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Following publication of the original article [1], it was brought to our attention of an error in the article title.
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BACKGROUND: Epigenetic modulation may play a role in anesthesia related phenotypes, such as cognitive impairment or memory loss, especially with exposure to anesthetics in the vulnerable phase of brain development. While isoflurane anesthesia can evoke neuroinflammation and neuroapoptosis in young animals, we investigated in a permanent hippocampal cell line (HT22) and in primary hippocampal neurons in an a priori in vitro analysis, whether isoflurane exposure 1) evokes DNA methylation changes in genes involved in apoptosis and inflammation, and 2) results observed in a permanent hippocampal cell line are comparable to primary hippocampal neurons. In case of methylation changes in specific genes, (3) mRNA analysis was performed to assess possible effects on gene expression. METHODS: HT22 cells and primary mouse hippocampal neurons were exposed to 3% isoflurane for 4 h and DNA (each 6 single experiments) and RNA (3 single independent experiments) were extracted. Methylation analysis (EpiTect Methyl II PCR Array Systems, Qiagen) included the methylation status of 66 genes involved in apoptosis, cytokine production, inflammatory response, and autoimmunity. Quantitative Real-Time PCR was performed using the Quantitect SYBR Green Kit on a Step One Plus. RESULTS: Methylation status was markedly different between immortalized HT22 cells and cultured primary hippocampal neurons without isoflurane exposure. Of 66 genes investigated, 29 were methylated to a significantly greater degree in HT22 cells compared to primary hippocampal neurons. In cultured primary hippocampal neurons, in contrast, there was a greater methylation in several genes involved in inflammation, accompanied with significant downregulation of C-X-C motif chemokine 12 with isoflurane exposure (p = 0.023). CONCLUSIONS: We demonstrate marked differences in gene methylation between HT22 cells and cultured primary hippocampal neurons without isoflurane exposure, with a greater methylation of several genes involved in inflammation upon isoflurane exposure and significant downregulation of Cxcl12 mRNA expression in primary hippocampal neurons. Accordingly, further investigations of anesthesia related DNA methylation should be performed with special consideration being given to the choice of cells targeted for such investigations.
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Anestésicos por Inhalación/administración & dosificación , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Isoflurano/administración & dosificación , Animales , Células Cultivadas , Metilación , Ratones , Modelos Animales , Neuronas/efectos de los fármacos , Neuronas/metabolismoRESUMEN
BACKGROUND: There is little knowledge, whether in patients with sepsis neutrophil extracellular trap (NET) formation and NET degrading nuclease activity are altered. Thus, we tested the hypotheses that 1) NET formation from neutrophils of septic patients is increased compared to healthy volunteers, both without stimulation and following incubation with mitochondrial DNA (mtDNA), a damage-associated molecular pattern, or phorbol 12-myristate 13-acetate (PMA; positive control) and 2) that serum nuclease activities are increased as well. METHODS: Following ethic committee approval, we included 18 septic patients and 27 volunteers in this prospective observational trial. Blood was withdrawn and NET formation from neutrophils was analyzed in vitro without stimulation and following incubation with mtDNA (10 µg/well) or PMA (25 nmol). Furthermore, serum nuclease activity was assessed using gel electrophoresis. RESULTS: In contrast to our hypothesis, in septic patients, unstimulated NET release from neutrophils was decreased by 46.3% (4.3% ± 1.8 SD vs. 8.2% ± 2.9, p ≤ 0.0001) and 48.1% (4.9% ± 2.5 vs. 9.4% ± 5.2, p = 0.002) after 2 and 4 h compared to volunteers. mtDNA further decreased NET formation in neutrophils from septic patients (4.7% ± 1.2 to 2.8% ± 0,8; p = 0.03), but did not alter NET formation in neutrophils from volunteers. Of note, using PMA, as positive control, we ensured that neutrophils were still able to form NETs, with NET formation increasing to 73.2% (±29.6) in septic patients and 91.7% (±7.1) in volunteers (p = 0.22). Additionally, we show that serum nuclease activity (range: 0-6) was decreased in septic patients by 39.6% (3 ± 2 vs 5 ± 0, median and ICR, p = 0.0001) compared to volunteers. CONCLUSIONS: Unstimulated NET formation and nuclease activity are decreased in septic patients. mtDNA can further reduce NET formation in sepsis. Thus, neutrophils from septic patients show decreased NET formation in vitro despite diminished nuclease activity in vivo. TRIAL REGISTRATION: DRKS00007694, german clinical trials database (DRKS). Retrospectively registered 06.02.2015.
