Characterisation of the biological response of Saccharomyces cerevisiae to the loss of an allele of the eukaryotic initiation factor 4A.

The translation initiation machinery is emerging as an important target for therapeutic intervention, with potential in the treatment of cancer, viral infections, and muscle wasting. Amongst the targets for pharmacological control of translation initiation is the eukaryotic initiation factor 4A (eIF4A), an RNA helicase that is essential for cap-dependent translation initiation. We set out to explore the system-wide impact of a reduction of functional eIF4A. To this end, we investigated the effect of deletion of TIF1, one of the duplicate genes that produce eIF4A in yeast, through synthetic genetic array interactions and system-wide changes in GFP-tagged protein abundances. We show that there is a biological response to deletion of the TIF1 gene that extends through the proteostasis network. Effects of the deletion are apparent in processes as distributed as chromatin remodelling, ribosome biogenesis, amino acid metabolism, and protein trafficking. The results from this study identify protein complexes and pathways that will make ideal targets for combination therapies with eIF4A inhibitors.

Identification of Targeting Peptides for Mucosal Delivery in Sheep and Mice.

In this study we identified and characterized a novel cyclic peptide that facilitates the rapid transportation of conjugated molecules across the epithelial layer of the small intestine. The peptide was initially selected from phage display libraries using a large animal experimental model, which employed consecutive in vitro and in vivo panning. The procedure was designed to enrich for peptides that facilitated transcytosis across the intestinal epithelium into the intestinal afferent lymphatic system. A small set of peptides was repeatedly isolated using this selection method; however, the cyclic nonamer CTANSSAQC, 13C, dominated. The activity of the putative targeting peptide 13C was then verified using a mouse model. These experiments showed that the 13C peptide as well as macromolecules conjugated to it were rapidly transported across the intestinal mucosa into distinct subsets of epithelial cells and CD11c+ cells located in the lamina propria and Peyer's Patches. Significant amounts of intact protein could be delivered into the systemic circulation after rectal and nasal application. Thus, peptide 13C is regarded as an attractive carrier candidate for mucosal delivery of large molecules. The preferential targeting to distinct intestinal cells may be utilized to deliver active biological drugs for the effective control of diseases of the gut.

Estrogen-provided cardiac protection following burn trauma is mediated through a reduction in mitochondria-derived DAMPs.

Mitochondria-derived danger-associated molecular patterns (DAMPs) play important roles in sterile inflammation after acute injuries. This study was designed to test the hypothesis that 17ß-estradiol protects the heart via suppressing myocardial mitochondrial DAMPs after burn injury using an animal model. Sprague-Dawley rats were given a third-degree scald burn comprising 40% total body surface area (TBSA). 17ß-Estradiol, 0.5 mg/kg, or control vehicle was administered subcutaneously 15 min following burn. The heart was harvested 24 h postburn. Estradiol showed significant inhibition on the productivity of H2O2 and oxidation of lipid molecules in the mitochondria. Estradiol increased mitochondrial antioxidant defense via enhancing the activities and expression of superoxide dismutase (SOD) and glutathione peroxidase (GPx). Estradiol also protected mitochondrial respiratory function and structural integrity. In parallel, estradiol remarkably decreased burn-induced release of mitochondrial cytochrome c and mitochondrial DNA (mtDNA) into cytoplasm. Further, estradiol inhibited myocardial apoptosis, shown by its suppression on DNA laddering and downregulation of caspase 1 and caspase 3. Estradiol's anti-inflammatory effect was demonstrated by reduction in systemic and cardiac cytokines (TNF-α, IL-1ß, and IL-6), decrease in NF-κB activation, and attenuation of the expression of inflammasome component ASC in the heart of burned rats. Estradiol-provided cardiac protection was shown by reduction in myocardial injury marker troponin-I, amendment of heart morphology, and improvement of cardiac contractility after burn injury. Together, these data suggest that postburn administration of 17ß-estradiol protects the heart via an effective control over the generation of mitochondrial DAMPs (mtROS, cytochrome c, and mtDNA) that incite cardiac apoptosis and inflammation.

