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We studied the capacity of isolated Bacteriodes fragilis outer membrane, B. fragilis NCTC9343 lipopolysaccharide (LPS; endotoxin), and B. fragilis NCTC9343 capsular polysaccharides to activate human umbilical vein endothelial cell (HUVEC) monolayers. To assess HUVEC activation, E-selectin expression was measured by enzyme-linked immunosorbent assay (ELISA), Northern blot analysis for E-selectin-specific mRNA, and electrophoretic gel mobility shift assay (EMSA) for NF-kappa B, a transcription factor necessary for E-selectin gene activation. Exposure of HUVECs to B. fragilis outer membrane fractions, separated from other components of the B. fragilis cell wall by isopycnic, sucrose gradient centrifugation, significantly increased surface expression of E-selectin and induced functional endothelial cell-dependent leukocyte adhesion. B. fragilis outer membranes induced translocation of NF-kappa B to HUVEC nuclei and accumulation of E-selectin mRNA in HUVEC cytoplasm. E-selectin expression induced by B. fragilis outer membranes was not blocked by polymixin B. In contrast, E-selectin expression induced by outer membrane fractions purified from E. coli was competitively inhibited by polymixin B. Neither purified B. fragilis LPS, a prominent constituent of the outer membrane, nor purified B. fragilis capsular polysaccharides induced HUVEC activation. Two different monoclonal antibodies directed against human CD14 completely inhibited B. fragilis outer membrane-induced NF-kappa B activation, E-selectin transcription, and E-selectin surface expression. We conclude that the outer membrane component of the B. fragilis cell wall contains a proinflammatory factor(s), that is not LPS, which induces human endothelial cell activation by a soluble CD14-dependent mechanism.

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We reviewed a series of deaths in which methamphetamine was detected in the decedent's blood. Analysis of postmortem whole blood was performed by gas chromatography/mass spectrometry with a limit of quantitation of 0.05 mg/L. Methamphetamine was detected in 146 cases; 52 were drug caused, i.e., a death in which the direct toxic effects of the drug caused or contributed to the death, 92 were classified as drug related, i.e., a death in which the drug was demonstrated in the blood, but did not directly cause death. A large proportion of the deaths resulted from homicidal (27%) or suicidal (15%) violence. An examination of methamphetamine concentrations in drug related deaths (n = 92), suggests that the range of concentrations in the recreational abusing population is substantial (0.05-9.30 mg/L) but with a median concentration of 0.42 mg/L, and with 90% of that population having concentrations less than 2.20 mg/L. There was substantial overlap in methamphetamine concentration between drug related deaths and drug caused deaths, although the highest concentrations were seen in the unintentional (accidental or undetermined) drug caused deaths. Methamphetamine related traffic deaths (n = 17) showed patterns of driving behavior consistent with reports elsewhere, and showed blood methamphetamine concentrations ranging from 0.05-2.60 mg/L (median 0.35 mg/L). The data show that most methamphetamine deaths occur with blood concentrations greater than 0.5 mg/L, but can occur with levels as low as 0.05 mg/L, though usually in conjunction with other drugs or significant natural disease. Neither apparently toxic nor therapeutic concentrations should be used in isolation to establish conclusively whether a death was caused by methamphetamine; proper classification of deaths involving methamphetamine requires complete death investigation, including investigation of the scene and circumstances of death, and a complete autopsy.

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Fifty-three suicides using plastic bags were identified in a review of cases within the jurisdiction of the King County Medical Examiner's Office, Seattle, Washington from 1984 to 1993. We found that this method was used at a greater frequency by individuals older than 50 in comparison with other methods. The most commonly identified stressor leading to the suicide in this population was failing health. The use of this method as a means of "self deliverance," as advocated by the Hemlock Society, could be inferred in only a small minority of cases where terminal illnesses were identified. This method may be preferred by those older than 50 years because of the ready availability of plastic bags and the relative nonviolence of the death. Analysis of the autopsy findings showed no specific features for this method of suicide. In particular, petechiae, which are often considered a marker of asphyxia, were present in only a small minority of cases (3%). Furthermore, the scene investigation rarely revealed specific features, other than the plastic bag in place. Thus, if the plastic bag were removed after death, the cause and manner of death would be obscure.

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During endothelial cell activation, the formation and expression of E-selectin require transcriptional activation of the E-selectin gene, mediated by the coordinated action of several transcription factors and cis-acting elements in its 5'-flanking region. It is reported that in vitro hypothermia (25 degrees C) transiently inhibits transcriptional activation and surface expression of E-selectin as well as neutrophil adherence to cultured human umbilical vein endothelial cells (HUVECs) treated with lipopolysaccharide (LPS), interleukin-1 (IL-1), or tumor necrosis factor (TNF). Rewarming HUVECs treated with LPS, IL-1, or TNF to 37 degrees C restores E-selectin transcript accumulation, E-selectin surface expression, and neutrophil adherence to HUVECs at levels equivalent to similarly treated HUVECs maintained at 37 degrees C continuously. Despite the absence of detectable E-selectin transcription at 25 degrees C, activation of the transcription factor NF-kappaB still occurred in HUVECs treated with LPS, IL-1, or TNF, indicating that signal transduction was not blocked by hypothermia. It is concluded that neutrophil adherence to activated endothelium mediated by E-selectin is reversibly inhibited by hypothermia. The protective effect of hypothermia clinically (e.g., cardiopulmonary bypass) may, in part, be mediated by transiently inhibiting the expression of an endothelial cell activation phenotype.