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Desoxirribonucleasas/sangre , Trampas Extracelulares , Sepsis/sangre , Sepsis/patología , Adulto , Anciano , ADN Mitocondrial/análisis , ADN Mitocondrial/metabolismo , Femenino , Voluntarios Sanos , Humanos , Masculino , Persona de Mediana Edad , Neutrófilos/química , Estudios Retrospectivos , Acetato de Tetradecanoilforbol/farmacología , Adulto JovenRESUMEN
BACKGROUND: Remote ischaemic preconditioning (RIPC) can attenuate myocardial ischaemia/reperfusion injury but its underlying mechanisms remain largely unknown. Recently, extracellular vesicles (EVs) containing microRNAs (miRNAs) were shown to mediate distant intercellular communication that may be involved in cardioprotection. We tested the hypothesis that RIPC in anaesthetized patients undergoing coronary artery bypass (CABG) surgery results in the release of EVs from the ischaemic/reperfused arm into the blood stream harbouring cardioprotective miRNAs. METHODS: In 58 patients randomised to RIPC (three 5/5 minutes episodes of left arm ischaemia/reperfusion by suprasystolic blood pressure cuff inflations/deflations) or Sham, a subprotocol comprising of parallel right radial artery and regional (left subclavian) venous blood sampling before (awake) and 5 and 60 minutes after RIPC/Sham during isoflurane/sufentanil anaesthesia could be completed. EVs were extracted by polymer-based precipitation methods, their concentrations measured, and their miRNA signature analysed. RESULTS: Five minutes after RIPC, regional venous EV concentrations downstream from the cuff increased and arterial concentrations increased after 60 minutes (fold change [fc]: RIPC: 1.33 ± 0.5, Sham: 0.91 ± 0.31; P = 0.003 for interaction). Already 5 minutes after RIPC, expression of 26 miRNAs (threshold fc: 3.0, P < 0.05) isolated from EVs including the cardioprotective miR-21 had increased. RIPC also decreased postoperative Troponin I concentrations (AUC RIPC: 336 ng/mL × 72 hours ± 306 vs Sham: 713 ± 1013; Pâ = â0.041). CONCLUSIONS: Remote ischaemic preconditioning increases serum EV concentrations, most likely by early EV release from the patients' left (RIPC) arm, alters their miRNA signature, and is associated with myocardial protection. Thus, an increased EV concentration with an altered miR-signature may mediate the RIPC effect.
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Puente de Arteria Coronaria , Vesículas Extracelulares , Precondicionamiento Isquémico Miocárdico/métodos , MicroARNs/sangre , Anciano , Anciano de 80 o más Años , Anestesia General , Anestésicos por Inhalación , Anestésicos Intravenosos , Método Doble Ciego , Femenino , Lesiones Cardíacas/sangre , Humanos , Isoflurano , Masculino , Persona de Mediana Edad , Complicaciones Posoperatorias/sangre , Sufentanilo , Troponina I/sangreRESUMEN
Hypoxia-inducible-factor-2α (HIF-2α) and HIF-2 degrading prolyl-hydroxylases (PHD) are key regulators of adaptive hypoxic responses i.e., in acute respiratory distress syndrome (ARDS). Specifically, functionally active genetic variants of HIF-2α (single nucleotide polymorphism (SNP) [ch2:46441523(hg18)]) and PHD2 (C/T; SNP rs516651 and T/C; SNP rs480902) are associated with improved adaptation to hypoxia i.e., in high-altitude residents. However, little is known about these SNPs' prevalence in Caucasians and impact on ARDS-outcome. Thus, we tested the hypotheses that in Caucasian ARDS patients SNPs in HIF-2α or PHD2 genes are (1) common, and (2) independent risk factors for 30-day mortality. After ethics-committee approval, 272 ARDS patients were prospectively included, genotyped for PHD2 (Taqman SNP Genotyping Assay) and HIF-2α-polymorphism (restriction digest + agarose-gel visualization), and genotype dependent 30-day mortality was analyzed using Kaplan-Meier-plots and multivariate Cox-regression analyses. Frequencies were 99.62% for homozygous HIF-2α CC-carriers (CG: 0.38%; GG: 0%), 2.3% for homozygous PHD2 SNP rs516651 TT-carriers (CT: 18.9%; CC: 78.8%), and 3.7% for homozygous PHD2 SNP rs480902 TT-carriers (CT: 43.9%; CC: 52.4%). PHD2 rs516651 TT-genotype in ARDS was independently associated with a 3.34 times greater mortality risk (OR 3.34, CI 1.09-10.22; p = 0.034) within 30-days, whereas the other SNPs had no significant impact (p = ns). The homozygous HIF-2α GG-genotype was not present in our Caucasian ARDS cohort; however PHD2 SNPs exist in Caucasians, and PHD2 rs516651 TT-genotype was associated with an increased 30-day mortality suggesting a relevance for adaptive responses in ARDS.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Prolina Dioxigenasas del Factor Inducible por Hipoxia/genética , Polimorfismo de Nucleótido Simple , Síndrome de Dificultad Respiratoria/genética , Adulto , Anciano , Femenino , Genotipo , Humanos , Estimación de Kaplan-Meier , Masculino , Persona de Mediana Edad , Modelos de Riesgos Proporcionales , Estudios Prospectivos , Síndrome de Dificultad Respiratoria/epidemiologíaRESUMEN