17ß-Estradiol reappropriates mass lost to the hypermetabolic state in thermally injured rats.

BACKGROUND: The hypermetabolic response to severe thermal injury is unlike any physiologic response seen in medicine. While some parallels can be drawn to shock and sepsis states, this response is typified by its intensity and duration. Our group has been interested in the myriad effects of estrogens after injury, specifically the ability of estrogens to reduce inflammatory responses. Given this, and the known link between severe inflammation and the hypermetabolic response, we examined the effects of a single dose of 17ß estradiol administered after a severe thermal injury in rats. METHODS: Twelve male Sprague-Dawley rats were subject to either a sham burn or a 40% total body surface area burn, followed by fluid resuscitation. Burned animals were divided into a vehicle and treatment group, with injections given 15 min after the injury. Animals were monitored for a period of 45 d, with markers of hypermetabolism (weight, fecal output, food intake, and serum insulin and glucose) measured daily. RESULTS: We identified a significant difference in daily measured weights between the burned groups. We observed a sparing of body mass during the acute phase lasting 2 wk after the injury and an improved recovery phase during the remainder of the study. Glucose and insulin levels during the first week of the study did not differ between the treatment groups. CONCLUSION: Estrogen may have a role in preserving body mass after severe thermal injury. Further studies are required to determine if this spared body mass composition.

The cellular target specificity of pateamine A.

The natural product pateamine A (pateamine) from the sponge Mycale hentscheli is active against a wide range of dividing cells and has been shown to inhibit the functions of the eukaryotic initiation factor 4A (eIF4A). We have identified that pateamine is additionally able to modulate the formation of actin filaments and microtubules in vitro but at higher concentrations than required for inhibition of eIF4A. Cell cycle analysis confirmed that actin and tubulin are not major mediators of the cellular activity of pateamine. The range of targets identified demonstrates the value of multiple approaches to determining the mode of action of biologically active compounds.

Specific inhibition of mitochondrial oxidative stress suppresses inflammation and improves cardiac function in a rat pneumonia-related sepsis model.

Using a mitochondria-targeted vitamin E (Mito-Vit-E) in a rat pneumonia-related sepsis model, we examined the role of mitochondrial reactive oxygen species in sepsis-mediated myocardial inflammation and subsequent cardiac contractile dysfunction. Sepsis was produced in adult male Sprague-Dawley rats via intratracheal injection of S. pneumonia (4 × 10(6) colony formation units per rat). A single dose of Mito-Vit-E, vitamin E, or control vehicle, at 21.5 µmol/kg, was administered 30 min postinoculation. Blood was collected, and heart tissue was harvested at various time points. Mito-Vit-E in vivo distribution was confirmed by mass spectrometry. In cardiac mitochondria, Mito-Vit-E improved total antioxidant capacity and suppressed H(2)O(2) generation, whereas vitamin E offered little effect. In cytosol, both antioxidants decreased H(2)O(2) levels, but only vitamin E strengthened antioxidant capacity. Mito-Vit-E protected mitochondrial structure and function in the heart during sepsis, demonstrated by reduction in lipid and protein oxidation, preservation of mitochondrial membrane integrity, and recovery of respiratory function. While both Mito-Vit-E and vitamin E suppressed sepsis-induced peripheral and myocardial production of proinflammatory cytokines (tumor necrosis factor-α, interleukin-1ß, and interleukin-6), Mito-Vit-E exhibited significantly higher efficacy (P < 0.05). Stronger anti-inflammatory action of Mito-Vit-E was further shown by its near-complete inhibition of sepsis-induced myeloperoxidase accumulation in myocardium, suggesting its effect on neutrophil infiltration. Echocardiography analysis indicated that Mito-Vit-E ameliorated cardiac contractility of sepsis animals, shown by improved fractional shortening and ejection fraction. Together, our data suggest that targeted scavenging of mitochondrial reactive oxygen species protects mitochondrial function, attenuates tissue-level inflammation, and improves whole organ activities in the heart during sepsis.

