An Assessment of Research Priorities to Dampen the Pendulum Swing of Burn Resuscitation.

On June 17 to 18, 2019, the American Burn Association, in conjunction with Underwriters Laboratories, convened a group of experts on burn resuscitation in Washington, DC. The goal of the meeting was to identify and discuss novel research and strategies to optimize the process of burn resuscitation. Patients who sustain a large thermal injury (involving >20% of the total body surface area [TBSA]) face a sequence of challenges, beginning with burn shock. Over the last century, research has helped elucidate much of the underlying pathophysiology of burn shock, which places multiple organ systems at risk of damage or dysfunction. These studies advanced the understanding of the need for fluids for resuscitation. The resultant practice of judicious and timely infusion of crystalloids has improved mortality after major thermal injury. However, much remains unclear about how to further improve and customize resuscitation practice to limit the morbidities associated with edema and volume overload. Herein, we review the history and pathophysiology of shock following thermal injury, and propose some of the priorities for resuscitation research. Recommendations include: studying the utility of alternative endpoints to resuscitation, reexamining plasma as a primary or adjunctive resuscitation fluid, and applying information about inflammation and endotheliopathy to target the underlying causes of burn shock. Undoubtedly, these future research efforts will require a concerted effort from the burn and research communities.

Serum Level of Musclin Is Elevated Following Severe Burn.

Muscle wasting induced by severe burn worsens clinical outcomes is associated with hyperglycemia. A novel muscle-specific secretory factor, musclin, was reported to regulate glucose metabolism with a homologous sequence of natriuretic peptides. The purpose of the study was to investigate musclin expression in response to burn injury in both human and animal models. Serum was collected from 13 adult burn patients and circulating levels of musclin protein were measured via elisa. The cytokine profile was measured by Bio-Plex multiple immunoassay. Following the clinical study, we used a burn rat model with 40% TBSA to study the time course of musclin expression till day 14. Rat serum and muscle tissue sample were harvested. Finally, an in vitro study was applied to investigate whether the muscle cell C2C12 myoblast expressed musclin under 10% burn serum stimulation. Pearson analysis showed that there was a significant positive correlation of musclin expression to total body surface area of burn in patients (P &= .038). Musclin expression was significantly positively correlated with IL-4, IL-7, IL-12, and IL-13 in burn patients' serum (P < .05). In the animal study, we found that the musclin level evaluated at 6 hours and 1 day in burn rat serum (P < .05). In vitro, musclin mRNA expression significantly increased with burn serum stimulation at 24 hours (P < .05). In conclusion, serum level of musclin elevated both in human patients and burn animals; musclin was correlated with the severity of burn injury as well as with an elevated cytokine profile in patients; burn serum-stimulated musclin expression in vitro further identified the resource of musclin expression after burn.

CEDRA: A Tool to Help Consumers Assess Risk for Ear Disease.

This article introduces the Consumer Ear Disease Risk Assessment (CEDRA) tool. CEDRA is a brief questionnaire designed to screen for targeted ear diseases. It offers an opportunity for consumers to self-screen for disease before seeking a hearing device and may be used by clinicians to help their patients decide the appropriate path to follow in hearing healthcare. Here we provide highlights of previously published validation in the context of a more thorough description of CEDRA's development and implementation. CEDRA's sensitivity and specificity, using a cut-off score of 4 or higher, was 90% and 72%, respectively, relative to neurotologist diagnoses in the initial training sample used to create the scoring algorithm (n = 246). On a smaller independent test sample (n = 61), CEDRA's sensitivity and specificity were 76% and 80%, respectively. CEDRA has readability levels similar to many other patient-oriented questionnaires in hearing healthcare, and informal reports from pilot CEDRA-providers indicate that the majority of patients can complete it in less than 10 min. As the hearing healthcare landscape changes and provider intercession is no longer mandated, CEDRA provides a measure of safety without creating a barrier to access.

A Retrospective Estimate of Ear Disease Detection Using the "Red Flags" in a Clinical Sample.

