significantly increased cytokine response, endotoxin tolerance is not caused by inhibition of protein synthesis, but to disturbances in the signal transduction pathway and its regulating mediators.
Asunto(s)
Citocinas/biosíntesis , Tolerancia Inmunológica , Leucocitos Mononucleares/inmunología , Lipopolisacáridos/farmacología , Traumatismo Múltiple/inmunología , Adolescente , Adulto , Anciano , Escherichia coli/inmunología , Femenino , Humanos , Inflamación/inmunología , Interferón gamma/biosíntesis , Interferón gamma/farmacología , Interleucina-12/biosíntesis , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Lipopolisacáridos/sangre , Masculino , Persona de Mediana Edad , Proteínas Recombinantes , Transducción de Señal/fisiologíaRESUMEN
BACKGROUND: Excessive release of proinflammatory cytokines has been involved in pathogenesis of acute respiratory distress syndrome. DESIGN: Since injured patients with chest trauma reveal a high risk for posttraumatic acute respiratory distress syndrome, local and systemic release of proinflammatory cytokines and their naturally occurring inhibitors were determined in the early posttraumatic period. MATERIALS AND METHODS: Proinflammatory and anti-inflammatory mediators were measured in plasma and bronchoalveolar lavage fluid (BALF) from 16 patients with multiple injuries including severe chest injury (Injury Severity Score of 34.4 +/- 2.3 points) and compared with healthy volunteers (n = 17). RESULTS: Tumor necrosis factor-alpha was detectable neither in plasma nor in BALF. Interleukin-1beta and interleukin-8 were significantly increased in BALF from injured patients, while plasma levels were similar in both groups. Soluble tumor necrosis factor receptors p55 and p75 and interleukin-1ra were markedly elevated in plasma (p < or = 0.01) and BALF (p < or = 0.001) from injured patients compared with controls. CONCLUSION: Highly increased concentrations of proinflammatory cytokines in BALF, but not in circulation, indicate a strong local inflammatory response early after multiple injuries combined with chest injury rather than severe systemic inflammation. In contrast, anti-inflammatory mechanisms seem to be activated locally and systemically.
Asunto(s)
Líquido del Lavado Bronquioalveolar/inmunología , Citocinas/sangre , Traumatismo Múltiple/inmunología , Traumatismos Torácicos/inmunología , Adulto , Citocinas/análisis , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Puntaje de Gravedad del Traumatismo , Proteína Antagonista del Receptor de Interleucina 1 , Interleucina-1/análisis , Interleucina-1/sangre , Interleucina-8/análisis , Interleucina-8/sangre , Masculino , Persona de Mediana Edad , Traumatismo Múltiple/clasificación , Traumatismo Múltiple/complicaciones , Neumonía/etiología , Sialoglicoproteínas/análisis , Sialoglicoproteínas/sangre , Traumatismos Torácicos/complicaciones , Factor de Necrosis Tumoral alfa/análisisRESUMEN
A mixed mesencephalic cell culture damaged by glutamate was used as a model to study the efflux of lactate dehydrogenase and neuron-specific enolase from neuronal cells into the culture medium. Glutamate toxicity was induced in sister cultures by 15 min exposure to 100 mumol/l glutamate in a Ca2+ containing salt solution. Cell injury was monitored 24 h later by measuring the lactate dehydrogenase activity and the neuron-specific enolase content in the cells and in the culture medium. The neuronal cell damage is reflected by an efflux of neuron-specific enolase and lactate dehydrogenase from the cells and an increase of lactate dehydrogenase catalytic activity concentration and neuron-specific enolase mass concentration in the culture medium. It was found that the efflux fraction calculated from estimations of the cells was clearly higher than the efflux fraction calculated from estimations of the amount of enzymes found in the culture medium. Calculations of the recovery of lactate dehydrogenase and neuron-specific enolase and experiments designed to study the efflux of lactate dehydrogenase and neuron-specific enolase during incubation and washing showed that higher amounts of neuron-specific enolase are released than lactate dehydrogenase. A close correlation was found between the glutamate-induced changes of the neuron-specific enolase efflux fraction, based on enzyme determinations of the cells, and the change of the microscopically counted neuron-specific enolase immunoreactive cell numbers. This indicates that the determination of the neuron-specific enolase efflux fraction (cells) is an accurate and sensitive marker of damaged neurons. The lactate dehydrogenase efflux fraction seems to be less sensitive for the quantitation of neuronal cell damage; in addition, it depends not only on the neuronal damage but also on the proportion of neurons in the cell culture.
