Joseph T. Murphy

Toxic epidermal necrolysis

Toxic epidermal necrolysis (TEN) is a poorly understood and devastating condition. It is usually diagnosed in a primary care setting. Treatment of severe cases by burn care personnel is usually by referral. In this review, we report excessive mortality rates associated with prolonged use of systemic steroid therapy and delayed referral (more than 1 week from diagnosis). Forty-four consecutive patients admitted to a regional burn center with the diagnosis of TEN over a 14-year period, (0.7% of all admissions) were included. Precipitating factors were identified in 30 cases. Twenty-one patients had known prehospital allergy conditions directly related to the inciting agent. The mean age of this population was 44.9 years, and the mean total body surface area (TBSA) injury was 52.4%. Eighty-four and one-half percent of all patients with TEN were admitted to the ICU. Twenty-four patients required ventilator support. Overall mortality rate was 36%. Nonsurviving patients had a mean age of 61.6 years, compared to 35.3 years for survivors. Nonsurvivors had a mean TBSA of 64.4%, survivors had a mean TBSA of 44%. TEN, although a nonthermal injury, is best managed by personnel experienced in the care of severe thermal injuries. Despite the availability of this expertise, delayed transfer of severe presentations continues to contribute to exceptionally high morbidity and mortality rates.

Mechanisms of pulmonary microvascular dysfunction during severe burn injury.

Even in the absence of inhalational injury, acute lung injury is a common cause of morbidity and mortality for patients sustaining severe burns. Other than general supportive measures, there are few therapeutic options for improving survival in these critically ill patients. Numerous clinical and laboratory studies have implicated tumor necrosis factor (TNF)-a and neutrophils as important participants in the pathogenesis of burn-induced lung injury. There is, however, little information regarding the mechanism by which these and other pro-inflammatory mediators affect the movement of fluid and protein across the microvascular barrier into the interstitium of the lung. In addition to reviewing the evidence implicating TNF-a and neutrophils in the pathophysiology of burn-induced lung injury, this report summarizes current theories regarding potential mechanisms by which these mediators may alter microvascular barrier function to lead ultimately to the development of pulmonary edema.

Smoke inhalation enhances early alveolar leukocyte responsiveness to endotoxin.

BACKGROUND Pulmonary dysfunction after smoke inhalation and thermal injury is associated with excessive morbidity and mortality. The purpose of this study was to evaluate alveolar leukocyte function after thermal injury and smoke inhalation. METHODS Twenty-one patients with thermal injury only (n = 8); thermal injury and smoke inhalation injury (n = 8); and nonburned controls (n = 5) were assessed by means of bronchoscopically directed lavage (bronchoalveolar lavage [BAL]) on the first and fourth days postinjury. BAL-isolated pulmonary leukocytes were assessed for number, composition, viability, and production of tumor necrosis factor (TNF)alpha, interleukin (IL)-8, and IL-6 in response to 100 ng/mL of lipopolysaccharide (LPS) (mean +/- SEM; significance at p < 0.05). RESULTS Six of eight Smoke patients had gross evidence of lung injury. On day 1, Smoke and Burn BAL isolates yielded greater cell counts than Control (10.6 vs. 4.5 vs. 2.4 x 10(6)/mL). Smoke macrophages on day 1 produced more TNFalpha (1.2 vs. 0.2 ng/mL), IL-6 (8.0 vs. 1.9 ng/mL), and IL-8 (85 vs. 32 ng/mL) after LPS stimulation compared with respective unstimulated (0 ng/mL of LPS) day-1 Smoke cells. LPS-stimulated Burn cells on day 1 produced more IL-8 (150 vs. 62 ng/mL) but not TNFalpha (0.4 vs. 0.25 ng/mL) or IL-6 (1.8 vs. 0.69 ng/mL), when compared with respective unstimulated Burn cells. By day 4, LPS-stimulated Smoke and Burn cells produced significantly more TNFalpha (Smoke, 0.41 vs. 0.16 ng/mL; Burn, 0.87 vs. 0.51 ng/mL) and IL-6 (Smoke, 2.5 vs. 0.47 ng/mL; Burn, 4.1 vs. 1.47 ng/mL), but not IL-8 (Smoke, 51.1 vs. 51.1 ng/mL; Burn, 54.4 vs. 55.6 ng/mL), compared with respective unstimulated day-4 cells. CONCLUSION Smoke inhalation induces a massive influx of alveolar leukocytes that are primed for an early, enhanced LPS-activated cytokine response compared with alveolar leukocytes isolated after burn injury alone or normal controls.

Effects of burn serum on myocardial inflammation and function.