OBJECTIVES: The purpose of this study was to evaluate the specificity and sensitivity of two red flag protocols in detecting ear diseases associated with changes in hearing. DESIGN: The presence of red-flag symptoms was determined in a chart review of 307 adult patients from the Mayo Clinic Florida Departments of Otorhinolaryngology and Audiology. Participants formed a convenience sample recruited for a separate study. Neurotologist diagnosis was the criterion for comparisons. RESULTS: Of the 251 patient files retained for analysis, 191 had one or more targeted diseases and 60 had age- or noise-related hearing loss. Food and Drug Administration red flags sensitivity was 91% (confidence interval [CI], 86 to 95%) and specificity was 72% (CI, 59 to 83%). American Academy of Otolaryngology-Head and Neck Surgery red flags sensitivity was 98% (CI, 95 to 99%) and specificity was 20% (CI, 11 to 32%). CONCLUSIONS: Stakeholders must determine which diseases are meaningful contraindications for hearing aid use and whether these red-flag protocols have acceptable levels of sensitivity and specificity. As direct-to-consumer models of hearing devices increase, a disease detection method that does not require provider intercession would be useful.

Severe Burn-Induced Inflammation and Remodeling of Achilles Tendon in a Rat Model.

Severe burn causes systemic inflammation and hypercatabolism, resulting in damage to multiple organs distant to the burn site, including the musculoskeletal system. Bone mass and muscle loss have been reported. However, tendon that connects bone and muscle has not been studied in comparable detail. Here we aimed to characterize the molecular and functional changes in Achilles tendon triggered by severe burn. Forty male Sprague-Dawley rats received 40% total body surface area scald burn. Achilles tendons were collected up to 14 days postburn. Sham-treated animals served as a control group. We analyzed tendons for changes in expression of IL-6, IL-1ß, TNF, MMP9, MMP13, TGFß1, Collagens I and III, and for morphological and biomechanical changes. Gene expression of IL-6 and IL-1ß as well as MMP9 and MMP13 increased in rat tendon 3 days after burn. Col3a1 increased at day 3 and col1a1 at day 7. At day 14, TGFß1 increased, whereas the protein ratio for collagens I/III decreased, indicating tendon remodeling. Histological analysis with H&E and Picrosirius red staining further revealed a decrease in organized collagen fibers 14 days after burn. Biomechanical analysis showed a decrease in stiffness and ultimate force of tendons in burn rats.We conclude that tendinopathy was observed in Achilles tendon 14 days after severe burn, via the induction of inflammation and remodeling. The present study provides a model of tendinopathy that may be used for the development of therapeutic approaches after burn.

Burn Serum Stimulates Myoblast Cell Death Associated with IL-6-Induced Mitochondrial Fragmentation.

BACKGROUND: Burn patients suffer muscle mass loss associated with hyperinflammation and hypercatabolism. The mitochondria are affected by this metabolic alteration. Mitochondrial fission activates a caspase cascade that ultimately leads to cell death. We postulate that burn-induced muscle loss is associated with increased mitochondrial fission and subsequent functional impairment. Further, we investigated whether the cytokine IL-6 plays a major role in mitochondrial fission-associated cell death after burn. METHODS: Murine myoblast C2C12 cells were treated with 10% serum isolated either from control rats or 40% total body surface area burned rats. Mitochondria were labeled with MitoTracker Green for live cell images. Mitochondrial function was assessed with an Enzo Mito-ID membrane potential cytotoxicity kit. Protein signals were detected by Western blot analysis. Moreover, recombinant IL-6 was applied to stimulate C2C12 to differentiate the role of cytokine IL-6; lastly, we treated burn serum-stimulated cells with IL-6 antibodies. RESULTS: Caspase 3 activity increased in C2C12 cells with burn serum stimulation, suggesting increased cell death in skeletal muscle after burn. Mitochondrial morphology shortened and mitochondrial membrane potential decreased in cells treated with burn serum. Western blot data showed that mitofusion-1 expression significantly decreased in burn serum-treated cells, supporting the morphologic observation of mitochondrial fission. Mitochondrial fragmentation increased with IL-6 stimulation, and IL-6 antibody decreased caspase 3 activity and mitochondrial membrane potential improved in burn serum-stimulated cells. CONCLUSION: Burn serum caused muscle cell death associated with increased mitochondrial fission and functional impairment. This alteration was alleviated with IL-6 antibody treatment, suggesting the cytokine plays a role in mitochondrial changes in muscle after systemic injury.

Targeting bacterial adherence inhibits multidrug-resistant Pseudomonas aeruginosa infection following burn injury.

