Fatal heat stroke in a child entrapped in a confined space.

We report the case of a child succumbing to heatstroke caused by confinement in an icebox. The post mortem examination found cyanosis and hematomas indicating that the child had tried to get out of the container. The temperature of the body was higher than it should have been considering the rigor and delay before post mortem examination. The autopsy showed no significant injury and toxicological tests were negative. A physiological study etablished that death resulted from heatstroke, not a lack of oxygen or CO2 poisoning. We conclude that heat stroke should be considered as a possible mechanism of death even in the absence of context of environmental hyperthermia. We recommend that in these situations involving confinement, establishing the mechanism of death should be done not only on the basis of a detailed post-mortem examination to rule out other causes of death, but also based on complete physiological investigations.

Distinct epithelial gene expression phenotypes in childhood respiratory allergy.

Epithelial cell contribution to the natural history of childhood allergic respiratory disease remains poorly understood. Our aims were to identify epithelial pathways that are dysregulated in different phenotypes of respiratory allergy. We established gene expression signatures of nasal brushings from children with dust mite-allergic rhinitis, associated or not associated with controlled or uncontrolled asthma. Supervised learning and unsupervised clustering were used to predict the different subgroups of patients and define altered signalling pathways. These profiles were compared with those of primary cultures of human nasal epithelial cells stimulated with either interleukin (IL)-4, IL-13, interferon (IFN)-α, IFN-ß or IFN-γ, or during in vitro differentiation. A supervised method discriminated children with allergic rhinitis from healthy controls (prediction accuracy 91%), based on 61 transcripts, including 21 T-helper cell (Th) type 2-responsive genes. This method was then applied to predict children with controlled or uncontrolled asthma (prediction accuracy 75%), based on 41 transcripts: nine of them, which were down-regulated in uncontrolled asthma, are directly linked to IFN. This group also included GSDML, which is genetically associated with asthma. Our data revealed a Th2-driven epithelial phenotype common to all children with dust mite allergic rhinitis. It highlights the influence of epithelially expressed molecules on the control of asthma, in association with atopy and impaired viral response.

Cyclosporin-A does not prevent cold ischemia/reperfusion injury of rat livers.

Cyclosporin-A (CsA) has been reported to protect livers from warm ischemia/reperfusion (I/R) injury. To study if CsA has also a protective effect on cold I/R injury, two models were used: the isolated perfused rat liver (IPRL) and the orthotopic rat liver transplantation (ORLT). (1) IPRL: Livers were preserved for 24 h (5°C) in University of Wisconsin (UW) solution alone (group 1), with CsA (400 nM) dissolved in dimethylsulfoxide (50 µM) (group 2), and with dimethylsulfoxide (DMSO) alone (group 3). Livers were reperfused for 60 min (37°C) (n = 8/group). Cell necrosis was evaluated by trypan blue uptake and apoptosis by laddering and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay, and by caspase-3 activation. Marked and similar sinusoidal endothelial cell necrosis was found in the three groups while apoptosis was found similarly deceased in groups 2 and 3 compared with group 1. (2) ORLT: Donors received either CsA (5 mg/kg) or corn oil 24 h before transplantation. Recipients were sacrificed after 240 min; cell necrosis and apoptosis were then evaluated. No difference was found between treated and control groups. The current data strongly suggest that CsA has no protective effect on hepatic cold I/R injury. Hepatocyte apoptosis can be reduced by antioxidants, as occurred with DMSO, but introduction of CsA does not provide additional protective effect.

Evaluation of impulse oscillometry during bronchial challenge testing in children.