BACKGROUND: Critically ill patients are at high risk to suffer from sepsis, even in the absence of an initial infectious source, but the molecular mechanisms for their increased sepsis susceptibility, including a suppressed immune system, remain unclear. Although microbes and pathogen-associated molecular pattern are accepted inducers of sepsis and septic immunosuppression, the role of endogenous Toll-like receptor (TLR) ligands, such as mitochondrial DNA (mtDNA), in altering the immune response is unknown. METHODS: Mitochondrial DNA serum concentrations of the mitochondrial genes D-Loop and adenosine triphosphatase 6 were determined (quantitative polymerase chain reaction) in 165 septic patients and 50 healthy volunteers. Furthermore, cytotoxic T-cell activity was analyzed in wild-type and TLR9 knockout mice, with/without previous mtDNA administration, followed by injection of an ovalbumin-expressing adenoviral vector. RESULTS: Mitochondrial DNA serum concentrations were increased in septic patients (adenosine triphosphatase 6, 123-fold; D-Loop, 76-fold, P < 0.0001) compared with volunteers. Furthermore, a single mtDNA injection caused profound, TLR9-dependent immunosuppression of adaptive T-cell cytotoxicity in wild-type but not in TLR9 knockout mice and evoked various immunosuppressive mechanisms including the destruction of the splenic microstructure, deletion of cross-presenting dendritic cells, and up-regulation of programmed cell death ligand 1 and indoleamine 2,3-dioxygenase. Several of these findings in mice were mirrored in septic patients, and mtDNA concentrations were associated with an increased 30-day mortality. CONCLUSIONS: The findings of this study imply that mtDNA, an endogenous danger associated molecular pattern, is a hitherto unknown inducer of septic immunoparalysis and one possible link between initial inflammation and subsequent immunosuppression in critically ill patients.
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ADN Mitocondrial/sangre , ADN Mitocondrial/inmunología , Inflamación/sangre , Inflamación/inmunología , Sepsis/sangre , Sepsis/inmunología , Adulto , Anciano , Animales , Enfermedad Crítica , Modelos Animales de Enfermedad , Femenino , Citometría de Flujo , Humanos , Inmunidad/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Estudios ProspectivosRESUMEN
BACKGROUND: Hypoxia-inducible-factor-1α (HIF-1α) and HIF-1 degrading prolyl-hydroxylases (PHD) are key regulators of the hypoxic-inflammatory response. Functionally active genetic variants in the HIF-1α (C/T; Single Nucleotide Polymorphism (SNP) rs11549465) and the PHD2 gene (EGLN1; C/T; SNP rs516651 and T/C; SNP rs480902) are associated with altered HIF-1α mRNA nuclear translocation and an altered adaptation to hypoxia. Furthermore, the HIF system is important in surviving inflammatory disorders and sepsis. Thus, we tested the hypotheses, that SNPs in the HIF-1α or PHD2 genes are (1) common in Caucasians, with 2) the HIF-1α genetic variant being associated with an altered HIF-1α mRNA expression; and 3) independent risk factors for 30-day mortality in severe sepsis. METHODS: After ethics approval, 128 septic patients (Caucasian descent) were included prospectively within 24 h after first diagnosing sepsis. Patients characteristics and severity of illness (simplified acute physiology score II), genotypes (Taqman assay), and their influence on leukocyte HIF-1α-mRNA-expression (Real-Time PCR) and 30-day mortality were determined. RESULTS: Frequencies were 0.8 % for homozygous HIF-1α TT-carriers (CT 17.6 %; CC 81.6 %), 2.5 % for homozygous PHD2 SNP rs516651 TT-allele carriers (CT 17.5 % and CC 80 %), and 9.4 % for homozygous PHD2 SNP rs480902 TT-allele carriers (CT 34.4 % and CC 56.3 %). While HIF-1α T-allele carriers had a borderline decrease in HIF-1α-mRNA-expression (p = 0.06) neither HIF-1α nor PHD2 SNPs were (independent) risk factors for 30-day mortality. CONCLUSIONS: Genetic variants in HIF-1α and PHD2 genes exist in Caucasians but do not appear to alter 30-day mortality in sepsis.
