Angiotensin II inhibitor DuP753 attenuates burn- and endotoxin-induced gut ischemia, lipid peroxidation, mucosal permeability, and bacterial translocation

OBJECTIVE: To investigate the role of angiotensin II as a mediator of burn- and sepsis-induced gut ischemia and reperfusion injury and to determine whether treatment with the angiotensin II inhibitor DuP753 can attenuate mucosal injury and bacterial translocation in a burn/endotoxemia porcine model. SUMMARY BACKGROUND DATA: Thermal injuries and endotoxemia have been shown to induce ischemia and reperfusion injury to the intestine, leading to increased mucosal permeability and bacterial translocation. Angiotensin II, the production of which has been reported to increase after burn, is thought to be one of the primary mediators of postburn mesenteric vasoconstriction. METHODS: An ultrasonic flow probe was inserted into the superior mesenteric artery and a catheter into the superior mesenteric vein in 21 female pigs. After 5 days, all animals were anesthetized, and 14 received 40% total body surface area third-degree burn. DuP753 was administered intravenously at 1 microg/kg to seven pigs immediately after burn. Eighteen hours after burn, 100 microg/kg Escherichia coli lipopolysaccharide (LPS) was intravenously administered. Systemic and splanchnic hemodynamics were measured and blood samples were drawn for blood gas analysis. Plasma conjugated dienes (PCDs), an index of lipid peroxidation, were measured every 6 hours. Intestinal permeability was assessed every 6 hours by measuring the lactulose/mannitol excretion ratio. At the end of the study (42 hours), tissue samples were harvested for bacteriologic cultures. RESULTS: Burn caused a significant decrease in mesenteric blood flow, to approximately 58% of baseline. Postburn endotoxemia significantly reduced the blood flow in the superior mesenteric artery to 53% of baseline. Treatment with DuP753 prevented postburn vasoconstriction and subsequently abrogated the impact of postburn endotoxemia on blood flow in the superior mesenteric artery. Mesenteric oxygen supply was significantly reduced after burn and endotoxin to 60% and 51% of baseline levels, respectively. DuP753 administration significantly improved mesenteric oxygen supply after both insults. Burn- and LPS-induced mesenteric hypoxia, as indicated by decreased mesenteric oxygen consumption, was also ameliorated by DuP753 treatment. PCD levels were significantly elevated 8 hours after burn. LPS caused a higher and prolonged increase in PCD levels. Treatment with DuP753 significantly reduced PCD levels after burn and after LPS. Intestinal permeability, as assessed by the lactulose/mannitol ratio, showed 6-fold and 12-fold increases after thermal injury and LPS, respectively. In contrast, the lactulose/mannitol ratio was only doubled in DuP753-treated animals. Bacterial translocation was significantly increased after burn and endotoxin. The incidence of bacterial translocation in the DuP753-treated animals was similar to that in the sham group. CONCLUSIONS: Angiotensin II appears to play a pivotal role in the burn- and endotoxin-induced intestinal ischemia and reperfusion injury, with subsequent increases in permeability and bacterial translocation. Postburn administration of the angiotensin II receptor antagonist DuP753 significantly reduces the extent of these events.     

Effects of interleukin-1alpha administration on intestinal ischemia and reperfusion injury,mucosal permeability,and bacterial translocation in burn and sepsis

OBJECTIVE: To evaluate the effect of interleukin-1alpha (IL-1alpha) on the mesenteric circulation, intestinal mucosal integrity, and bacterial translocation in a burn/endotoxemia chronic porcine model. SUMMARY BACKGROUND DATA: Major burn and sepsis are associated with a high mortality, ischemia/reperfusion injury to the intestine, and an increased rate of bacterial translocation. Pathologic alterations of IL-1 synthesis, degradation, and binding to receptors have been reported. Manipulation of IL-1-mediated effects might be of therapeutic utility. METHODS: Twenty-one female pigs were instrumented with an ultrasonic flow probe on the superior mesenteric artery and a catheter into the superior mesenteric vein. After 5 days, all animals were anesthetized, and 14 received 40% total body surface area third-degree burn. IL-1alpha was administered intravenously at 1,000 ng/kg to seven pigs immediately after burn. Eighteen hours after burn, 100 microg/kg lipopolysaccharide (LPS) was administered intravenously. Systemic and splanchnic hemodynamics were measured and blood samples were drawn for blood gas analysis. Intestinal permeability was assessed every 6 hours by measuring the lactulose/mannitol (L/M) excretion ratio. At the end of the study (42 hours), tissue samples were harvested for bacteriologic cultures. RESULTS: Mesenteric blood flow was significantly decreased after burn and endotoxin. Administration of IL-1alpha significantly improved mesenteric blood flow postburn and post-LPS. Mesenteric oxygen supply and consumption showed a significant reduction after burn. In contrast, animals treated with IL-1alpha showed an increase in postburn mesenteric oxygen supply and consumption. LPS-induced mesenteric hypoxia was also ameliorated by IL-1alpha treatment. Intestinal permeability, as assessed by the L/M ratio, showed a 7- and 10-fold elevation after thermal injury and LPS, respectively. In contrast, IL-1alpha-treated animals showed an increase of only three- and fourfold in the L/M ratio, respectively. Bacterial translocation was significantly increased in the burn/endotoxin group. IL-1alpha significantly reduced the rates of bacterial translocation. CONCLUSIONS: IL-1alpha treatment attenuates mesenteric ischemia and reperfusion injury induced by thermal injury and endotoxemia by improving mesenteric blood flow and oxygenation. Subsequently, IL-1alpha reduces intestinal permeability and bacterial translocation after burn and sepsis.     