Regulation of VEGF-induced endothelial cell migration by mitochondrial reactive oxygen species.

Endothelial migration is a crucial aspect of a variety of physiologic and pathologic conditions including atherosclerosis and vascular repair. Reactive oxygen species (ROS) function as second messengers during endothelial migration. Multiple intracellular sources of ROS are regulated by cellular context, external stimulus, and the microenvironment. However, the predominant source of ROS during endothelial cell (EC) migration and the mechanisms by which ROS regulate cell migration are incompletely understood. In this study, we tested the hypothesis that mitochondria-derived ROS (mtROS) regulate EC migration. In cultured human umbilical vein endothelial cells, VEGF increased mitochondrial metabolism, promoted mtROS production, and induced cell migration. Either the targeted mitochondrial delivery of the antioxidant, vitamin E (Mito-Vit-E), or the depletion of mitochondrial DNA abrogated VEGF-mediated mtROS production. Overexpression of mitochondrial catalase also inhibited VEGF-induced mitochondrial metabolism, Rac activation, and cell migration. Furthermore, these interventions suppressed VEGF-stimulated EC migration and blocked Rac1 activation in endothelial cells. Constitutively active Rac1 reversed Mito-Vit-E-induced inhibition of EC migration. Mito-Vit-E also attenuated carotid artery reendothelialization in vivo. These results provide strong evidence that mtROS regulate EC migration through Rac-1.

Intraspecific epitopic variation in a carbohydrate antigen exposed on the surface of Trichostrongylus colubriformis infective L3 larvae.

The carbohydrate larval antigen, CarLA, is present on the exposed surface of all strongylid nematode infective L3 larvae tested, and antibodies against CarLA can promote rapid immune rejection of incoming Trichostrongylus colubriformis larvae in sheep. A library of ovine recombinant single chain Fv (scFv) antibody fragments, displayed on phage, was prepared from B cell mRNA of field-immune sheep. Phage displaying scFvs that bind to the surface of living exsheathed T. colubriformis L3 larvae were identified, and the majority of worm-binding scFvs recognized CarLA. Characterization of greater than 500 worm surface binding phage resulted in the identification of nine different anti-CarLA scFvs that recognized three distinct T. colubriformis CarLA epitopes based on blocking and additive ELISA. All anti-CarLA scFvs were specific to the T. colubriformis species of nematode. Each of the three scFv epitope classes displayed identical Western blot recognition patterns and recognized the exposed surface of living T. colubriformis exsheathed L3 larvae. Surprisingly, each of the anti-CarLA scFvs was able to bind to only a subset of worms. Double-labelling indirect immunofluorescence revealed that the three classes of anti-CarLA scFvs recognize distinct, non-overlapping, T. colubriformis sub-populations. These results demonstrate that individual T. colubriformis L3 larvae display only one of at least three distinct antigenic forms of CarLA on their surface at any given time, and suggest that antigenic variation within CarLA is likely a mechanism of immune evasion in strongylid nematodes.

Burn serum causes a CD14-dependent mitochondrial damage in primary cardiomyocytes.

