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BACKGROUND: Diagnosing urinary tract infections (UTIs) in nursing home residents is complex, as specific urinary symptoms are often absent and asymptomatic bacteriuria (ASB) is prevalent. The aim of this study was to assess the sensitivity of blood C-reactive protein (CRP) and procalcitonin (PCT), measured by point-of-care tests (PoCTs), to diagnose UTIs in this setting. METHODS: Elderly residents (≥65 years old) with a suspected UTI were recruited from psychogeriatric, somatic, or rehabilitation wards across 13 participating nursing homes. CRP and PCT were tested simultaneously in the same study participants. To assess the tests' sensitivities, a stringent definition of "true" UTI was used that included the presence of symptoms, urinary leucocytes, a positive urine culture, and symptom resolution during antibiotic treatment covering isolated uropathogen(s). The original sample size was 440 suspected UTI episodes, in order to detect a clinically relevant sensitivity of at least 65% when calculated using the matched analysis approach to compare both PoCTs. RESULTS: After enrollment of 302 episodes (68.6% of the planned sample size), an unplanned and funder-mandated interim analysis was done, resulting in premature discontinuation of the study for futility. For 247 of 266 eligible episodes, all mandatory items required for the true UTI definition (92.9%) were available. In total, 49 episodes fulfilled our stringent UTI definition (19.8%). The sensitivities of CRP (cut-off, 6.5 mg/L) and PCT (cut-off, 0.025 ng/mL) were 52.3% (95% confidence interval [CI], 36.7-67.5%) and 37.0% (95% CI, 23.2-52.5%), respectively. CONCLUSIONS: Our results indicate that CRP and PCT are not suitable tests for distinguishing UTI and ASB in nursing home residents. CLINICAL TRIALS REGISTRATION: Netherlands Trial Registry NL6293.
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Pró-Calcitonina , Infecções Urinárias , Idoso , Proteína C-Reativa/análise , Estudos Transversais , Humanos , Casas de Saúde , Testes Imediatos , Infecções Urinárias/diagnóstico , Infecções Urinárias/tratamento farmacológicoRESUMO
PURPOSE: A patient was identified with severe metabolic acidosis, a high anion gap and 5-oxoproline accumulation, probably caused by the simultaneous use of paracetamol (acetaminophen) and flucloxacillin. We wanted to investigate the necessity to control the interaction between both drugs with an automatic alert system. METHODS: To investigate the relevance of the interaction of paracetamol and flucloxacillin, a retrospective study was conducted. Data on paracetamol and flucloxacillin prescriptions and laboratory data (pH, Na+, HCO3-, Cl-, albumin and 5-oxoproline levels) were combined to assess the prevalence of acidosis, calculate the anion gap and analyse 5-oxoproline levels in clinically admitted patients using both drugs simultaneously. RESULTS: In the 2-year study period, approximately 53,000 admissions took place in our hospital. One thousand and fifty-seven patients used paracetamol and flucloxacillin simultaneously, of which 51 patients (4.8%) had a serum pH ≤ 7.35. One patient, the same patient as presented in the case report, had a high anion gap and a toxic level of 5-oxoproline. CONCLUSION: The prevalence of metabolic acidosis is very low and the only patient identified with the interaction was recognised during normal clinical care. We conclude that automatic alerts based on simultaneous use of paracetamol and flucloxacillin will generate too many signals. To recognise patients earlier and prevent severe outcomes, a warning system (clinical rule) based on paracetamol, flucloxacillin and pH measurement may be helpful. Early calculation of the anion gap can narrow the differential diagnosis of patients with metabolic acidosis and measurement of 5-oxoproline can explain acidosis due the interaction of paracetamol and flucloxacillin.