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Hypothermia frequently accompanies cardiopulmonary bypass (CPB) and myocardial protection strategies during cardiac surgery. With CPB, the blood/artificial surface interface activates components of the humoral and cellular inflammatory cascades and may contribute to postoperative end organ dysfunction including the heart or multiple other organ systems. The endothelial cell (EC) monolayer normally mediates components of solute transport, vasomotor function, coagulation, cell differentiation/growth, and immune/inflammatory processes. E-selectin is a vascular adhesion molecule that mediates neutrophil adherence and that is inducible in ECs by inflammatory mediators such as cytokines. Tissue factor (TF) is similarly an inducible procoagulant factor in ECs that contributes to thrombosis. The induction, transcription, and expression of both molecules were studied in cultured human umbilical vein cells at normothermic (37 degrees), hypothermic (25 degrees), and rewarmed (37 degrees) conditions after stimulation with the cytokines tumor necrosis factor alpha and interleukin-1. Hypothermia reversibly inhibits the transcription and expression but not the induction of both E-selectin and TF.

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The role of protein kinase C (PKC) in interleukin-1 beta- (II-1 beta)-, tumor necrosis factor-alpha- (TNF-alpha)-, and lipopolysaccharide- (LPS)-induced vascular cell adhesion molecule-1 (VCAM-1) expression on human umbilical vein endothelial cells (HUVEC) was studied. PKC inhibition or downregulation diminished VCAM-1 mRNA accumulation and protein expression. Interleukin-1 beta, TNF-alpha, and LPS induce nuclear factor (NF)-kappa B-like binding activity, which precedes VCAM-1 transcription. PKC inhibition did not prevent NF-kappa B-like binding activity, indicating that this is PKC-independent, and NF-kappa B-like binding activity is insufficient for transcription of VCAM-1.

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Reprioritization of hepatic protein synthesis, a process involving accelerated production of acute-phase proteins at the expense of constitutive proteins, accompanies major trauma. The impact of isocaloric, isonitrogenous total enteral nutrition (TEN) versus total parenteral nutrition (TPN) on hepatic reprioritization was investigated in a prospective, randomized trial. Of the 59 patients with an abdominal trauma index (ATI) greater than 15 but not more than 40, 45 evaluable patients were followed. Results from 36 (18 TEN, 18 TPN) evaluable patients revealed that mean serum levels of acute-phase proteins increased, whereas mean serum levels increased to a greater extent in the TPN group. The maximal increase from baseline for the acute-phase response in both groups occurred at postinjury day 5 and was significantly higher for alpha 1-antitrypsin (alpha 1AT, p = 0.03) and orosomucoid (p = 0.02) in the TPN group. Nonacute-phase proteins reached a nadir at day 10 in the TPN group and increased in the TEN group; significant differences between TEN and TPN groups appeared for albumin (p = 0.004) and retinol-binding protein (RBP, p = 0.03); alpha 2-macroglobulin (alpha 2M) approached significance at day 10 (p = 0.07). When change from baseline values was compared, day 10 increases in alpha 2M were significantly higher (p = 0.04) in the TEN group. These data suggest that postinjury TEN attenuates reprioritization of hepatic protein synthesis in patients sustaining major trauma.

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The size and depth of burn and patient age are useful early prognostic indicators in burned patients, but have limited value in predicting which patients in a given cohort are likely to die. The objective of this study was to identify additional variables in the first 10 days of burn injury which could better predict patient outcome. Variables consisting of demographic information, routine laboratory data, and clinical observations on 89 burned patients (63 survivors and 26 nonsurvivors) were analyzed. Compared to survivors, nonsurvivors had significantly larger burns (58 +/- 23% vs. 37 +/- 17%; p less than 0.0002) and a higher incidence of Gram-negative septicemia (20 of 26 [77%] vs. 24 of 63 [38%]; p less than 0.001). Potential prognostic variables were subjected to multivariate logistic regression analysis for each of the first 10 days following burn injury in order to identify a combination of parameters which predicted patient outcome. The regression analyses revealed that, as previously demonstrated, patient age and burn size were significant predictors of mortality on admission and throughout the first 10 days postburn. In addition, absolute monocyte count (AMC), absolute lymphocyte count (ALC), maximum daily temperature (Tmax), and BUN were also significant predictors (p less than 0.05). These data indicate that logistic regression models can identify simple prognostic variables in burned patients which may improve clinicians' ability to identify high-risk patients early in the course of their burn injuries.

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