Asunto(s)
Ácido Glutámico/farmacología , L-Lactato Deshidrogenasa/efectos de los fármacos , Mesencéfalo/enzimología , Fosfopiruvato Hidratasa/efectos de los fármacos , Animales , Células Cultivadas , Femenino , L-Lactato Deshidrogenasa/metabolismo , Mesencéfalo/citología , Mesencéfalo/efectos de los fármacos , Neuronas/efectos de los fármacos , Neuronas/enzimología , Fosfopiruvato Hidratasa/metabolismo , Embarazo , Ratas , Ratas WistarRESUMEN
This study examines the effects of high K+ concentration on the growth and development of mesencephalic cells and their glutamate vulnerability. Mesencephalic cell cultures obtained from Wistar rat embryos on the 14th gestational day were maintained for 14 days in medium with either normal (4.2 mM) or elevated (24.2 mM) potassium concentration. There was no significant difference due to various K+ concentration in cell growth and survival up to day in vitro (DIV) 13-14. In order to test the glutamate (Glu) vulnerability, cultures were treated with 100 mu M Glu for 15 min in salt solution on the DIV 3,6,8 and 13. Glu-induced neuronal damage was estimated 24 h later by measuring the neuron-specific enolase (NSE) content in the culture medium and by counting the number of tyrosine hydroxylase-immunoreactive (TH-IR) neurons. Glu had no damaging effect on the cells on DIV 3, but became pronounced beyond DIV 6. Elevated potassium concentration 24.2 mM) in the culture medium during development significantly increased neuronal vulnerability to Glu treatment, indicated by a higher increase of NSE content in the medium and by a more pronounced Glu-induced decrease of the number of TH-IR cells. The Glu-induced decrease of the number of TH-IR cells and of NSE-IR cells let us conclude that dopaminergic neurons are more vulnerable to glutamate than other neurons from mesencephalic culture.
Asunto(s)
Dopamina/metabolismo , Ácido Glutámico/farmacología , Mesencéfalo/efectos de los fármacos , Neuronas/efectos de los fármacos , Potasio/farmacología , Animales , Células Cultivadas , Relación Dosis-Respuesta a Droga , Femenino , Inmunohistoquímica , Fosfopiruvato Hidratasa/metabolismo , Embarazo , Ratas , Ratas WistarRESUMEN
OBJECTIVE: To determine whether interleukin (IL)-10, besides its potent anti-inflammatory properties, causes depression of splenocyte functions in a murine model of gram-negative endotoxemia. DESIGN: Mice (strain C3H/HeN) were injected intravenously with 1 mg of Escherichia coli lipopolysaccharide at 15 minutes after intravenous injection of either 200 U of recombinant murine IL-10 or saline solution. Serum levels of tumor necrosis factor alpha, IL-6, and IL-1 alpha were determined at 90 minutes and 12 hours after lipopolysaccharide challenge. In addition, splenocyte proliferation and lymphokine release (IL-2, IL-6, and interferon gamma) were measured. RESULTS: Pretreatment with IL-10 markedly reduced (P < .05) serum levels of tumor necrosis factor alpha (-79%), IL-6 (-94%), and IL-1 alpha (-69%), but it significantly inhibited splenocyte proliferation (-32%) and IL-2 (-40%), IL-6 (-49%), and interferon gamma (-54%) release of splenocytes. CONCLUSIONS: Interleukin-10 prevents E coli lipopolysaccharide-induced cytokinemia but dampens antigen-driven cellular immune responses. Although IL-10 protects against the detrimental effects of proinflammatory cytokines by deactivation of macrophages, its immunosuppressive effect may augment susceptibility to repeated or continuous invasion of microorganisms, as it is observed during clinical sepsis.