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Variación Genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Prolina Dioxigenasas del Factor Inducible por Hipoxia/genética , Sepsis/fisiopatología , Anciano , Femenino , Genotipo , Humanos , Masculino , Persona de Mediana Edad , Polimorfismo de Nucleótido Simple , Estudios Prospectivos , ARN Mensajero/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Factores de Riesgo , Sepsis/genética , Sepsis/mortalidad , Índice de Severidad de la Enfermedad , Población Blanca/genéticaRESUMEN
Like other physiological responses, immune functions are the subject of behavioural conditioning. Conditioned immunosuppression can be induced by contingently pairing a novel taste with an injection of the immunosuppressant cyclosporine A (CsA) in an associative learning paradigm. This learned immunosuppression is centrally mediated by the insular cortex and the amygdala. However, the afferent mechanisms by which the brain detects CsA are not understood. In this study we analysed whether CsA is sensed via the chemosensitive vagus nerve or whether CsA directly acts on the brain. Our experiments revealed that a single peripheral administration of CsA increases neuronal activity in the insular cortex and the amygdala as evident from increased electric activity, c-Fos expression and amygdaloid noradrenaline release. However, this increased neuronal activity was not affected by prior vagal deafferentation but rather seems to partially be induced by direct action of CsA on cortico-amygdaloid structures and the chemosensitive brainstem regions area postrema and nucleus of the solitary tract. Together, these data indicate that CsA as an unconditioned stimulus may directly act on the brain by a still unknown transduction mechanism.
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Amígdala del Cerebelo/fisiología , Aprendizaje por Asociación/fisiología , Corteza Cerebral/fisiología , Ciclosporina/farmacología , Terapia de Inmunosupresión/métodos , Nervio Vago/fisiología , Amígdala del Cerebelo/efectos de los fármacos , Animales , Aprendizaje por Asociación/efectos de los fármacos , Corteza Cerebral/efectos de los fármacos , Masculino , Ratas , Nervio Vago/efectos de los fármacosRESUMEN
BACKGROUND: It is widely accepted that SARS-CoV-2 causes a dysregulation of immune and coagulation processes. In severely affected patients, viral sepsis may result in life endangering multiple organ dysfunction. Furthermore, most therapies for COVID-19 patients target either the immune system or coagulation processes. As the exact mechanism causing SARS-CoV-2-induced morbidity and mortality was unknown, we started an in-depth analysis of immunologic and coagulation processes. METHODS: 127 COVID-19 patients were treated at the University Hospital Essen, Germany, between May 2020 and February 2022. Patients were divided according to their maximum COVID-19 WHO ordinal severity score (WHO 0-10) into hospitalized patients with a non-severe course of disease (WHO 4-5, n = 52) and those with a severe course of disease (WHO 6-10, n = 75). Non-infected individuals served as healthy controls (WHO 0, n = 42). Blood was analyzed with respect to cell numbers, clotting factors, as well as pro- and anti-inflammatory mediators in plasma. As functional parameters, phagocytosis and inflammatory responses to LPS and antigen-specific stimulation were determined in monocytes, granulocytes, and T cells using flow cytometry. FINDINGS: In the present study, immune and coagulation systems were analyzed simultaneously. Interestingly, many severe COVID-19 patients showed an upregulation of pro-inflammatory mediators and at the same time clear signs of immunosuppression. Furthermore, severe COVID-19 patients not only exhibited a disturbed immune system, but in addition showed a pronounced pro-coagulation phenotype with impaired fibrinolysis. Therefore, our study adds another puzzle piece to the already complex picture of COVID-19 pathology implying that therapies in COVID-19 must be individualized. CONCLUSION: Despite years of research, COVID-19 has not been understood completely and still no therapies exist, fitting all requirements and phases of COVID-19 disease. This observation is highly reminiscent to sepsis. Research in sepsis has been going on for decades, while the disease is still not completely understood and therapies fitting all patients are lacking as well. In both septic and COVID-19 patients, immune activation can be accompanied by immune paralysis, complicating therapeutic intervention. Accordingly, therapies that lower immune activation may cause detrimental effects in patients, who are immune paralyzed by viral infections or sepsis. We therefore suggest individualizing therapies and to broaden the spectrum of immunological parameters analyzed before therapy. Only if the immune status of a patient is understood, can a therapeutic intervention be successful.
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The COVID-19 pandemic has caused more than 6 million deaths worldwide since its first outbreak in December 2019 and continues to be a major health problem. Several studies have established that the infection by SARS-CoV-2 can be categorized in a viremic, acute and recovery or severe phase. Hyperinflammation during the acute pneumonia phase is a major cause of severe disease progression and death. Treatment of COVID-19 with directly acting antivirals is limited within a narrow window of time between first clinical symptoms and the hyperinflammatory response. Therefore, early initiation of treatment is crucial to assure optimal health care for patients. Molecular diagnostic biomarkers represent a potent tool to predict the course of disease and thus to assess the optimal treatment regimen and time point. Here, we investigated miRNA-200c as a potential marker for the prediction of the severity of COVID-19 to preventively initiate and personalize therapeutic interventions in the future. We found that miRNA-200c correlates with the severity of disease. With retrospective analysis, however, there is no correlation with prognosis at the time of hospitalization. Our study provides the basis for further evaluation of miRNA-200c as a predictive biomarker for the progress of COVID-19.