Studies from animal models suggest that myocardial mitochondrial damage contributes to cardiac dysfunction after burn injury. In this report, we used an ex vivo model of primary cardiomyocyte culture to investigate the mechanisms of burn-induced mitochondrial impairment. Briefly, blood serum was collected from Sprague-Dawley (SD) rats subjected to 40% total body surface area burn and added (10% vol/vol) to primary cardiomyocytes prepared from SD rats. The effect of the burn serum on mitochondrial function and membrane integrity in the myocytes was analyzed. Exposure of myocytes to burn serum doubled the mitochondrial membrane damage measured by two independent assays. This treatment also significantly elevated mitochondrial oxidative stress, indicated by a more than 30% increase in lipid oxidation. Downregulation of mitochondrial antioxidant defense was also evident since the activities of the antioxidant enzymes superoxide dismutase and glutathione peroxidase were reduced by about 30% and 50%, respectively. Burn serum also induced deficiency of mitochondrial metabolism, indicated by a 30% decrease in the activity of cytochrome c oxidase. These mitochondrial dysfunctions appear to be generated by oxidative stress because burn serum induced a significant increase of mitochondrial oxygen species (mtROS) in cardiomyocytes, and pretreatment of cardiomyocytes with the antioxidant N-acetyl-cysteine prevented the mitochondrial damages induced by burn serum. Remarkably, the increase in mtROS was abolished by an antibody-mediated blockade of CD14. Furthermore, burn injury-induced mitochondrial damage in cardiomyocytes was prevented in CD14 knockout mice. Taken together, these data suggested that burn injury produces CD14-dependent mitochondrial damage via oxidative stress in myocardium.

Estrogen treatment following severe burn injury reduces brain inflammation and apoptotic signaling.

BACKGROUND: Patients with severe burn injury experience a rapid elevation in multiple circulating pro-inflammatory cytokines, with the levels correlating with both injury severity and outcome. Accumulations of these cytokines in animal models have been observed in remote organs, however data are lacking regarding early brain cytokine levels following burn injury, and the effects of estradiol on these levels. Using an experimental animal model, we studied the acute effects of a full-thickness third degree burn on brain levels of TNF-alpha, IL-1beta, and IL-6 and the protective effects of acute estrogen treatment on these levels. Additionally, the acute administration of estrogen on regulation of inflammatory and apoptotic events in the brain following severe burn injury were studied through measuring the levels of phospho-ERK, phospho-Akt, active caspase-3, and PARP cleavage in the placebo and estrogen treated groups. METHODS: In this study, 149 adult Sprague-Dawley male rats received 3rd degree 40% total body surface area (TBSA) burns. Fifteen minutes following burn injury, the animals received a subcutaneous injection of either placebo (n = 72) or 17 beta-estradiol (n = 72). Brains were harvested at 0.5, 1, 2, 4, 6, 8, 12, 18, and 24 hours after injury from the control (n = 5), placebo (n = 8/time point), and estrogen treated animals (n = 8/time point). The brain cytokine levels were measured using the ELISA method. In addition, we assessed the levels of phosphorylated-ERK, phosphorylated-Akt, active caspase-3, and the levels of cleaved PARP at the 24 hour time-point using Western blot analysis. RESULTS: In burned rats, 17 beta-estradiol significantly decreased the levels of brain tissue TNF-alpha (approximately 25%), IL-1beta (approximately 60%), and IL-6 (approximately 90%) when compared to the placebo group. In addition, we determined that in the estrogen-treated rats there was an increase in the levels of phospho-ERK (p < 0.01) and Akt (p < 0.05) at the 24 hour time-point, and that 17 beta-estradiol blocked the activation of caspase-3 (p < 0.01) and subsequent cleavage of PARP (p < 0.05). CONCLUSION: Following severe burn injury, estrogens decrease both brain inflammation and the activation of apoptosis, represented by an increase in the levels of phospho-Akt and inhibition of caspase-3 activation and PARP cleavage. Results from these studies will help further our understanding of how estrogens protect the brain following burn injury, and may provide a novel, safe, and effective clinical treatment to combat remote secondary burn injury in the brain and to preserve cognition.

Alpaca (Lama pacos) as a convenient source of recombinant camelid heavy chain antibodies (VHHs).