Classical antimicrobial drugs target proliferation and therefore place microbes under extreme selective pressure to evolve resistance. Alternative drugs that target bacterial virulence without impacting survival directly offer an attractive solution to this problem, but to date few such molecules have been discovered. We previously discovered a widespread group of bacterial adhesins, termed Multivalent Adhesion Molecules (MAMs) that are essential for initial binding of bacteria to host tissues and virulence. Thus, targeting MAM-based adherence is a promising strategy for displacing pathogens from host tissues and inhibiting infection. Here, we show that topical application of polymeric microbeads functionalized with the adhesin MAM7 to a burn infected with multidrug-resistant Pseudomonas aeruginosa substantially decreased bacterial loads in the wound and prevented the spread of the infection into adjacent tissues. As a consequence, the application of this adhesion inhibitor allowed for vascularization and wound healing, and maintained local and systemic inflammatory responses to the burn. We propose that MAM7-functionalized microbeads can be used as a topical treatment, to reduce bacterial attachment and hence prevent bacterial colonization and infection of wounds. As adhesion is not required for microbial survival, this anti-infective strategy has the potential to treat multidrug-resistant infections and limit the emergence of drug-resistant pathogens.

Caspase inhibition reduces cardiac myocyte dyshomeostasis and improves cardiac contractile function after major burn injury.

In the heart, thermal injury activates a group of intracellular cysteine proteases known as caspases, which have been suggested to contribute to myocyte inflammation and dyshomeostasis. In this study, Sprague-Dawley rats were given either a third-degree burn over 40% total body surface area plus conventional fluid resuscitation or sham burn injury. Experimental groups included 1) sham burn given vehicle, 400 microl DMSO; 2) sham burn given Q-VD-OPh (6 mg/kg), a highly specific and stable caspase inhibitor, 24 and 1 h prior to sham burn; 3) burn given vehicle, DMSO as above; 4) burn given Q-VD-OPh (6 mg/kg) 24 and 1 h prior to burn. Twenty-four hours postburn, hearts were harvested and studied with regard to myocardial intracellular sodium concentration, intracellular pH, ATP, and phosphocreatine (23Na/31P nuclear magnetic resonance); myocardial caspase-1, -3,and -8 expression; myocyte Na+ (fluorescent indicator, sodium-binding benzofurzan isophthalate); myocyte secretion of TNF-alpha, IL-1beta, IL-6, and IL-10; and myocardial performance (Langendorff). Burn injury treated with vehicle alone produced increased myocardial expression of caspase-1, -3, and -8, myocyte Na+ loading, cytokine secretion, and myocardial contractile depression; cellular pH, ATP, and phosphocreatine were stable. Q-VD-OPh treatment in burned rats attenuated myocardial caspase expression, prevented burn-related myocardial Na+ loading, attenuated myocyte cytokine responses, and improved myocardial contraction and relaxation. The present data suggest that signaling through myocardial caspases plays a pivotal role in burn-related myocyte sodium dyshomeostasis and myocyte inflammation, perhaps contributing to burn-related contractile dysfunction.

Cardiac molecular signaling after burn trauma.

Research using mammalian burn models has defined significant cardiac deficits after burn injury. The physiologic response to burn and burn complicated by sepsis, including the cardiac dysfunction associated with these insults, remains a very complex physiologic process which, despite active study, remains unclear. The well-characterized inflammatory mediators such as tumor necrosis factor-alpha, interleukin-1beta, and interleukin-6 continue to play an active role in mediating cardiac dysfunction. However, perhaps of greater interest are the late mediators, high mobility group box 1 and macrophage migration inhibitory factor, because they offer a very realistic window for therapeutic intervention for controlling the inflammatory response. In addition, several other mediators of cardiac dysfunction have been identified and include the heat shock proteins, apoptosis, and the inflammatory caspases. These new mediators provide opportunities for therapeutic intervention, but further research is needed to clarify the importance of their mechanisms of action and the complex interactions between these various signaling pathways.

Tumor necrosis factor-alpha-induced caspase activation mediates endotoxin-related cardiac dysfunction.