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RATIONALE: The immune profile of sepsis patients is incompletely understood and hyperinflammation and hypoinflammation may occur concurrently or sequentially. Immune checkpoint inhibition (ICI) may counter hypoinflammation but effects are uncertain. We tested the reactivity of septic whole blood to bacteria, Toll-like receptor (TLR) ligands and to ICI. METHODS: Whole blood assays of 61 patients' samples within 24h of meeting sepsis-3 criteria and 12 age and sex-matched healthy volunteers. Measurements included pattern/danger-associated molecular pattern (P/DAMP), cytokine concentrations at baseline and in response to TLR 2, 4, and 7/8 ligands, heat-inactivated Staphylococcus aureus or Escherichia coli, E.coli lipopolysaccharide (LPS), concentration of soluble and cellular immune checkpoint molecules, and cytokine concentrations in response to ICI directed against programmed-death receptor 1 (PD1), PD1-ligand 1, or cytotoxic T-lymphocyte antigen 4, both in the absence and presence of LPS. MAIN RESULTS: In sepsis, concentrations of P/DAMPs and inflammatory cytokines were increased and the latter increased further upon incubation ex vivo. However, cytokine responses to TLR 2, 4, and 7/8 ligands, heat-inactivated S. aureus or E. coli, and E. coli LPS were all depressed. Depression of the response to LPS was associated with increased in-hospital mortality. Despite increased PD-1 expression on monocytes and T-cells, and monocyte CTLA-4 expression, however, addition of corresponding checkpoint inhibitors to assays failed to increase inflammatory cytokine concentrations in the absence and presence of LPS. CONCLUSION: Patients first meeting Sepsis-3 criteria reveal 1) depressed responses to multiple TLR-ligands, bacteria, and bacterial LPS, despite concomitant inflammation, but 2) no response to immune checkpoint inhibition.
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Sepsis , Receptor Toll-Like 2 , Citocinas/metabolismo , Escherichia coli/metabolismo , Humanos , Inhibidores de Puntos de Control Inmunológico , Ligandos , Lipopolisacáridos , Monocitos/metabolismo , Sepsis/metabolismo , Staphylococcus aureus/metabolismo , Receptor Toll-Like 2/metabolismo , Receptores Toll-Like/metabolismoRESUMEN
Immune-to-brain communication is essential for an individual to aptly respond to challenging internal and external environments. However, the specificity by which the central nervous system detects or 'senses' peripheral immune challenges is still poorly understood. In contrast to post-mortem c-Fos mapping, we recorded neural activity in vivo in two specific cortico-limbic regions relevant for processing visceral inputs and associating it with other sensory signalling, the amygdala (Am) and the insular cortex (IC). Adult rats were implanted with deep-brain monopolar electrodes and electrical activity was monitored unilaterally before and after administration of two different immunogens, the T-cell-independent antigen lipopolysaccharide (LPS) or the T-cell-dependent antigen staphylococcal enterotoxin B (SEB). In addition, the neural activity of the same individuals was analysed after single as well as repeated antigen administration, the latter inducing attenuation of the immune response. Body temperature and circulating cytokine levels confirmed the biological activity of the antigens and the success of immunization and desensitization protocols. More importantly, the present data demonstrate that neural activity of the Am and IC is not only specific for the type of immune challenge (LPS versus SEB) but seems to be also sensitive to the different immune state (naive versus desensitization). This indicates that the forebrain expresses specific patterns of electrical activity related to the type of peripheral immune activation as well as to the intensity of the stimulation, substantiating associative learning paradigms employing antigens as unconditioned stimuli. Overall, our data support the view of an intensive immune-to-brain communication, which may have evolved to achieve the complex energetic balance necessary for mounting effective immunity and improved individual adaptability by cognitive functions.