Intestinal permeability is increased in burn patients shortly after injury

There is increasing direct experimental and indirect clinical evidence to indicate that under certain conditions intestinal barrier function may be lost in trauma victims. No direct measurements, however, have been performed in patients to determine whether intestinal permeability is increased shortly after a major thermal injury in the absence of infection. Fifteen hemodynamically stable burn patients with burns on more than 20% of their body surface (39% +/- 12%) had their intestinal permeability measured within 24 hours of injury with use of the two nonmetabolizable sugars lactulose and mannitol as permeability markers. Lactulose absorption was fourfold higher in the patients (223 +/- 54 mumol) than in the controls (58 +/- 11 mumole; p less than 0.02), whereas the lactulose/mannitol ratio was threefold higher (5.2 vs 1.7; p less than 0.05). Thus intestinal permeability was increased in patients with moderate to major burn injuries shortly after injury.     

U-74,500A inhibition of burn-induced cardiac dysfunction.

Our previous studies suggest that oxygen-derived free radicals, particularly the hydroxyl radical, play a major role in cardiac dysfunction which is characteristic of burn injury. In this present study, we examined the effects of U-74,500A (U7), a 21-aminosteroid, nonglucocorticoid on ventricular contraction and relaxation recovery from burn injury. Parameters measured included left ventricular pressure (LVP) and the maximal rate of LVP rise (+dP/dt max) and fall (-dP/dt max). Full-thickness burns comprising 45% of the total body surface area (burn groups, N = 69) or 0% for controls (Group 1, N = 8) were produced in guinea pigs. In Group 2, 20 burned guinea pigs were not fluid resuscitated (vehicle only) and served as untreated burns; in Group 3, 11 burned guinea pigs received U7 alone (2.5 mg/kg in 0.01 N HCl iv). Eleven burned guinea pigs were resuscitated with vehicle plus 4 ml lactated Ringer's (LR)/kg/% burn for 24 hr (Group 4); in Group 5, 14 guinea pigs were treated with U7 as described for Group 3 followed immediately by LR for 24 hr as described for Group 4. In Group 6, U7 was administered immediately postburn as described for Group 3; and LR resuscitation, begun 1 hr postburn, was continued for 24 hr (N = 14). Compared to controls, untreated burn injury significantly impaired cardiac function as indicated by a fall in LVP (74 +/- 3 vs 60 +/- 4 mm Hg, P less than 0.05) and +/- dP/dt max (1126 +/- 51 vs 1011 +/- 39 and 1159 +/- 53 vs 993 +/- 59 mm Hg/sec, P less than 0.05, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)     

Attenuated metabolic response to surgery in tumor-bearing rats

BACKGROUND: During cancer, proteins are chronically wasted, including proteins of the gut. Surgical stress acutely increases protein breakdown of the gut. Surgery in cancer patients may thus have a double effect on the gut and lead to exhaustion and functional loss of the gut. METHODS: Female Lewis rats (+/-200 g) were studied bearing a subcutaneous tumor or after sham implantation. Hysterectomy was performed in half of the rats as a standardized operative procedure. Postoperative protein kinetics of the gut were determined using a primed constant infusion of L-[2,6-(3)H]-phenylalanine. Gut function was assessed by testing its permeability for sugar probes lactulose and L-rhamnose. Villus height and crypt depth were measured and polyamine concentrations were measured as markers for mucosal proliferation and differentiation. RESULTS: In control rats, gut protein breakdown increased from 6 +/- 3 to 32 +/- 8 nmol phenylalanine x 100 g body wt x min after hysterectomy. This was accompanied by increased amino acid membrane transport rates and metabolic shunting. In tumor-bearing rats, increased protein breakdown in response to surgery was attenuated (8 +/- 4 vs 17 +/- 4 nmol x 100 g body wt x min). Surgery increased the lactulose/L-rhamnose recovery ratio, indicating increased gut permeability. In the presence of a tumor gut permeability also increased and it increased further after surgery. No changes in villus height or polyamine levels could explain the increased permeability of the gut. CONCLUSION: The study shows that a mild surgical trauma increases protein breakdown of the gut and simultaneously increases gut permeability. In the presence of a tumor the metabolic response to surgery is attenuated. Gut barrier loss was highest in the combined presence of cancer and the surgical insult.     