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Acetaminofen/efeitos adversos , Acidose/induzido quimicamente , Analgésicos não Narcóticos/efeitos adversos , Antibacterianos/efeitos adversos , Floxacilina/efeitos adversos , Idoso , Interações Medicamentosas , Humanos , MasculinoRESUMO
Currently, approximately 19 million people with a migration background live in Germany. The majority of those descend from regions where the population has a genetically different distribution of HLA antigens when compared to the HLA frequencies usually found in North Western Europe. In case of severe haematological disorders of these individuals, allogeneic stem cell transplantation may be the treatment of choice. However, finding appropriate histocompatible hematopoietic stem cell donors continues to be a major challenge. If no matching sibling donors are available, there are only few suitable donors with a similar genetic background available in international blood stem cell donor registries. The "BluStar.NRW" project aimed to recruit new blood and hematopoietic stem cell donors with a migration background and to noticeably increase the number of suitable donors for patients within this group. Since December 2017, a total number of 9100 blood and stem cell donors with a migration background were recruited and typed for this project. HLA typing for HLA-A, -B, -C, -DRB1, -DQB1, and -DPB1 was performed by Next Generation Sequencing. We assessed the proportion of rare alleles according to HLA frequency tables, as defined by a frequency of <1:1000. The rare HLA allele frequencies according to HLA frequency tables of the BluStar.NRW cohort were compared with a matched control donor cohort: Rare HLA-A, -B, -C, -DRB1 and -DQB1 alleles occurred three times more frequent than in the control group, but rare HLA-DPB1 alleles occurred more frequently in the control cohort. This difference was highly significant for all HLA alleles (p < 0.0001 for HLA-A, -B, -C, -DRB1, -DPB1; p = 0.0002 for HLA-DQB1). In addition, the distribution of rare alleles differed between the two groups. To date, 29 work-ups were initiated, 12 PBSC, one BM and three DLI were collected so far out of the BluStar.NRW cohort. The apheresis probability is twofold higher (0.18% vs. 0.07%) compared to the control group which clearly shows a serious medical need. However, 13 work-ups were cancelled in the BluStar.NRW donor cohort which represents an almost twice as higher cancellation rate (45% vs. 25%). This single registry analysis with a large sample cohort clearly indicates that hematopoietic stem cell donors with a migration background represent an adequate donor pool to serve patients of comparable ethnicity.
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Transplante de Células-Tronco Hematopoéticas , Refugiados , Migrantes , Humanos , Etnicidade/genética , Doadores de Tecidos , Antígenos de Histocompatibilidade Classe I/genética , Células-Tronco Hematopoéticas , Frequência do Gene , Antígenos HLA-A/genética , Alelos , Teste de Histocompatibilidade , HaplótiposRESUMO
Most methods for quality control of white blood cell (WBC) depletion in blood products are based on flow cytometric techniques. Nearly all commercial kits are based on propidium iodide staining of the DNA and subsequently counting those DNA based events as residual WBC. Here, we could show that a substantial proportion of those events are derived from nucleated red blood cells and therefore not specific for WBCs (e.g. in erythrocyte products 30%). We developed a flow cytometric method for residual WBC counting applying simultaneous DNA- and WBC-specific surface staining to enable this.
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Transfusão de Componentes Sanguíneos/métodos , Preservação de Sangue/métodos , Núcleo Celular/metabolismo , Leucócitos/citologia , Bancos de Sangue/normas , Coagulação Sanguínea , Plaquetas/citologia , Separação Celular/métodos , DNA/análise , Eritrócitos/citologia , Citometria de Fluxo/métodos , Humanos , Contagem de Leucócitos , Controle de Qualidade , Propriedades de SuperfícieRESUMO
A successful implantation of a mammalian embryo into the maternal endometrium depends on a highly synchronized fetal-maternal dialogue involving chemokines, growth factors, and matrix-modifying enzymes. A growing body of evidence suggests an important role for proteinases playing a role in matrix degeneration and enhancing the embryo's invasive capacity and influencing the mother's immunological status in favor of the conceptus. This study focused on the expression of cathepsin S (CTSS) and its inhibitors in the murine fetal-maternal interface as well as the detection of the cellular sources of either proteinase and inhibitors. Nested RT-PCR for detection of embryonic mRNAs, immunohistochemistry of maternal and fetal tissues in B6C3F1 mice, and FACS analysis for determination of immunocompetent cell population were applied. This study shows that the cysteine proteinase CTSS is upregulated in the stroma of the implantation site, and that pregnancy induces an influx of CTSS-positive uterine natural killer cells. Compared to maternal tissues, the CTSS inhibitors cystatin F and C, but not the proteinase itself, are expressed in blastocysts. In conclusion, CTSS underlies a hormonal regulation in the maternal tissue and therewith most likely supports the embryonic implantation. The invading embryo regulates the depth of its own invasion through the expression of the cathepsin inhibitors and furthermore, interleukin-6 to activate CTSS in maternal tissues. Additionally, the observed decrease in CD3(+) cells leads to the hypothesis that cells of the cytotoxic T-cell group are down-regulated in the decidua to support the implantation and ensure the survival of the embryo.