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Amígdala del Cerebelo/fisiología , Corteza Cerebral/fisiología , Enterotoxinas/farmacología , Lipopolisacáridos/farmacología , Vías Nerviosas/inmunología , Staphylococcus/fisiología , Animales , Mapeo Encefálico , Citocinas , Estudios Longitudinales , Masculino , Vías Nerviosas/efectos de los fármacos , Vías Nerviosas/metabolismo , RatasRESUMEN
The amygdala, a group of nuclei located in the medial temporal lobe, is a key limbic structure involved in mood regulation, associative learning, and modulation of cognitive functions. Functional neuroanatomical studies suggest that this brain region plays also an important role in the central integration of afferent signals from the peripheral immune system. In the present study, intracerebral electroencephalography and microdialysis were employed to investigate the electrophysiological and neurochemical consequences of systemic immune activation in the amygdala of freely moving rats. Intraperitoneal administration of bacterial lipopolysaccharide (100 µg/kg) induced with a latency of about 2 h a significant increase in amygdaloid neuronal activity and a substantial rise in extracellular noradrenaline levels. Activated neurons in the amygdaloid complex, identified by c-Fos immunohistochemistry, were mainly located in the central nucleus and, to a lesser extent, in the basolateral nucleus of the amygdala. Gene expression analysis in micropunches of the amygdala revealed that endotoxin administration induced a strong time-dependent increase in IL-1ß, IL-6, and TNF-α mRNA levels indicating that these cytokines are de novo synthesized in the amygdala in response to peripheral immune activation. The changes in amygdaloid activity were timely related to an increase in anxiety-like behavior and decreased locomotor activity and exploration in the open-field. Taken together, these data give novel insights into different features of the acute amygdaloid response during experimental inflammation and provides further evidence that the amygdala integrates immune-derived information to coordinate behavioral and autonomic responses.
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Amígdala del Cerebelo/fisiopatología , Inflamación/fisiopatología , Lipopolisacáridos/farmacología , Neuronas/metabolismo , Norepinefrina/metabolismo , Amígdala del Cerebelo/inmunología , Amígdala del Cerebelo/metabolismo , Animales , Inflamación/inducido químicamente , Inflamación/inmunología , Inflamación/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Masculino , Microdiálisis , Actividad Motora/fisiología , Vías Nerviosas/metabolismo , Vías Nerviosas/fisiopatología , Neuronas/inmunología , Proteínas Proto-Oncogénicas c-fos/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
BACKGROUND: G protein-coupled receptor kinase 6 (GRK6) is part of the G protein-coupled receptor kinase family, whose members act as key regulators of seven-transmembrane receptor signalling. GRK6 seems to play a role in regulation of inflammatory processes, but mechanisms of transcriptional regulation of GRK6 expression in inflammatory cell lines have not been characterized. Protein kinase C (PKC) signalling is also involved in inflammatory regulation and an impact of PKC activation on GRK6 protein expression was described previously. Thus, the aim of this study was to 1) characterize the GRK6 promoter, and 2) investigate a potential influence of PKC on GRK6 expression. METHODS: Five deletion constructs of the GRK6 promoter were cloned. After transient transfection into a human T cell line, promoter activity was assessed using luciferase reporter gene assays. Putative transcription factor binding sites were identified, mutated, and binding was investigated using electrophoretic mobility shift assays (EMSA). Following stimulation with a PKC activator, GRK6 expression on mRNA and protein levels was assessed by reverse transcriptase qPCR and Western blots. RESULTS: Investigation of the GRK6 promoter revealed a putative cAMP responsive element (CRE), whose mutation led to decreased promoter activity (p = 0.0006). Functionality of the CRE binding protein (CREB) binding site was verified in EMSA blots. Stimulation with a PKC activator resulted in decreased GRK6 promoter activity (p = 0.0027), mRNA (p = 0.04) and protein expression. CONCLUSION: We characterized the human GRK6 promoter and identified promoter activity to be influenced by a CREB binding site. PKC might be one determinant contributing to altered GRK6 expression.
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Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Quinasas de Receptores Acoplados a Proteína-G/genética , Elementos de Respuesta/genética , Secuencia de Bases , Sitios de Unión , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/química , Ensayo de Cambio de Movilidad Electroforética , Quinasas de Receptores Acoplados a Proteína-G/química , Quinasas de Receptores Acoplados a Proteína-G/metabolismo , Humanos , Células Jurkat , Mutagénesis Sitio-Dirigida , Regiones Promotoras Genéticas , Unión Proteica , Proteína Quinasa C/metabolismo , ARN Mensajero/metabolismoRESUMEN
Pavlovian conditioning is one of the major neurobiological mechanisms of placebo effects, potentially influencing the course of specific diseases and the response to a pharmacological therapy, such as immunosuppression. In our study with behaviorally conditioned rats, a relevant taste (0.2% saccharin) preceded the application of the immunosuppressive drug cyclosporin A (CsA), a specific calcineurin (CaN) inhibitor. Our results demonstrate that through pavlovian conditioning the particular pharmacological properties of CsA can be transferred to a neutral taste, i.e., CaN activity was inhibited in splenocytes from conditioned rats after reexposure to the gustatory stimulus. Concomitant immune consequences were observed on ex vivo mitogenic challenge (anti-CD3). Particularly, Th1-cytokine, but not Th2-cytokine, production and cell proliferation were impeded. Appropriate pharmacological and behavioral controls certify that all these changes in T-lymphocyte reactivity are attributable to mere taste reexposure. Furthermore, the underlying sympathetic-lymphocyte interaction was revealed modeling the conditioned response in vitro. CaN activity in CD4(+) T lymphocytes is reduced by beta-adrenergic stimulation (terbutaline), with these effects antagonized by the beta-adrenoreceptor antagonist nadolol. In summary, CaN was identified as the intracellular target for inducing conditioned immunosuppression by CsA, contributing to our understanding of the intracellular mechanisms behind "learned placebo effects."