BACKGROUND: The impulse oscillation system (IOS) allows easy measurement of respiratory system impedance (Zrs). The aim of this retrospective study was to evaluate the accuracy of IOS parameters obtained during methacholine challenge by comparison with "the gold standard" forced expiratory volume in the first second (FEV1). METHODS: Measurements of FEV1 and resistances at 5 and 20 Hz, reactance at 5 Hz, impedance at 5 Hz and resonant frequency were performed in 227 children with suspected asthma, before and during methacholine challenge. Data were analyzed in the overall population and in three subgroups according to the final diagnosis: asthma (n = 72), chronic cough and nonspecific respiratory symptoms (n = 122), allergic rhinitis (n = 33). RESULTS: All IOS parameters changed significantly during the tests but only changes in X5 were significantly different between responders and nonresponders. Moreover, changes in IOS parameters were not correlated with changes in FEV1 apart from a weak correlation for X5. The receiver operating characteristic (ROC) curve for changes in X5 (to predict a 20% decrease in FEV1 showed a best decision level for a 50% decrease in X5 with a sensitivity of 36% and a specificity of 85%. Results were not different in the asthma group. CONCLUSION: The accuracy of measurements by IOS during methacholine bronchial challenge in children was not suitable when compared with FEV1 . It could be assumed that spirometry and IOS, while both providing indirect indices of airway patency, are exploring different mechanisms, each with its own methodological potentials and limitations.

Relationship between impulse oscillometry and spirometric indices in cystic fibrosis children.

BACKGROUND: The aim of our retrospective study was to determine the relationship between impulse oscillometry (IOS) data and spirometric tests in cystic fibrosis (CF) children. methods: Thirty CF children aged 4-19 years have performed lung function tests (LFT). A subset of 15 patients repeated LFT on five separate occasions. IOS parameters were respiratory resistance (Rrs), reactance (Xrs) and impedance at 5 Hz (R5, X5, Zr) and the resonant frequency (Fres). Spirometry indices (SI) included forced expiratory volume in 1 sec (FEV(1)), forced expiratory flow during the middle half of FVC (FEF(25-75)) and forced vital capacity (FVC). RESULTS: An inverse relationship was observed between raw values of R5, Zr, Fres and SI respectively, and X5 correlated positively with SI. Although significant, these correlations were poor. Receiver operating characteristic curves (ROC) were constructed to identify cutoff points for IOS parameters to discriminate between children according to predefined FEV(1) thresholds (percent predicted), generally used to categorize the level of lung function impairment. No acceptable cutoff points can be found for IOS parameters. Trends analyses in the subgroup of 15 patients showed a significant decline of FEV(1) between the first and the fifth evaluation. None of the IOS indices demonstrated a consistent tendency, apart from a slight decrease of Fres. CONCLUSION: IOS measurements presented an insufficient sensitivity to detect and follow bronchial obstruction in CF patients.

Effect of cold ischemia-warm reperfusion on the cirrhotic rat liver.

Cirrhosis is known to induce capillarization of the sinusoidal endothelial cells (SECs) and collagenization of the space of Disse, resulting in a reduced access of plasma and plasma-dissolved substances to hepatocytes due to their limited diffusion in the extravascular space. The aim of the present study was to use a well known effect of cold ischemia-warm reperfusion (CI-WR) on liver SECs, that is, their retraction and detachment, progressing to a denudation of the SEC lining. The disappearance of the capillarized SEC lining would improve the access of plasma and plasma-dissolved substances to the hepatocytes and consequently might improve the metabolic function of cirrhotic livers. This study was performed using the isolated perfused rat liver model subjected to 24-hour CI followed by a 60-minute WR in thioacetamide-induced cirrhosis. Liver microcirculation was evaluated using the multiple indicator dilution curve (MIDC) technique. Hepatocyte, SEC, and Kupffer cell functions were evaluated using specific elimination processes. As occurs in normal livers, CI-WR induced extensive SEC necrosis with a marked reduction of the hyaluronic acid elimination. By contrast, the hepatic microcirculation was not modified: vascular, extravascular, and the cellular spaces were similar before and following CI-WR. In addition, the hepatic metabolic functions, as evaluated by propranolol and taurocholate hepatic uptake, were neither improved nor decreased, as were Kupffer cell functions. The present data strongly suggest that capillarization of SECs plays a lesser role than collagenization of the space of Disse in the reduced exchange between sinusoids and hepatocytes in thioacetamide-induced cirrhotic rat livers, which appear to be quite resistant to CI-WR.

Matrix metalloproteinase inhibition protects rat livers from prolonged cold ischemia-warm reperfusion injury.