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Catepsinas/antagonistas & inibidores , Cistatina C/fisiologia , Cistatinas/fisiologia , Citoproteção/genética , Embrião de Mamíferos/metabolismo , Animais , Catepsinas/metabolismo , Catepsinas/fisiologia , Cistatina C/genética , Cistatina C/metabolismo , Cistatinas/genética , Cistatinas/metabolismo , Implantação do Embrião/genética , Embrião de Mamíferos/fisiologia , Desenvolvimento Embrionário/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Relações Materno-Fetais , Camundongos , Camundongos Endogâmicos C57BL , GravidezRESUMO
The identification of radiological sources by analysis of a gamma spectrum usually relies on the location of the set of radionuclide-specific electron energies corresponding to the incident photons interacting by photoelectric absorption in the detection medium. The challenge in low-level detection applications is the identification of these "photopeaks" above the background counts registered in the detector from the natural radiation environment and system noise. For source detection decisions, regions of the gamma spectrum other than at the photopeak energies may provide additional information about the presence of a source and allow for a higher rate of correct identification of a weak source. A statistical algorithm utilizing low-fidelity spectral data partitioned into three distinct regions and employing a binomial discriminator was tested in a laboratory setting against the traditional approach of source identification by exceeding a decision threshold within the photopeak region of interest. For an unshielded Cs source with no significant scatter between the source and the detector, the traditional peak identification method performs as well or better than most algorithm settings for various source strengths. However, an algorithm which also includes information in the energy range of Compton scattered photons provides improved detection capabilities for shielded weak sources. Such algorithms, including higher-fidelity developments, could be deployed to improve current tools for the search for orphan radiological sources and in the characterization of low-level environmental contamination.
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Algoritmos , Método de Monte Carlo , Radiometria/instrumentação , Espalhamento de Radiação , Simulação por Computador , Elétrons , Raios gama , HumanosRESUMO
INTRODUCTION: Ammonia is a metabolite of protein catabolism that, when elevated, may be toxic for tissues, especially for the central nervous system. Elevated ammonia in blood is an indicator and a prognostic factor for hepatic and kidney disease or inherited metabolic disorders in nitrogen metabolism. The accuracy of ammonia determination is influenced by sampling condition, handling, storage and assay itself. Our and other laboratories have been experiencing high frequencies sample error flags while measuring ammonia with glutamate dehydrogenase method on Roche Cobas 8000 platform. To reduce the number of error flags we adapted Roche NH3L protocol by incorporation of an additional onboard routine step for sample pre-dilution. MATERIAL AND METHODS: The AMC NH3L is an adaptation of Roche protocol that uses four fold pre-dilution of the sample in the rerun prior to the analysis. It was assessed for 1.occurrence of absorbance error flags, 2.precision, 3.correlation with Roche method and 4.interference by hemolysis, icterus and lipemia. RESULTS: The AMC NH3L adaptation demonstrates acceptable within-run and total precision. Comparison studies show no differences between the Roche rerun application and AMC NH3L adaptation. The AMC NH3L adaptation solves 78% of absorbance errors and for samples with high ammonia concentration is less affected by interferences from icterus and hemolysis than the Roche rerun application. CONCLUSION: The AMC NH3L adaptation is less prone to instrument error flags and for samples with high ammonia concentration, is more robust to endogenous interferences. The AMC NH3L adaptation is viable alternative to the Roche protocol for the ammonia measurement.
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BACKGROUND: Increased systemic levels of myeloperoxidase (MPO) have been reported in patients with acute myocardial ischemia. We studied the association between exercise-induced myocardial ischemia measured by myocardial perfusion scintigraphy (MPS) and the magnitude and time course of changes in MPO levels in humans. METHODS: One hundred and twenty six patients underwent symptom limited exercise MPS. Myocardial ischemia was assessed semi-quantitatively. Plasma samples were taken before the start of exercise (baseline), at maximum exercise and every hour up to 6 h after maximum exercise. RESULTS: Myocardial ischemia was present in 42 (33%) patients. MPO levels rapidly increased during exercise in patients with and without ischemia (to 131% (106-172%) and 145% (121-199%) of baseline, respectively). MPO levels and absolute changes in MPO did not differ between patients with and without ischemia at any time point. None of the patient characteristics, including presence of ischemia, was independently predictive of the absolute increase in MPO levels during exercise. CONCLUSIONS: Exercise related immediate increases in MPO levels do not reflect myocardial ischemia.