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Inhibidores de la Calcineurina , Condicionamiento Clásico/efectos de los fármacos , Ciclosporina/farmacología , Inmunosupresores/farmacología , Bazo/efectos de los fármacos , Animales , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Condicionamiento Clásico/fisiología , Masculino , Ratas , Ratas Endogámicas , Sacarina/farmacología , Bazo/citología , Edulcorantes/farmacología , Subgrupos de Linfocitos T/efectos de los fármacos , Subgrupos de Linfocitos T/inmunología , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Gusto/efectos de los fármacosRESUMEN
Dysfunction of the central dopaminergic system is associated with neurodegenerative disorders and mental illnesses such as Parkinson's disease and schizophrenia. Patients suffering from these diseases were reported to exhibit altered immune functions compared to healthy subjects and imbalance of the central dopaminergic system has been suggested as one causative factor for the immune disturbances. However, it is unclear whether the observed immune changes are primary or secondary to the disease. Here we demonstrate that central dopamine (DA) depletion in a rat model of Parkinson's disease induced transient changes in blood leukocyte distribution and cytokine production that were apparent until four weeks after bilateral intrastriatal administration of the neurotoxin 6-hydroxydopamine (6-OHDA). Eight weeks after treatment, no differences in blood immune parameters were anymore evident between neurotoxin-treated and control animals. Nevertheless, animals with a widespread damage of dopaminergic neurons in the nigrostriatal system showed an exacerbated pro-inflammatory response following in vivo challenge with bacterial lipopolysaccharide. Our data indicate that peripheral immune perturbations in the early phase after intrastriatal 6-OHDA administration might have been related to the neurodegenerative process itself whereas the increased sensitivity to the inflammatory stimulus seems to have resulted from an impaired dopaminergic control of prolactin (PRL) and corticosterone (CORT) secretion. The findings demonstrate that the brain dopaminergic system is involved in peripheral immune regulation and suggest that central dopaminergic hypoactivity bears the risk of excessive inflammation, e.g., during infection or tissue injury.
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Cuerpo Estriado/metabolismo , Citocinas/sangre , Dopamina/metabolismo , Enfermedad de Parkinson/metabolismo , Sustancia Negra/metabolismo , Inhibidores de Captación Adrenérgica/farmacología , Análisis de Varianza , Animales , Recuento de Células , Cuerpo Estriado/efectos de los fármacos , Corticosterona/sangre , Desipramina/farmacología , Modelos Animales de Enfermedad , Citometría de Flujo , Inmunohistoquímica , Leucocitos/metabolismo , Lipopolisacáridos/administración & dosificación , Masculino , Oxidopamina/toxicidad , Prolactina/sangre , Ratas , Ratas Endogámicas F344 , Sustancia Negra/efectos de los fármacos , Factores de Tiempo , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
During the last 30 years of psychoneuroimmunology research the intense bi-directional communication between the central nervous system (CNS) and the immune system has been demonstrated in studies on the interaction between the nervous-endocrine-immune systems. One of the most intriguing examples of such interaction is the capability of the CNS to associate an immune status with specific environmental stimuli. In this review, we systematically summarize experimental evidence demonstrating the behavioural conditioning of peripheral immune functions. In particular, we focus on the mechanisms underlying the behavioural conditioning process and provide a theoretical framework that indicates the potential feasibility of behaviourally conditioned immune changes in clinical situations.
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Aprendizaje por Asociación/fisiología , Sistema Nervioso Central/fisiología , Condicionamiento Psicológico/fisiología , Sistema Nervioso Periférico/inmunología , Animales , Conducta Animal , Humanos , PsiconeuroinmunologíaRESUMEN
Frequent or chronic stress as a result of repeated or persistent exposure to social challenges has been shown to affect the glucocorticoid (GC) responsiveness of immune cells in mice. Lipopolysaccharide-stimulated splenocytes of mice that were repeatedly subjected to social disruption were less sensitive to the anti-inflammatory actions of GC as evident from an increased production of pro-inflammatory cytokines and enhanced cell survival. The development of functional GC resistance was accompanied by the accumulation of GC-insensitive CD11b(+) cells in the spleen. These cells were shown to exhibit impaired nuclear translocation of the GC receptor and lack of GC-induced suppression of NF-kappaB. Similar impairments in GC receptor function have been reported after in vitro treatment of various cell lines with interleukin (IL)-1. The aim of this study was to elucidate whether IL-1 is a critical factor for the development of GC resistance in socially stressed mice. In the first experiment, we investigated if repeated social stress alters plasma levels and tissue gene expression of IL-1alpha and IL-1beta. It revealed that recurrent stressor exposure significantly increased splenic and hepatic mRNA expression and the plasma protein level of IL-1beta, and hepatic mRNA expression of IL-1alpha. In the second experiment, IL-1 receptor type 1 (IL1R1)-deficient mice were subjected to the stressor and both the tissue distribution of CD11b(+) cells and the GC sensitivity of the splenocytes were compared to wildtype mice. Mice lacking the IL1R1 exhibited adrenal hypertrophy, thymic involution, and elevated serum corticosterone levels in response to the stressor but did not show splenic accumulation of CD11b(+) cells and failed to develop GC resistance. These findings suggest that IL-1 plays a critical role in the development of the social stress-associated GC resistance in the murine spleen.