UNLABELLED: Matrix metalloproteinases (MMPs) have been implicated in the hepatic injury induced after cold ischemia-warm reperfusion (CI-WR), by altering the extracellular matrix (ECM), but their precise role remains unknown. The hepatic MMP expression was evaluated after 2 conditions of CI (4 degrees C for 24 and 42 hours: viable and nonviable livers) followed by different periods of WR, using isolated perfused rat livers. CI-WR induced moderate changes in hepatic MMP transcript levels not influenced by CI duration, whereas gelatinase activities accumulated in liver effluents. Therefore, the protective effect of a new phosphinic MMP inhibitor, RXP409, was tested after prolonged CI. RXP409 (10 microM) was added to the University of Wisconsin solution, and livers were preserved for 42 hours (4 degrees C), then reperfused for 1 hour in Krebs solution (37 degrees C), containing 20% erythrocytes. Liver viability parameters were recorded, and the extent of cell necrosis was evaluated on liver biopsies, using trypan blue nuclear uptake. Treatment with RXP409 significantly improved liver function (transaminase release and bile secretion) and liver injury. In particular, the MMP inhibitor significantly modified the extent of cell death from large clusters of necrotic hepatocytes as found in control livers (2%-60% of liver biopsies; mean, 26% +/- 9%) to isolated necrotic hepatocytes as found in treated livers (0.2%-12%; mean, 3% +/- 2%) (P < 0.05). CONCLUSION: These data demonstrate that MMPs, by altering the ECM, play a major role in liver CI-WR injury leading to extensive hepatocyte necrosis and that their inhibition might prove to be a new strategy in improving preservation solutions.

Warm ischemia-reperfusion injury is decreased by tacrolimus in steatotic rat liver.

Ischemia-reperfusion (I-R) injury is poorly tolerated by fatty livers, most probably secondary to reduced cellular adenosine triphosphate (ATP) levels. We investigated the effectiveness of tacrolimus pretreatment on fatty liver I-R injury in obese Zucker rats. Tacrolimus (0.3 mg/kg, intravenously) was injected 24 hours before a 75-minute ischemic period and rats were sacrificed 6 hours later. Tacrolimus modified the response to I-R observed in obese Zucker rats, when compared to nontreated obese rats: a significant reduction in hepatocyte necrosis was associated with a significant increase in hepatocyte apoptosis. In addition, cell necrosis and apoptosis were significantly and inversely correlated in lean nontreated and treated obese Zucker rats following I-R. Tacrolimus also significantly increased the hepatic ATP levels, reduced in nontreated obese rats, toward values found in lean Zucker rat livers. This protective effect of tacrolimus was further confirmed in vivo by a significantly improved survival following pretreatment with tacrolimus, 24 hours prior to ischemia. In conclusion, in obese Zucker rat livers, tacrolimus pretreatment reversed the I-R injury toward the one found in lean Zucker rats. The correlations between ATP levels and the opposite changes in necrosis and apoptotic pathways strongly suggest a cause-effect relationship between tacrolimus and changes in ATP levels.

Hypoxia-reoxygenation-induced chemokine transcription is not prevented by preconditioning or intermittent hypoxia, in mice hepatocytes.

Prolonged ischemia used in liver surgery and/or transplantation causes cellular damage resulting in apoptosis and necrosis. Ischemia-reperfusion (I/R) led Kupffer cells to pro-inflammatory cytokines secretion [tumor necrosis factor (TNF)-alpha, interleukin-1] which involve chemokines secretion by hepatocytes. These chemokines have neutrophil chemotactic properties and neutrophils are involved in the development of I/R-induced necrosis. The aim of this study was to specify the consequence of partial oxygen pressure variation on hepatocyte chemokines synthesis and to verify if intermittent hypoxia and/or preconditioning could decrease it. It was performed on primary cultured mice hepatocytes and Kupffer cells, subjected to continuous, intermittent hypoxia or preconditioning phases, mimicking surgical processes. The chemokine secretion was evaluated by RNase protection assay and enzyme-linked immunosorbent assay method. Only monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-2 (MIP-2) mRNA formation were observed, especially after 1-h hypoxia followed by 10-h (for MCP-1) or 24-h reoxygenation (for MIP-2). In conclusion, TNF-alpha and coculture with Kupffer cells increased hepatocyte chemokines mRNA transcription, whereas surgical split up protocols (intermittent hypoxia and preconditioning) had no significant effect.