Recombinant single domain antibody fragments (VHHs) that derive from the unusual camelid heavy chain only IgG class (HCAbs) have many favourable properties compared with single-chain antibodies prepared from conventional IgG. As a result, VHHs have become widely used as binding reagents and are beginning to show potential as therapeutic agents. To date, the source of VHH genetic material has been camels and llamas despite their large size and limited availability. Here we demonstrate that the smaller, more tractable and widely available alpaca is an excellent source of VHH coding DNA. Alpaca sera IgG consists of about 50% HCAbs, mostly of the short-hinge variety. Sequencing of DNA encoding more than 50 random VHH and hinge domains permitted the design of PCR primers that will amplify virtually all alpaca VHH coding DNAs for phage display library construction. Alpacas were immunized with ovine tumour necrosis factor alpha (TNFalpha) and a VHH phage display library was prepared from a lymph node that drains the sites of immunizations and successfully employed in the isolation of VHHs that bind and neutralize ovine TNFalpha.

Three surface antigens dominate the mucosal antibody response to gastrointestinal L3-stage strongylid nematodes in field immune sheep.

Although gastrointestinal nematode parasites are a major human and veterinary health problem, little is known about how the host is sometimes able to mount an effective immune rejection response. In previous work, we identified a carbohydrate larval surface antigen (CarLA) as the target of mucosal antibodies that can elicit rejection of Trichostrongylus colubriformis L3s in sheep. Here we characterise the natural mucosal antibody responses to L3s from three major strongylid gastrointestinal parasites of sheep, Trichostrongylus colubriformis, Haemonchus contortus and Teladorsagia circumcincta. The mucosal antibody repertoire of naturally field-immune sheep was displayed on bacteriophage as single-chain antibodies (scFvs) and phage were selected for the ability to bind to the surface of living L3s of the three nematode species. All nematode-binding scFvs were found to recognize one of three different antigen classes that are each found in the three strongylid species. These three antigen classes appear to represent all of the major antigens recognized on Western blots by pooled mucosal antibodies from field-immune sheep. One of the antigen classes is a heterogeneous, high molecular weight molecule that is protease-sensitive. The scFvs recognizing this surface antigen also recognize a similar antigen in all strongylids tested. A second antigen class is a protease-insensitive, low molecular weight antigen found only in sheaths and scFvs recognizing this antigen cross-react with a similar molecule found in all strongylids tested. The third surface antigen class is CarLA and all of the anti-CarLA scFvs obtained from the field-immune sheep repertoire were specific to L3s of only one species and often recognized only a subset of the worms. Thus three different L3-stage surface antigens, two that lack a protein component, dominate the natural mucosal antibody response to L3-stage gastrointestinal strongylid nematodes in sheep.

Cardiac mitochondrial damage and loss of ROS defense after burn injury: the beneficial effects of antioxidant therapy.

Mechanisms of burn-related cardiac dysfunction may involve defects in mitochondria. This study determined 1) whether burn injury alters myocardial mitochondrial integrity and function; and 2) whether an antioxidant vitamin therapy prevented changes in cardiac mitochondrial function after burn. Sprague-Dawley rats were given a 3 degrees burn over 40% total body surface area and fluid resuscitated. Antioxidant vitamins or vehicle were given to sham and burn rats. Mitochondrial and cytosolic fractions were prepared from heart tissues at several times postburn. In mitochondria, lipid peroxidation was measured to assess oxidative stress, mitochondrial outer membrane damage and cytochrome-c translocation were determined to estimate mitochondrial integrity, and activities of SOD and glutathione peroxidase were examined to evaluate mitochondrial antioxidant defense. Cardiac function was measured by Langendorff model in sham and burn rats given either vitamins or vehicle. Twenty-four hours postburn, mitochondrial outer membrane damage was progressively increased to approximately 50%, and cytosolic cytochrome-c gradually accumulated to approximately three times more than that measured in shams, indicating impaired mitochondrial integrity. Maximal decrease of mitochondrial SOD activity occurred 8 h postburn ( approximately 63.5% of shams), whereas maximal decrease in glutathione peroxidase activity persisted 2-24 h postburn ( approximately 60% of shams). In burn animals, lipid peroxidation in cardiac mitochondria increased 30-50%, suggesting burn-induced oxidative stress. Antioxidant vitamin therapy prevented burn-related loss of membrane integrity and antioxidant defense in myocardial mitochondria and prevented cardiac dysfunction. These data suggest that burn-mediated mitochondrial dysfunction and loss of reactive oxygen species defense may play a role in postburn cardiac dysfunction.