OBJECTIVE: Sepsis-induced cardiac dysfunction is a serious clinical syndrome characterized by hypotension, decreased systemic vascular resistance, and elevated cardiac index. Although cytokines such as tumor necrosis factor (TNF)-alpha have been shown to play a significant role early in this response, the downstream effects of TNF-alpha signaling on cardiac function, specifically its relationship to apoptosis, have not been fully elucidated. DESIGN: Previous studies from our laboratory have identified endotoxin-induced apoptosis in cardiac cells in vitro. To further determine the role of lipopolysaccharide-induced apoptosis in vivo, mice were injected intraperitoneally with lipopolysaccharide (4 mg/kg), and cardiac apoptosis was detected and inhibited using a broad-spectrum caspase inhibitor. SETTING: University research laboratory. SUBJECTS: Adult male wild-type (B6:129PF1/J) and TNF receptor 1/receptor 2 (TNFR-1/2) knockout mice (B6;129S-Tnfrsf1aTnfrsf1b). INTERVENTIONS: We sought to determine the dependence of cardiac apoptosis on TNF-alpha signaling and determine the physiologic role of caspase activation on lipopolysaccharide-induced cardiac dysfunction. MEASUREMENTS AND MAIN RESULTS: Cardiac apoptosis was determined at baseline and at 2, 4, 8, and 24 hrs by detection of capase-3 and -8 activity, cytoplasmic levels of Bax/Bcl-2, cleaved caspase-3 immunohistochemistry, and terminal deoxynucleotidyl transferase UTP nick-end labeling (TUNEL) staining of histologic sections in wild-type and TNFR-1/2 knockout mice. To determine the role of caspase activation in lipopolysaccharide-induced cardiac dysfunction, a broad-spectrum caspase inhibitor Z-Val-Ala-Asp (ome)-FMK (sad) was given, and cardiac function was determined in isolated beating hearts (Langendorff preparation). Our experiments determined that caspase-3-dependent apoptosis was active in cardiac tissue by 2 hrs and that this activation was completely mediated by TNFR-1/2. The Bax/Bcl-2 ratios supported the finding and time course of apoptosis, whereas TUNEL staining of cardiac tissue sections identified sporadic apoptotic ventricular cells. The administration of zVAD significantly inhibited myocardial caspase-3 activity and preserved cardiac physiologic function (Langendorff preparation). CONCLUSIONS: Endotoxin induces a TNF-alpha-dependent apoptotic cascade in the myocardium, which contributes to the development of cardiac dysfunction.

A three-component dicamba O-demethylase from Pseudomonas maltophilia, strain DI-6: purification and characterization.

Dicamba O-demethylase is a multicomponent enzyme that catalyzes the conversion of the herbicide 2-methoxy-3,6-dichlorobenzoic acid (dicamba) to 3,6-dichlorosalicylic acid (DCSA). The three components of the enzyme were purified and characterized. Oxygenase(DIC) is a homotrimer (alpha)3 with a subunit molecular mass of approximately 40 kDa. FerredoxinDIC and reductaseDIC are monomers with molecular weights of approximately 14 and 45 kDa, respectively. EPR spectroscopic analysis suggested the presence of a single [2Fe-2S](2+/1+) cluster in ferredoxinDIC and a single Rieske [2Fe-2S](2+; 1+) cluster within oxygenaseDIC. Consistent with the presence of a Rieske iron-sulfur cluster, oxygenaseDIC displayed a high reduction potential of E(m,7.0) = -21 mV whereas ferredoxinDIC exhibited a reduction potential of approximately E(m,7.0) = -171 mV. Optimal oxygenaseDIC activity in vitro depended on the addition of Fe2+. The identification of formaldehyde and DCSA as reaction products demonstrated that dicamba O-demethylase acts as a monooxygenase. Taken together, these data suggest that oxygenaseDIC is an important new member of the Rieske non-heme iron family of oxygenases.

Macrophage migration inhibitory factor mediates late cardiac dysfunction after burn injury.

We have recently demonstrated that macrophage migration inhibitory factor (MIF) is a myocardial depressant protein and that MIF mediates late, prolonged cardiac dysfunction after endotoxin challenge in mice. Because many factors, including endotoxin, have been implicated in the pathogenesis of cardiac dysfunction after burn injury, we tested the hypothesis that MIF might also be the mediator of prolonged cardiac dysfunction in this model. At 4 h after 40% total body surface area burn in anesthetized mice, serum MIF levels increased significantly compared with baseline (2.2-fold). This increase was accompanied by a significant decrease in cardiac tissue MIF levels (2.1-fold decrease compared with controls). This pattern was consistent with MIF release from preformed cytoplasmic stores in the heart and other organs. To determine whether MIF mediates cardiac dysfunction after burn injury, mice were pretreated with anti-MIF neutralizing monoclonal antibodies or isotype control antibodies. Beginning 4 h after burn injury (and continuing through 48 h), burned mice demonstrated a significantly depressed left ventricular shortening fraction of 38.6 +/- 1.8%, compared with the normal controls (56.0 +/- 2.6%). Mice treated with anti-MIF displayed an initial depression of cardiac function similar to nontreated animals but then showed rapid restoration of cardiac function with complete recovery by 24 h, which persisted for the duration of the protocol. This study is the first to demonstrate that MIF mediates late, prolonged cardiac dysfunction after burn injury and suggests that MIF blockade should be considered a therapeutic target for the treatment of burn injury.