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Doença da Artéria Coronariana/diagnóstico por imagem , Doença da Artéria Coronariana/enzimologia , Exercício Físico , Isquemia Miocárdica/enzimologia , Peroxidase/sangue , Idoso , Doença da Artéria Coronariana/sangue , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Isquemia Miocárdica/diagnóstico por imagem , Isquemia Miocárdica/fisiopatologia , Fatores de Tempo , Tomografia Computadorizada de Emissão de Fóton ÚnicoRESUMO
In this study, we provide a molecular signature of highly enriched CD34+ cells from bone marrow of untreated patients with chronic myelogenous leukemia (CML) in chronic phase in comparison with normal CD34+ cells using microarrays covering 8746 genes. Expression data reflected several BCR-ABL-induced effects in primary CML progenitors, such as transcriptional activation of the classical mitogen-activated protein kinase pathway and the phosphoinositide-3 kinase/AKT pathway as well as downregulation of the proapoptotic gene IRF8. Moreover, novel transcriptional changes in comparison with normal CD34+ cells were identified. These include upregulation of genes involved in the transforming growth factorbeta pathway, fetal hemoglobin genes, leptin receptor, sorcin, tissue inhibitor of metalloproteinase 1, the neuroepithelial cell transforming gene 1 and downregulation of selenoprotein P. Additionally, genes associated with early hematopoietic stem cells (HSC) and leukemogenesis such as HoxA9 and MEIS1 were transcriptionally activated. Differential expression of differentiation-associated genes suggested an altered composition of the CD34+ cell population in CML. This was confirmed by subset analyses of chronic phase CML CD34+ cells showing an increase of the proportion of megakaryocyte-erythroid progenitors, whereas the proportion of HSC and granulocyte-macrophage progenitors was decreased in CML. In conclusion, our results give novel insights into the biology of CML and could provide the basis for identification of new therapeutic targets.
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Perfilação da Expressão Gênica , Regulação Leucêmica da Expressão Gênica , Células-Tronco Hematopoéticas/química , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Leucemia Mieloide de Fase Crônica/patologia , Proteínas de Neoplasias/análise , Células-Tronco Neoplásicas/química , Antígenos CD34/análise , Apoptose/genética , Adesão Celular/genética , Diferenciação Celular/genética , Divisão Celular/genética , DNA Complementar/genética , DNA de Neoplasias/genética , Proteínas de Fusão bcr-abl/análise , Proteínas de Fusão bcr-abl/genética , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/biossíntese , Peptídeos e Proteínas de Sinalização Intercelular/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Leucemia Mieloide de Fase Crônica/genética , Leucemia Mieloide de Fase Crônica/metabolismo , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , RNA Mensageiro/genética , RNA Neoplásico/genética , Receptores de Superfície Celular/biossíntese , Receptores de Superfície Celular/genética , Receptores de Fatores de Crescimento/biossíntese , Receptores de Fatores de Crescimento/genética , Receptores para Leptina , Transdução de Sinais/genética , Regulação para CimaRESUMO
The behaviour of Mn, Fe, Co, Zn, Cd, Pb and Ni has been studied during early diagenesis in three different riverine sediments (Spierre, Lys and Sheldt). For that purpose (1) pore waters were extracted from sediment cores by centrifugation under nitrogen and further analyzed for the determination of total dissolved metal concentrations and (2) DET and DGT probes have been deployed in situ for the determination of high resolution profiles of labile and total dissolved metal concentrations. Furthermore, sulfidization processes have been examined; they revealed a production of pyrite near the water-sediment interface at Helkijn and Wervik sampling sites, probably due to a partial re-oxidation of reduced sulphur species. In Spierre sediments, where Eh values are the most negative, pyrite production should be mainly due to strict anaerobic processes. Concentrations of AVS in Spierre sediments are also very high and result in low TI values and low trace metal concentrations in the pore waters. Otherwise, in Wervik sediments, the low pH values combined to a TI value close to 0 results in the highest observed dissolved trace metal levels. DOS remains low at the three sites, since it does not exceed 0.4. In Wervik and Helkijn, the limitation is probably due to low sedimentary inputs of sulphate. In Spierre, sulphate is never exhausted in the pore water, suggesting a limitation of the DOS by a lack of bio-degradable organic matter. Values of Cd, Cu and Pb DGT concentrations remain low in pore waters whatever the site, due to their strong affinity with the reduced sulphur pool. It has also been demonstrated that the labile fractions of Pb and Cd are the lowest and do not exceed 0.5, while Co and Ni are the most available metals.