Bactericidal/permeability increasing protein attenuates the myocardial inflammation/dysfunction that occurs with burn complicated by subsequent infection.

Intubation and mechanical ventilation after burn contribute to pneumonia-related infection. Although postburn presence or absence of endotoxin has been described, inactivation of Toll-like receptor 4 signaling has been shown to improve postburn organ function, suggesting that LPS participates in burn-related susceptibility to infection. We hypothesized that bactericidal/permeability-increasing protein (rBPI) given postburn would attenuate myocardial inflammation/dysfunction associated with postburn septic challenge given 7 days postburn. Rats were given burn over 40% total body surface area, lactated Ringer 4 ml.kg(-1).% burn(-1); burns received either vehicle or rBPI, 1 mg.kg(-1).h(-1) for 48 h postburn. Postburn day 7, subgroups of burns and shams were given intratracheal Klebsiella pneumoniae, 4 x 10(6) CFU to produce burn complicated by sepsis; additional sham and burn subgroups received intratracheal vehicle to produce sham sepsis. Vehicle-treated groups: 1) sham burn + sham sepsis 2) sham burn + sepsis, 3) burn + sham sepsis, 4) burn + sepsis. rBPI-treated groups: 5) sham burn + sham sepsis, 6) sham burn + sepsis, 7) burn + sham sepsis, 8) burn + sepsis. Cardiomyocyte cytokine secretion and myocardial function were studied 24 h after septic challenge, postburn day 8. Pneumonia-related infection 8 days after vehicle-treated burn produced myocyte cytokine secretion (pg/ml), indicated by increased myocyte TNF-alpha, 549 +/- 46; IL-1beta, 50 +/- 8; IL-6, 286 +/- 3 levels compared with levels in sham myocytes (TNF-alpha, 88 +/- 11; IL-1beta, 7 +/- 1; IL-6, 74 +/- 10; P < 0.05). Contractile dysfunction was evident from lower left ventricular pressure +/-dP/dt values in this group compared with sham. rBPI attenuated myocyte cytokine responses to septic challenge and improved contractile function, suggesting that burn-related mobilization of microbial-like products contribute to postburn susceptibility to infection.

Reducing susceptibility to bacteremia after experimental burn injury: a role for selective decontamination of the digestive tract.

We proposed that selective decontamination of the digestive tract (SDD) initiated after experimental burn injury would decrease myocardial inflammation and dysfunction after a second insult such as septic challenge. Rats were divided into eight experimental groups. Groups included sham burn plus sham sepsis, burn alone, sepsis alone, and burn plus sepsis given either water by oral gavage for 5 days after burn (or sham burn) or given oral antibiotics (polymyxin E, 15 mg; tobramycin, 6 mg; 5-flucytosin, 100 mg given by oral gavage, 2x daily for 5 days after burn or sham burn). Cardiac function and inflammation were studied 24 h after septic challenge. In the absence of SDD, burn alone, sepsis alone, or burn plus septic challenge promoted cardiac myocyte secretion of TNF-alpha (burn, 174+/-11; sepsis, 269+/-19; burn+sepsis, 453+/-14 pg/ml), IL-1beta (burn, 35+/-2; sepsis, 29+/-1; burn+sepsis, 48+/-7 pg/ml), and IL-6 (burn, 143+/-18; sepsis, 116+/-3; burn+sepsis, 248+/-12 pg/ml) compared with values measured in sham (TNF-alpha, 3+/-1; IL-1beta, 1+/-0.4; IL-6, 6+/-1.5 pg/ml) (P<0.05). Impaired ventricular contraction and relaxation responses were evident in the absence of SDD [burn+sepsis: left ventricular pressure (LVP), 65+/-4 mmHg; rate of LVP rise (+dP/dt), 1,320+/-131 mmHg/s compared with values measured in sham: LVP, 96+/-4 mmHg; +dP/dt, 2,095+/-99 mmHg/s, P<0.05]. SDD treatment of experimental burn attenuated septic challenge-related inflammatory responses and improved myocardial contractile responses, producing cardiac TNF-alpha, IL-1beta, and IL-6 levels, LVP, +dP/dt, and rate of LVP fall (-dP/dt) values that were significantly better (P<0.05) than values measured in burn plus sepsis in the absence of SDD. This work confirms that endogenous gut organisms contribute to sensitivity to subsequent infectious challenge.