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Dominación-Subordinación , Glucocorticoides/fisiología , Receptores Tipo I de Interleucina-1/metabolismo , Bazo/metabolismo , Estrés Psicológico/metabolismo , Glándulas Suprarrenales/anatomía & histología , Animales , Antígeno CD11b/metabolismo , Enfermedad Crónica , Corticosterona/sangre , Modelos Animales de Enfermedad , Interleucina-1alfa/genética , Interleucina-1alfa/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Leucocitos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Tamaño de los Órganos , ARN Mensajero/genética , Receptores Tipo I de Interleucina-1/genética , Medio Social , Bazo/anatomía & histología , Bazo/citología , Estadísticas no Paramétricas , Estrés Psicológico/inmunología , Timo/anatomía & histologíaRESUMEN
Coronary artery-bypass-graft (CABG) surgery is associated with myocardial damage and increased blood concentrations of circulating microRNAs (miRNA). However, whether and to what extent these miRNAs relate to cardiac tissue miRNA expression have not yet been explored. Since plasma miRNA quantification in samples from cardiopulmonary bypass (CPB) patients is severely hampered by heparin, we established and validated successfully a protocol to reliably measure miRNA in 49 heparinized patients undergoing CABG so as to investigate the relationship between circulating and right atrial miRNAs. Plasma and right atrial expression of miR-1, miR-133a, miR-423-5p, and miR-499 were measured before and after CPB, as well as miRNAs in plasma 24 h thereafter. All plasma miRNAs increased significantly with surgery while cardiac tissue expression of only miR-133a (1.4-fold; p = 0.003) and miR-423-5p (1.3 fold; p = 0.025) increased as well. Right atrial and plasma miR-133a expression correlated positively before CPB (r = 0.288, p = 0.045) but miR-499 expression inversely (r = -0.484, p = 0.0004). There was a strong association between plasma miR-133a and miR-499 concentrations and postoperative troponin I concentrations, the marker for myocardial damage. Increased myocardial miR-133a and miR-423-5p expression together with unchanged miR-1 and miR-499 expression might suggest active release of these miRNAs rather than their origin from damaged cells.
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Puente de Arteria Coronaria/efectos adversos , Liasa de Heparina/metabolismo , MicroARNs/sangre , MicroARNs/genética , Miocardio/química , Anciano , Femenino , Regulación de la Expresión Génica , Humanos , MasculinoRESUMEN
The G-protein-coupled receptor kinase 2 (GRK2) plays a major role in cardiovascular diseases, and its expression is increased in heart failure. However, only little is known about factors being involved in up-regulation of GRK2 expression through transcriptional regulation of its promoter. Since the transcription factor early-growth response 1 (EGR-1) is also up-regulated in patients with heart failure, we tested the hypothesis that EGR-1 regulates GRK2 transcription. Stimulation of immortalized rat cardiomyocytes (H9c2) with phorbol 12-myristate 13-acetate (PMA) resulted in up-regulation of Egr-1 and subsequently of Grk2 mRNA expression, with maximum Grk2 expression (p = 0.008) 5 hr after PMA stimulation and being abolished by actinomycin D, indicating a transcriptional mechanism. To identify naturally occurring variants affecting promoter transcriptional activity, we identified a novel G(-43)A polymorphism (rs182084609), which surrounded a putative EGR-1-binding site. While the minor A allele frequency was rare (0.02), this variant was used to explore regulation by EGR-1 and promoter construct with altered alleles at nt-43 were subjected of reporter assays in human embryonic kidney cells (Hek293). Here, EGR-1 over-expression resulted in a more than twofold increase in GRK2 promoter activity but only in the presence of the G-allele (p = 0.04). In electrophoretic mobility shift assays, EGR-1 over-expression resulted in a specific binding of transcription factors only to the G oligonucleotide. Finally, EGR-1 over-expression resulted in increased GRK2 mRNA expression (p = 0.03). We identified EGR-1 as a regulator of GRK2 transcription. Suppression of GRK2 expression by inhibition of EGR-1 binding to GRK2 might be a promising approach to mitigate adrenergic desensitization.