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Monitoramento Ambiental/métodos , Sedimentos Geológicos/análise , Metais Pesados/análise , Rios/química , Oligoelementos/análise , Poluentes Químicos da Água/análise , França , Metais Pesados/química , Tamanho da Partícula , Solubilidade , Oligoelementos/química , Poluentes Químicos da Água/químicaRESUMO
High resolution profiles of Mn, Tl and Fe concentrations have been assessed in the pore waters of river Leie sediments at Warneton and Menen (at the border of Belgium and France) by DET (Diffusive Equilibrium in Thin Films) and DGT (Diffusive Gradients in Thin Films) techniques. The oxidized, solid Mn (IV), Tl (III) and Fe (III) compounds were reduced in the suboxic (+255 to -20 mV versus Standard Hydrogen Electrode (SHE)) riverine sediments and since these reduced species are much more soluble also they are released into the pore waters. The highest DET (total dissolved) concentrations of Fe (76 mg l(-1)), Mn (2 mg l(-1)) were observed at the station of Menen, while Tl maxima differed only slightly between the 3 surveys (21 to 27 microg l(-1)). The average ratios of Fe/Mn/Tl in the pore waters at the 3 sampling stations are fairly constant for both the DET and DGT samplings. However, the results indicate that compared to Fe and Tl a greater proportion of the Mn measured by DET is accumulated by DGT, reflecting the ready supply of Mn from solid phase to solution.
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Monitoramento Ambiental/métodos , Sedimentos Geológicos/análise , Ferro/análise , Manganês/análise , Tálio/análise , Poluentes Químicos da Água/análise , Bélgica , Difusão , França , Oxirredução , Porosidade , Sensibilidade e EspecificidadeRESUMO
The techniques of DET (diffusive equilibrium in thin films) and DGT (diffusive gradients in thin films) were applied to obtain high-resolution vertical profiles of trace metals in freshwater sediments. In the framework of the EU-Interreg project Stardust (http://www.vliz.be/projects/stardust/) between France and Belgium, in which the mobility of sediment bound metals is investigated, sediment samples were collected from the Upper Scheldt River (at Helkijn, Belgium) and the Leie River (at Warneton, located at the Belgian-French border). Intra- and inter-laboratory comparisons of the gel techniques were carried out between the two laboratories involved. In general, a good agreement was observed, taking sediment heterogeneity into account. At both stations, metal pore water profiles show more or less similar tendencies although the sediment at Warneton was more anoxic than at Helkijn. A strong correlation between Fe and Co was found at Helkijn as well as at Warneton. The metal gradients at the water/sediment interface were calculated from the high resolution profiles and the conventional, low resolution profiles. Significant differences were observed.
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Arsênio/análise , Monitoramento Ambiental/métodos , Sedimentos Geológicos/análise , Metais Pesados/análise , Poluentes Químicos da Água/análise , Bélgica , França , Sedimentos Geológicos/química , Oxirredução , Porosidade , Reprodutibilidade dos Testes , RiosRESUMO
OBJECTIVES: Venous thromboembolism (VTE) is a major potential complication following orthopaedic surgery. Subcutaneously administered enoxaparin has been used as the benchmark to reduce the incidence of VTE. However, concerns have been raised regarding the long-term administration of enoxaparin and its possible negative effects on bone healing and bone density with an increase of the risk of osteoporotic fractures. New oral anticoagulants such as rivaroxaban have recently been introduced, however, there is a lack of information regarding how these drugs affect bone metabolism and post-operative bone healing. METHODS: We measured the migration and proliferation capacity of mesenchymal stem cells (MSCs) under enoxaparin or rivaroxaban treatment for three consecutive weeks, and evaluated effects on MSC mRNA expression of markers for stress and osteogenic differentiation. RESULTS: We demonstrate that enoxaparin, but not rivaroxaban, increases the migration potential of MSCs and increases their cell count in line with elevated mRNA expression of C-X-C chemokine receptor type 4 (CXCR4), tumor necrosis factor alpha (TNFα), and alpha-B-crystallin (CryaB). However, a decrease in early osteogenic markers (insulin-like growth factors 1 and 2 (IGF1, IGF2), bone morphogenetic protein2 (BMP2)) indicated inhibitory effects on MSC differentiation into osteoblasts caused by enoxaparin, but not by rivaroxaban. CONCLUSIONS: Our findings may explain the adverse effects of enoxaparin treatment on bone healing. Rivaroxaban has no significant impact on MSC metabolism or capacity for osteogenic differentiation in vitro.Cite this article: Dr H. Pilge. Enoxaparin and rivaroxaban have different effects on human mesenchymal stromal cells in the early stages of bone healing. Bone Joint Res 2016;5:95-100. DOI: 10.1302/2046-3758.53.2000595.