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 mitochondrial damage and inflammation responses in sepsis.

BACKGROUND AND PURPOSE: Studies in sepsis suggest that mitochondria mediate multiple organ dysfunction, including cardiac failure; however, the underlying molecular mechanisms remain elusive. This study examined changes in mitochondrial membrane integrity, antioxidant activities, and oxidative stress in the heart after infectious challenge (intratracheal Streptococcus pneumoniae, 4 x 10(6) colony-forming units). Inflammation responses also were examined. METHODS: Cardiac tissues were harvested from Sprague-Dawley rats 4, 8, 12, and 24 h after bacterial challenge (or intratracheal vehicle for sham-treated animals) and homogenized, followed by preparation of subcellular fractions (mitochondrial, cytosol, and nuclei) or whole-tissue lysate. We examined mitochondrial outer membrane damage and cytochrome C translocation to evaluate mitochondrial integrity, mitochondrial lipid and protein oxidation to assess oxidative stress, and mitochondrial superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities to estimate antioxidant defense. In addition, we measured nuclear factor-kappa B (NF-kappaB) activation in myocardium and cytokine production to investigate inflammatory responses to septic challenge. RESULTS: Oxidation of mitochondrial protein and lipid was evident 4 h through 24 h after bacterial challenge. Mitochondrial outer membrane damage and cytochrome C release were accompanied by down-regulation of mitochondrial SOD and GPx activity. After bacterial challenge, systemic and myocardial cytokine production increased progressively, and NF-kappaB was activated gradually. CONCLUSION: Sepsis impaired cardiac mitochondria by damaging membrane integrity, increasing oxidative stress, and altering defenses against reactive oxygen species. These alterations occur earlier than or simultaneously with inflammatory responses in myocardium after infectious challenge, suggesting that mitochondria play a role in modulating inflammation in sepsis.

Sepsis and burn complicated by sepsis alter cardiac transporter expression.

UNLABELLED: Sepsis alone and burn complicated by sepsis produce significant cardiac dysfunction. Since calcium handling by the cardiomyocyte is essential for cardiac function, one mechanism for cardiac abnormalities may be calcium dyshomeostasis. We hypothesized that sepsis and burn plus sepsis alter cardiac calcium transporter expression. Sprague-Dawley rats were divided into: (1) control, (2) sepsis (intratracheal S. Pneumoniae, 4x10(6) CFU), and (3) burn (40% TBSA) plus sepsis. Myocyte [Ca(2+)](i) and [Na(+)](i) were quantified with Fura-2 AM and SBFI, respectively. Western blot analysis of rat hearts used antibodies against the sarcoplasmic reticular Ca(2+) ATPase (SERCA), the L-type calcium channel, the Na(+)/Ca(2+) exchanger or the Na(+)/K(+) ATPase. RESULTS: Sepsis in the presence and absence of burn trauma increased [Ca(2+)](i) and [Na(+)](i). SERCA expression was decreased in the sepsis and burn plus sepsis groups while calcium channel expression was transiently increased in these sepsis groups. Na(+)/Ca(2+) exchanger expression exhibited a biphasic pattern of altered expression. Sepsis and burn plus sepsis reduced Na(+)/K(+) ATPase protein levels. These data suggest that altered transporter expression produce cardiomyocyte calcium and sodium loading and may contribute to sepsis-mediated cardiac contractile dysfunction.