Large cutaneous burns are clearly recognized to produce acute myocardial contractile dysfunction. This study used a model of burn serum challenge in either primary cardiomyocyte cultures or isolated perfused hearts to examine several aspects of burn-serum-related contractile dysfunction as well as myocardial inflammatory responses. Despite the absence of detectable LPS in burn serum, pretreating isolated cells or perfused hearts with recombinant bactericidal permeability-increasing protein (rBPI21) prevented both the inflammatory cytokine cascade and the cardiac contractile dysfunction induced by burn serum treatment of myocytes or ventricular muscle preparations. Our finding that anti-TNF strategies applied to isolated myocytes or hearts before burn serum challenge prevented myocardial inflammation and contractile dysfunction suggested that LPS or LPS-like factors may require the action of second messengers such as TNF-α and IL-1β to mediate LPS-related myocardial depressant effects. Our finding that experimental approaches neutralizing circulating LPS provided cardioprotection suggested that bacterial endotoxin or LPS-like molecules contribute, in part, to burn-related myocardial contractile dysfunction.

Reactive oxygen species mediate endotoxin-induced human dermal endothelial NF-κB Activation

Abstract Background. Microvascular endothelial cell “activation” by endotoxin is an early and critical phenomenon underlying organ dysfunction related to sepsis. Dermal endothelial cells contribute to many of the manifestations of septic shock, such as cutaneous interstitial edema and loss of peripheral vasomotor regulation. Human dermal endothelial cell activation by endotoxin (lipopolysaccharide [LPS]) is characterized by the generation of reactive oxygen species (ROS) that enhance nuclear translocation of the transcription factor kappa-B (NF-κB). Methods. We tested our hypothesis by stimulating human dermal microvascular endothelial cells (HMEC.1) with endotoxin and assaying for endothelial generation of ROS and nuclear translocation of NF-κB subunits. HMEC.1 cultures were treated individually with LPS, hydrogen peroxide, or xanthine, xanthine oxidase, and ferrous sulfate (xanthine/XO/Fe2+). Nuclear proteins were isolated and consensus sequence binding was assessed by electrophoretic mobility shift assay (EMSA). 2′,7′-Dichlorofluorescin diacetate and confocal microscopy were used to examine ROS production in LPS-stimulated HMEC.1. Results. Nuclear translocation of the p65/p50 NF-κB heterodimer was detectable 30 min after stimulation with LPS alone or the xanthine/XO/Fe2+ combination, but not with hydrogen peroxide. Antioxidant N-acetylcysteine (NAC) inhibited LPS-stimulated ROS production in HMEC.1. Antioxidant prior to or simultaneously with LPS exposure, but not following LPS, also prevented NF-κB activation. NAC was ineffective at inhibiting NF-κB translocation at increased LPS concentrations. Conclusions. Dermal endothelial cells, a microvascular cell type that may contribute to the systemic response to blood-borne endotoxemia, generate ROS in the absence of other inflammatory cells. These LPS-activated endothelial cells, in turn, rapidly translocate transcription factor NF-κB to cell nuclei, a process regulated in part by intracellular ROS.

HUMAN CORONARY ENDOTHELIAL CELL ACTIVATION BY ENDOTOXIN IS CHARACTERIZED BY NF-κB ACTIVATION AND TNF-α SYNTHESIS

Abstract Tumor necrosis factor-alpha (TNF-alpha), an early inflammatory mediator typically regulated by nuclear factor kappa B (NF-kappa B), plays a critical role in the development of cardiovascular dysfunction in sepsis. While several myocardial cell types synthesize TNF-alpha, the importance of the myocardial endothelium in sepsis-related cardiac cytokine production is unclear. To determine the role of the human coronary artery endothelial cell (HCA-EC) in the cytokine response to endotoxin we measured in vitro TNF-alpha synthesis, TNF-alpha mRNA, and the associated NF-kappa B response to LPS. To determine the magnitude of the HCA-EC response we assessed the TNF-alpha and NF-kappa B response to LPS in a human monocytic cell line (THP-1) as well. We observed an increase in supernatant TNF-alpha from LPS-stimulated HCA-EC (12 h) that was ablated by the proteosome inhibitor, ALLN (N-acetyl-Leu-Leu-norleucinal). Similarly, ALLN-sensitive TNF-alpha was produced by monocytes following LPS, although at concentrations 100-fold higher than HCA-EC. TNF-alpha mRNA from HCA-EC was detected at 60 min in LPS-stimulated cells, but not in unstimulated cells or cells pretreated with ALLN. NF-kappa B p50/p65 subunits were detectable in endothelial nuclear protein 60 min following LPS. In contrast, NF-kappa B subunits from monocytes were detected at 15 min. Also, while ALLN only attenuated endothelial NF-kappa B translocation, monocyte NF-kappa B translocation was completely inhibited. These data suggest endotoxin-stimulated human coronary endothelial cells express TNF-alpha, which is regulated in part by NF-kappa B activation, in a manner and degree distinct from human monocytes.