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BACKGROUND: Microdialysis is a well-established technology that can be used for continuous blood glucose monitoring. We determined point and trend accuracy, and reliability of a microdialysis-based continuous blood glucose-monitoring device (EIRUS(®)) in critically ill patients. METHODS: Prospective study involving patients with an expected intensive care unit stay of ≥48 h. Every 15 min, device readings were compared with blood glucose values measured in arterial blood during blocks of 8 h per day for a maximum of 3 days. The Clarke error grid, Bland-Altman plot, mean absolute relative difference and glucose prediction error analysis were used to express point accuracy and the rate error grid to express trend accuracy. Reliability testing included aspects of the device and the external sensor, and the special central venous catheter (CVC) with a semipermeable membrane for use with this device. RESULTS: We collected 594 paired values in 12 patients (65 [26-80; 8-97] (median [IQR; total range]) paired values per patient). Point accuracy: 93.6 % of paired values were in zone A of the Clarke error grid, 6.4 % were in zone B; bias was 4.1 mg/dL with an upper limit of agreement of 28.6 mg/dL and a lower level of agreement of -20.5 mg/dL in the Bland-Altman analysis; 93.6 % of the values ≥75 mg/dL were within 20 % of the reference values in the glucose prediction error analysis; the mean absolute relative difference was 7.5 %. Trend accuracy: 96.4 % of the paired values were in zone A, and 3.3 and 0.3 % were in zone B and zone C of the rate error grid. Reliability: out of 16 sensors, 4 had to be replaced prematurely; out of 12 CVCs, two malfunctioned (one after unintentional flushing by unsupervised nurses of the ports connected to the internal microdialysis chamber, causing rupture of the semipermeable membrane; one for an unknown reason). Device start-up time was 58 [56-67] min; availability of real-time data was 100 % of the connection time. CONCLUSIONS: In this study in critically ill patients who had no hypoglycemic episodes and a limited number of hyperglycemic excursions, point accuracy of the device was moderate to good. Trend accuracy was very good. The device had no downtimes, but 4 out of 16 external sensors and 2 out of 12 CVCs had practical problems.
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Tissue damage due to apoptotic or necrotic cell death typically initiates distinct cellular responses, leading either directly to tissue repair and regeneration or to immunological processes first, to clear the site, for example, of potentially damage-inducing agents. Mesenchymal stem cells (MSC) as well as immature dendritic cells (iDC) and monocytes migrate to injured tissues. MSC have regenerative capacity, whereas monocytes and iDC have a critical role in inflammation and induction of immune responses, including autoimmunity after tissue damage. Here, we investigated the influence of apoptotic and necrotic cell death on recruitment of MSC, monocytes and iDC, and identified hepatocyte growth factor (HGF) and the alarmin high mobility group box 1 (HMGB1) as key factors differentially regulating these migratory responses. MSC, but not monocytes or iDC, were attracted by apoptotic cardiomyocytic and neuronal cells, whereas necrosis induced migration of monocytes and iDC, but not of MSC. Only apoptotic cell death resulted in HGF production and HGF-mediated migration of MSC towards the apoptotic targets. In contrast, HMGB1 was predominantly released by the necrotic cells and mediated recruitment of monocytes and iDC via the receptor of advanced glycation end products. Moreover, necrotic cardiomyocytic and neuronal cells caused an HMGB1/toll-like receptor-4-dependent inhibition of MSC migration towards apoptosis or HGF, while recruitment of monocytes and iDC by necrosis or HMGB1 was not affected by apoptotic cells or HGF. Thus, the type of cell death differentially regulates recruitment of either MSC or monocytes and iDC through HGF and HMGB1, respectively, with a dominant, HMGB1-mediated role of necrosis in determining tropism after tissue injury.