Malondialdehyde-acetaldehyde haptenated protein binds macrophage scavenger receptor(s) and induces lysosomal damage.

There is evidence that the chemical modification of proteins (haptens) with malondialdehyde-acetaldehyde (MAA) and the immune response to these haptenated proteins is associated with the initiation and/or progression of alcohol liver disease. Experimentally, proteins modified with MAA induce antibody and T cell responses, which are mediated by scavenger receptor(s). Moreover, macrophages have been shown to play an important role in processing and presenting MAA-haptenated proteins in vitro. In vitro, MAA-modified proteins have been shown to induce both apoptosis and necrosis in a dose- and cell-type-dependent manner. Natural ligands modified by oxidative stress, such as oxidized LDL, similarly initiate not only antibody responses, but also cause cell death by disrupting lysosomes after binding to scavenger receptors and internalization. We therefore investigated the binding, internalization, and lysosomal integrity in a macrophage cell line to a MAA-haptenated protein. We demonstrate for the first time that MAA-haptenated proteins are preferentially bound by scavenger receptors on macrophages, which internalize the ligands and shuttle them to lysosomes. Moreover, MAA-haptenated proteins are demonstrated to be associated with a rapid dose-dependent disruption in lysosomal integrity, resulting in leakage and caspase activation. Similarly, as hen egg lysozyme (HEL)-MAA concentrations increased (>31.3 microg/ml), increased levels of apoptosis and a G1/S cell cycle checkpoint inhibition were identified. This study identifies mechanisms by which MAA-haptenated proteins are taken up by a representative antigen-presenting cell and may delineate steps by which MAA-haptenated proteins induce cell death and induce their immunogenicity to the carrier protein.

Macrophage migration inhibitory factor is a cardiac-derived myocardial depressant factor.

Macrophage migration inhibitory factor (MIF) is a pluripotent proinflammatory cytokine that is ubiquitously expressed in organs, including the heart. However, no specific role for MIF in modulating cardiac performance has yet been described. Therefore, we examined cardiac MIF expression in mice after LPS challenge (4 mg/kg) and tested the hypothesis that MIF is a mediator of LPS-induced cardiac dysfunction. Western blots of whole heart lysates, as well as immunohistochemistry, documented constitutive MIF protein expression in the heart. Cardiac MIF protein levels significantly decreased after LPS challenge, reaching a nadir at 12 h, and then returned to baseline by 24 h. This pattern was consistent with MIF release from cytoplasmic stores after endotoxin challenge. After release of protein, MIF mRNA levels increased 24-48 h postchallenge. To determine the functional consequences of MIF release, we treated LPS-challenged mice with anti-MIF neutralizing antibodies or isotype control antibodies. Anti-MIF-treated animals had significantly improved cardiac function, as evidenced by a significant improvement in left ventricular (LV) fractional shortening percentage at 8, 12, 24, and 48 h after endotoxin challenge. In support of these findings, perfusion of isolated beating mouse hearts (Langendorff preparation) with recombinant MIF (20 ng/ml) led to a significant decrease in both systolic and diastolic performance [LV pressure (LVP), positive and negative first derivative of LVP with respect to time, and rate of LVP rise at developed pressure of 40 mmHg]. This study demonstrates that MIF mediates LPS-induced cardiac dysfunction and suggests that MIF should be considered a pharmacological target for the treatment of cardiac dysfunction in sepsis and potentially other cardiac diseases.

Molecular and pharmacological approaches to inhibiting nitric oxide after burn trauma.