Rat liver ischemia-reperfusion-induced apoptosis and necrosis are decreased by FK506 pretreatment.

The aim of this study was to demonstrate that tacrolimus (FK506) has a hepatoprotective effect by reducing ischemia-reperfusion-induced apoptosis and necrosis, both of which lead to post-surgical liver dysfunction. An ischemia-reperfusion model and primary cultured rat hepatocytes subjected to hypoxic and reoxygenation phases, mimicking the surgical process, were used. c-Jun N-terminal kinase 1/stress-activated protein kinase 1 (JNK1/SAPK1) activation leads to caspase 3 activation, a trigger of apoptosis. The activation status of JNK1/SAPK1 was evaluated by immunoprecipitation or Western-blotting experiments. Apoptosis was assessed by measuring caspase activation and by TUNEL (terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate-biotin nick-end labeling) reaction. Necrosis was assessed histologically. Tacrolimus improved the survival rate of rats subjected to ischemia-reperfusion. After FK506 pretreatment, the liver necrosis rate was reduced, and ischemia-reperfusion-induced JNK1/SAPK1 activation and apoptosis were significantly decreased. In hypoxia-reoxygenation-subjected hepatocytes, tacrolimus reduced JNK1/SAPK1 and caspase 3 activation. In the liver, tacrolimus prevented ischemia-reperfusion-induced apoptosis and necrosis.

Mapacalcine specifically blocks hypoxia-induced calcium influx in rat hepatocytes.

Post ischaemic cell calcium invasion has been described as one of the main causes of graft failure. Protective effects of calcium antagonists have been investigated but are not convincing and their mechanisms of action remain unclear. In this work we tested the protective effect of a new calcium inhibitor described to block a calcium current insensitive to all known calcium blockers. Specific mapacalcine receptors were first characterized on rat hepatocytes membranes using the 125I-labeled mapacalcine. 45Ca fluxes were then measured on cultured hepatocytes submitted (or not) to an hypoxic period. The action of mapacalcine was investigated on the ischaemia-induced calcium influx. We demonstrate here that: (a) there are specific receptors for mapacalcine in rat hepatocytes; (b) Mapacalcine is able to specifically block ischaemia-induced calcium influx with an IC50 of 0.3 micro m and does not significantly interact with the basal calcium flux. Our work demonstrates that the mapacalcine receptor is a cellular structure directly involved in the phenomenon of postischaemic cell invasion by calcium. Specific block of ischaemia-induced Ca2+ influx by mapacalcine suggests that the development of a panel of pharmacological drugs acting on this receptor could lead to the discovery of therapeutic agents able to protect cells against one of the events responsible for organ failure after transplantation or simply after an ischaemic period. Moreover, identification of the cellular protein which binds mapacalcine may become an important step in the research of mechanisms involved in postischaemic cell invasion by calcium.

Diltiazem reduces apoptosis in rat hepatocytes subjected to warm hypoxia-reoxygenation.

Interruption of hepatic blood flow is necessary in surgery, but the liver is sensitive to ischemia and reperfusion. Hypoxia induces an increase in intracellular calcium concentration. In previous studies, we have shown that hypoxia-reoxygenation (H/R) increased calcium influx and induced JNK(1)/SAPK(1) activation which was involved in the triggering of apoptosis. The aim of this study was to demonstrate that diltiazem, a calcium inhibitor, reduced JNK(1)/SAPK(1) activation and consequently could decrease H/R-induced apoptosis. Experiments were performed, in the presence of diltiazem, on primary cultured rat hepatocytes, subjected to warm H/R phases and in a liver ischemia-reperfusion model. The activation status of JNK(1)/SAPK(1) was evaluated by immunoprecipitation and immunohistolocalisation experiments, while apoptosis was assessed by measuring caspase activity and by TUNEL labeling. Diltiazem inhibited H/R-induced JNK(1)/SAPK(1) activation and decreased apoptosis. It could be used to improve postoperative liver function.