Thrombin-mediated permeability of human microvascular pulmonary endothelial cells is calcium dependent

BACKGROUND In response to inflammation, endothelial cytoskeleton rearrangement, cell contraction, and intercellular gap formation contribute to a loss of capillary barrier integrity and resultant interstitial edema formation. The intracellular signals controlling these events are thought to be dependent on intracellular calcium concentration ([Ca2+]i). We hypothesized that, in human pulmonary microvascular endothelial cells, a thrombin-induced increase in permeability to albumin would be dependent on Ca2+i and subsequent actin cytoskeleton rearrangements. METHODS Human lung microvascular endothelial cells, grown on 0.4 micromol/L pore membranes, were activated with 10 nmol/L human thrombin in Hanks balanced salt solution/0.5% fetal bovine serum. Select cultures were pretreated (45 minutes) with 4 micromol Fura-2/AM to chelate Ca2+i. Permeability was assessed as diffusion of bovine serum albumin/biotin across the monolayer. Similarly treated cells were stained with rhodamine-phalloidin to demonstrate actin cytoskeletal morphology. Separately, cells loaded 2 micromol Fura-2/AM were assessed at OD340/380nm after thrombin exposure to detect free Ca2+i. RESULTS Intracellular Ca2+ levels increased 15-fold (2 seconds) and fell to baseline (10 minutes) after thrombin. Permeability increased 10-fold (30 minutes), and a shift from cortical to actin stress fiber morphology was observed. Chelation of Ca2+i diminished permeability to baseline and reduced the percentage of cells exhibiting stress fiber formation. CONCLUSION Thrombin stimulates pulmonary capillary leak by affecting the barrier function of activated pulmonary endothelial cells. These data demonstrate a thrombin-stimulated increase in monolayer permeability, and cytoskeletal F-actin stress fibers were, in part, regulated by endothelial Ca2+i. This early, transient rise in Ca2+i likely activates downstream pathways that more directly affect the intracellular endothelial structural changes that control vascular integrity.

ZO-1 redistribution and F-actin stress fiber formation in pulmonary endothelial cells after thermal injury.

BACKGROUND In response to isolated inflammatory stimuli, changes in endothelial cell morphology that enhance paracellular flow of solutes result from F-actin stress fiber formation, myosin phosphorylation, and actin anchoring protein (ZO-1) modifications. We hypothesized that myosin light chain kinase inhibition would diminish burn-enhanced endothelial monolayer permeability by secondarily preventing F-actin and actin anchoring protein rearrangements. METHODS Human pulmonary microvascular endothelial cells were treated for 4 hours with 20% human burn serum (isolated from patients with > 45% total body surface area thermal injury or healthy volunteers). Select cultures were pretreated with myosin light chain kinase inhibitors (ML-9). Permeability was assessed by migration of bovine serum albumin across cell monolayers. Cells were stained with rhodamine-phalloidin and anti-ZO-1 antisera and examined by means of confocal microscopy. RESULTS Burn serum significantly enhanced monolayer permeability to albumin, whereas pretreatment with ML-9 limited this effect. Control cells maintained cortical F-actin and peripheral ZO-1 distributions (1a, b), whereas burn serum induced transcellular F-actin stress fiber formation and a diffuse ZO-1 staining (2a, b). ML-9 prevented burn-induced actin rearrangements, but not the diffuse redistribution of ZO-1. CONCLUSION These data demonstrate that endothelial F-actin stress fiber formation and ZO-1 redistribution contribute to postburn loss of pulmonary endothelial monolayer integrity. Although myosin phosphorylation appears to be required for endothelial F-actin stress fiber formation, redistribution of actin-membrane anchoring proteins appears to be regulated independently after thermal injury.

Renal autotransplantation after horseshoe kidney injury: A case report and literature review

We present a patient with lap seatbelt trauma to a previously unsuspected horseshoe kidney. Preoperative single film intravenous pyelography did not suggest the presence of this renal anomaly or define the extent of renal injury. Because of the severity of the injury, nephrectomy was life-saving, but inadvertently rendered the patient anephric. Recognition of horseshoe kidney anatomy, microvascular back-bench reconstruction, and renal autotransplantation allowed the salvage of both the patient and her renal function.

Prehospital cardiopulmonary resuscitation in the pediatric trauma patient

PURPOSE Children requiring prehospital cardiopulmonary resuscitation (CPR) after traumatic injury have been shown to have poor survival. However, outcome of children still receiving CPR on-arrival by emergency medical service to the emergency department (ED) has not been demonstrated in a published clinical series. METHODS An 11-year retrospective analysis from a level I pediatric trauma center of the outcomes of children requiring prehospital CPR after traumatic injury was undertaken. Outcome variables were stratified by survival, death, and CPR on-arrival. RESULTS Of 169 children requiring prehospital CPR, there were 28 survivors and 141 deaths. Of 69 children requiring CPR on-arrival to the ED, there were no survivors. There were 70 females and 99 males. Mean age of survivors was 3.4 years; nonsurvivors, 8.8 years; and 4.6 years for CPR on-arrival. Thirty-nine percent of all injuries were sustained in motor vehicle collisions; 20%, motor pedestrian collisions; 19%, assaults; 7%, falls; 4%, all terrain vehicle/motorcycle/bicycle; and 4%, gunshot wounds. Forty-two percent of all patients expired in the ED, whereas 34% expired in the intensive care unit. Eighty-seven percent of CPR on-arrival patients expired in the ED. Fifty-five percent of survivors had full neurologic recovery. CONCLUSION Although mortality was extremely high for children requiring CPR in the field After traumatic injury, it was absolute for those arriving at the ED still undergoing CPR.