Echocardiography assessment of myocardial function after burn injury.

INTRODUCTION: Echocardiography provides noninvasive and clinically relevant assessment of left ventricle (LV) function in injury and disease. In this present study, we hypothesized that application of transthoracic echocardiography in awake mice would provide in vivo assessment of myocardial performance that correlates with in vitro assessment of LV function using isolated perfused hearts (Langendorff). METHODS: Burn over 40% of the total body surface area (or sham burn) was given in C57/BL6 mice 22 to 24 g; lactated Ringer fluid resuscitation was given intraperitoneally. Transthoracic echocardiography was performed at baseline and at designated intervals over 7 days postburn. Subgroups of mice were killed at intervals and hearts perfused to assess LV pressure and +/-dP/dt max responses to inotropic challenge (n = 8/group per time). RESULTS: Burn produced myocardial depression, evidenced by a fall in fractional shortening from 69% +/- 3% at baseline to 50% +/- 2% (12 h postburn), 58% +/- 3% (24 h), 59% +/- 2% (48 h), and 62% +/- 2% (72 h) (P < 0.05). Burn-related changes in in vivo LV performance were paralleled by in vitro evidence of myocardial contractile depression; LV pressure progressively fell from 97 +/- 2 mmHg at baseline to 84 +/- 7 mmHg (12 h), 63 +/- 2 (24 h), 79 +/- 4 mmHg (48 h postburn) (P < 0.05). Analysis of variance and multiple comparison procedure. Myocardial recovery occurred by day 8 postburn. CONCLUSIONS: Echocardiography provides measures of cardiac function in a conscious animal, which correlate with contractile depression measured in vitro using Langendorff preparations. Echocardiography is a valuable tool for noninvasive assessment of postburn myocardial function.

Effect of aspiration pneumonia-induced sepsis on post-burn cardiac inflammation and function in mice.

INTRODUCTION: Numerous studies have found that burn injury alters immune function, predisposing the subject to infectious complications. We developed a mouse model of burn injury complicated by either gram-positive or gram-negative infection and hypothesized that post-burn infection would exacerbate the myocardial cytokine responses and contractile dysfunction characteristic of either sepsis alone or burn alone. METHODS: Adult C57 BL6 mice were given burn injury over 40% of the total body surface area and conventional fluid resuscitation (lactated Ringer's solution, 4 mL/kg/% burn) followed on day 7 by intratracheal administration of 1 x 10(5) cfu of either Streptococcus pneumoniae or Klebsiella pneumoniae or saline. Mice received fluid resuscitation (2 mL of lactated Ringer's intraperitoneally) again after bacterial challenge. Cardiomyocyte cytokine secretion and the contractile function of isolated hearts (Langendorff perfusion) were examined in vitro 24 h after bacterial challenge. RESULTS: Infectious challenge seven days after burn injury exaggerated the inflammatory cytokine responses over those observed with either burn alone or gram-positive or gram-negative infection alone (tumor necrosis factor-alpha: sham, 72 +/- 9 pg/mL; burn alone, 176 +/- 6 pg/mL, Klebsiella pneumoniae alone, 337 +/- 8 pg/mL; Streptococcus pneumoniae alone, 184 +/- 2 pg/mL; burn + Klebsiella, 476 +/- 14 pg/mL; burn + Streptococcus, 351 +/- 6 pg/mL). Myocardial contractile depression was evident in the burn alone, infection alone, and burn plus infection groups, regardless of the organism selected to produce pneumonia-related sepsis. CONCLUSIONS: Gram-negative or gram-positive infection exacerbated the myocardial inflammation seen with burn alone or infection alone. The availability of a mouse model of burn injury complicated by pneumonia-related sepsis will allow use of genetically engineered mice to examine further the mechanisms by which burn injury increases susceptibility to infection.