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Apoptose , Células Dendríticas/fisiologia , Proteína HMGB1/metabolismo , Fator de Crescimento de Hepatócito/metabolismo , Células-Tronco Mesenquimais/fisiologia , Monócitos/fisiologia , Necrose , Animais , Quimiotaxia , Humanos , Inflamação , Masculino , Camundongos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/fisiologia , Neurônios/metabolismo , Neurônios/fisiologia , RegeneraçãoRESUMO
A 17-year-old patient had a progressive hypokinetic-rigid syndrome and several other signs and symptoms that indicated central nervous system involvement. Biochemical studies revealed a reduced form of nicotinamide-adenine dinucleotide dehydrogenase deficiency in skeletal muscle. Clinical signs and symptoms, and their association with an established defect of energy metabolism, led us to classify this disorder as a mitochondrial encephalomyopathy of Leigh's type.
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Encefalopatias Metabólicas/enzimologia , Redutases do Citocromo/deficiência , Doença de Leigh/enzimologia , Mitocôndrias Musculares/enzimologia , Doenças Musculares/enzimologia , NADH Desidrogenase/deficiência , Adolescente , Metabolismo Energético , Humanos , Doença de Leigh/fisiopatologia , Masculino , Doenças Musculares/fisiopatologiaRESUMO
A method is presented which allows the investigation of almost the complete mitochondrial content of small human skeletal muscle biopsy specimens. Thorough mechanical disruption with a chopper apparatus results in the release of about 50% of the mitochondrial content. Subsequent treatment of the 600 x g sediment with trypsin releases another 30% of the total mitochondrial population. The biochemical characteristics of the two mitochondrial fractions obtained in these two successive steps have been compared. No obvious differences could be established. The procedure is well suited for biochemical investigation of muscle biopsy specimens from patients suspected of suffering from a mitochondrial myopathy.
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Fracionamento Celular/métodos , Mitocôndrias Musculares/metabolismo , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Adolescente , Adulto , Criança , Humanos , Pessoa de Meia-Idade , Mitocôndrias Musculares/ultraestrutura , Doenças Musculares/metabolismo , Piruvatos/metabolismo , Ácido PirúvicoRESUMO
Assay procedures are described for the detection of defects in the process of NADH oxidation by the respiratory chain in human skeletal muscle biopsy specimens. The procedures allow determination of rotenone-sensitive NADH: O2 oxidoreductase and NADH: ubiquinone-1 oxidoreductase activity not only in isolated mitochondria but also in post-nuclear supernatants. The use of ferricyanide as electron acceptor for estimation of NADH dehydrogenase activity is inadequate when only applied on a disrupted mitochondrial preparation.
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Mitocôndrias Musculares/enzimologia , NAD/metabolismo , Grupo dos Citocromos c/metabolismo , Humanos , NAD(P)H Desidrogenase (Quinona) , NADH Desidrogenase/metabolismo , NADH NADPH Oxirredutases/metabolismo , Oxirredução , Quinona Redutases/metabolismo , Rotenona/farmacologia , SonicaçãoRESUMO
Procedures are described for the estimation of the succinate:ubiquinone oxidoreductase and succinate:phenazine methosulfate oxidoreductase activities in post-nuclear supernatants of human skeletal muscle homogenates using 2,6-dichlorophenol indophenol as the terminal electron acceptor. The influence of ionic strength and of sucrose upon these assays and upon the succinate:cytochrome c oxidoreductase activity has been investigated. Sucrose markedly interferes with the activation of the succinate dehydrogenase complex. Succinate:cytochrome c oxidoreductase activity and succinate:phenazine methosulfate oxidoreductase activity are inhibited by increasing concentrations of ions and of sucrose. Our results lead us to propose the existence of a single acceptor site for phenazine methosulfate at the succinate dehydrogenase complex, not involved in the physiological electron flux across ubiquinone. Estimation of the enzymatic activities mentioned above allows differential investigation of the functional integrity of a large part of the respiratory chain in patients suspected of suffering from a neuromuscular disorder.