Whereas controversial, several studies have suggested that nitric oxide (NO) alters cardiac contractility via cGMP, peroxynitrite, or poly(ADP ribose) synthetase (PARS) activation. This study determined whether burn-related upregulation of myocardial inducible NO synthase (iNOS) and NO generation contributes to burn-mediated cardiac contractile dysfunction. Mice homozygous null for the iNOS gene (iNOS knockouts) were obtained from Jackson Laboratory. iNOS knockouts (KO) as well as wild-type mice were given a cutaneous burn over 40% of the total body surface area by the application of brass probes (1 x 2 x 0.3 cm) heated to 100 degrees C to the animals' sides and back for 5 s (iNOS/KO burn and wild-type burn). Additional groups of iNOS KO and wild-type mice served as appropriate sham burn groups (iNOS/KO sham and wild-type sham). Cardiac function was assessed 24 h postburn by perfusing hearts (n = 7-10 mice/group). Burn trauma in wild-type mice impaired cardiac function as indicated by the lower left ventricular pressure (LVP, 67 +/- 2 mmHg) compared with that measured in wild-type shams (94 +/- 2 mmHg, P < 0.001), a lower rate of LVP rise (+dP/dtmax, 1,620 +/- 94 vs. 2,240 +/- 58 mmHg/s, P < 0.001), and a lower rate of LVP fall (-dP/dtmax, 1,200 +/- 84 vs. 1,800 +/- 42 mmHg/s, P < 0.001). Ventricular function curves confirmed significant contractile dysfunction after burn trauma in wild-type mice. Burn trauma in iNOS KO mice produced fewer cardiac derangements compared with those observed in wild-type burns (LVP: 78 +/- 5 mmHg; +dP/dt: 1,889 +/- 160 mmHg/s; -dP/dt: 1,480 +/- 154 mmHg/s). The use of a pharmacological approach to inhibit iNOS (aminoguanidine, given ip) in additional wild-type shams and burns confirmed the iNOS KO data. Whereas the absence of iNOS attenuated burn-mediated cardiac contractile dysfunction, these experiments did not determine the contribution of cardiac-derived NO versus NO generated by immune cells. However, our data indicate a role for NO in cardiac dysfunction after major trauma.

I kappa B overexpression in cardiomyocytes prevents NF-kappa B translocation and provides cardioprotection in trauma.

This study examined the effects of either IkappaBalpha overexpression (transgenic mice) or N-acetyl-leucinyl-leucinyl-norleucinal (ALLN) administration (proteosome inhibitor in wild-type mice) on cardiomyocyte secretion of tumor necrosis factor-alpha (TNF-alpha) and on cardiac performance after burn trauma. Transgenic mice were divided into four experimental groups. IkappaBalpha overexpressing mice were given a third-degree scald burn over 40% of the total body surface area or wild-type littermates were given either a scald or sham burn to provide appropriate controls. Pharmacological studies included ALLN (20 mg/kg) administration in either burned wild-type mice or wild-type shams. Burn trauma in wild-type mice promoted nuclear factor-kappaB (NF-kappaB) nuclear translocation, cardiomyocyte secretion of TNF-alpha, and impaired cardiac performance. IkappaBalpha overexpression or ALLN treatment of burn trauma prevented NF-kappaB activation in cardiac tissue, prevented cardiomyocyte secretion of TNF-alpha, and ablated burn-mediated cardiac contractile dysfunction. These data suggest that NF-kappaB activation and inflammatory cytokine secretion play a significant role in postburn myocardial abnormalities.

Burn plasma mediates cardiac myocyte apoptosis via endotoxin.

Thermal trauma is associated with cardiac myocyte apoptosis in vivo. To determine whether cardiac myocyte apoptosis could be secondary to burn-induced cytokines or inflammatory mediators, we investigated the effects of tumor necrosis factor-alpha (TNF-alpha) and burn plasma on a murine cardiac myocyte cell line and primary culture myocytes. HL-1 cells were exposed to plasma isolated from burned or sham rats. Burn, but not sham plasma, induced significant increases in caspase-3 activity and DNA fragmentation. Similar results were obtained in primary culture rat myocytes. A dose-dependent increase in caspase-3 activity was observed when HL-1 cells were incubated with increasing concentrations of TNF-alpha. Even though TNF-alpha increased apoptosis, enzyme-linked immunosorbent assay detected no TNF-alpha in burn plasma. Burn plasma also failed to induce TNF-alpha mRNA, eliminating an autocrine mechanism of TNF-alpha secretion and binding. Also, treatment of burn plasma containing rhuTNFR:Fc failed to inhibit apoptosis. To examine the possibility that endotoxin within burn plasma might account for the apoptotic effect, burn plasma was preincubated with rBPI(21). Caspase-3 activity was reduced to control levels. These data indicate that burn plasma induces apoptosis in cardiac myocytes via an endotoxin-dependent mechanism and suggest that systemic inhibition of endotoxin may provide a therapeutic approach for treatment of burn-associated cardiac dysfunction.