Increased intestinal permeability associated with infection in burn patients

Thermal injury may be associated with disruption of normal gut barrier integrity. To test this hypothesis, we assessed intestinal permeability with the nonmetabolizable, poorly absorbed disaccharide lactulose, which is efficiently excluded by the normal intestinal mucosa. Permeability studies were performed in 15 burned patients (aged 18 to 67 years; mean burn size, 40%) and 11 healthy controls. Lactulose, 10 g, was administered enterally, together with 5 g of mannitol as a control, and urinary excretion rates were determined. Lactulose excretion and the lactulose/mannitol excretion ratio increased threefold (160 +/- 30 vs 57 +/- 7 mumol and 0.113 +/- 0.033 vs 0.035 +/- 0.005) in the infected patients (sepsis score, 10 +/- 2; burn size, 38% +/- 6%). In contrast, noninfected burn patients (sepsis score, 0) had permeability values similar to those of controls (66 +/- 10 mumol and 0.036 +/- 0.007). Permeability increased as the severity of infection increased. Infection in burn patients is associated with increased bowel permeability. The intestine may be a primary source of sepsis. Alternatively, the systemic response to infection may alter gut barrier function, which could facilitate translocation of bacteria and absorption of endotoxin.     

Hypertonic saline dextran resuscitation fails to improve cardiac function in neonatal and senescent burned guinea pigs.

Our previous studies suggested that the greater diminution in burn-induced cardiac contractile function which occurs in young and elderly subjects compared with adult subjects is related to differences in intracellular calcium availability to the myofilaments. We recently showed that improved cardiac function after hypertonic saline dextran (HSD) resuscitation from burn injury in adults was related to enhanced intracellular calcium content. In the study presented here, 126 hearts isolated from neonatal, adult, and senescent guinea pigs were used to evaluate age-related differences in cardiac contractile response to HSD resuscitation from burn injury. Scald burn was induced in 30 adult, 18 neonatal, and 30 senescent guinea pigs; within each age group, half of the burned animals were resuscitated with lactated Ringer's (Parkland formula, 4 mL/kg/% burn for 24 hours); half received an initial bolus of HSD (4 mL/kg, IV) plus lactated Ringer's (1 mL/kg/% burn for 24 hours). An additional 16 animals from each age group served as sham burn controls.(ABSTRACT TRUNCATED AT 250 WORDS)     

Burn-induced transcriptional regulation of small intestinal ornithine decarboxylase.

The mechanisms responsible for gut repair after burn injury have not been established. Polyamines are required for eukaryotic cell growth and differentiation. The enzyme ornithine decarboxylase (ODC) catalyzes the rate-limiting step in polyamine biosynthesis. The role of ODC activity in repair of injured small bowel mucosa after burns has not been investigated. This study examined the effects of burn injury on gut mucosal mass and regulation of ODC gene expression and ODC activity in small bowel mucosa. After an overnight fast, 18 male Sprague-Dawley rats (250 to 300 g) were randomized into sham, 20% burn, or 60% burn groups. We measured ODC activity, mucosal weight, deoxyribonucleic acid (DNA) content, and protein content in proximal and distal small bowel mucosa at postburn intervals of 0, 3, 12, 24, and 48 hours. Gut mucosal ODC messenger ribonucleic acid (mRNA) levels were determined. Burn injury caused significant atrophy of the gut mucosa by 12 hours postburn; restoration was evident by 48 hours after burn. ODC activity was increased in the proximal small bowel at 12 and 24 hours after burn in the rats in both the 20% burn and 60% burn groups; by contrast, only rats in the 60% burn group had increased ODC activity in the distal small bowel. ODC mRNA levels increased in the proximal gut mucosa as early as 3 hours after the burn and returned to control values after 24 hours. These data show that mucosal restoration begins soon after burn injury and that the induction of ODC mRNA and ODC activity are important events.     

Synthetic immunomodulators for prevention of fatal infections in a burned guinea pig model.

Individuals who have suffered severe trauma, such as burns, have a high incidence of infection associated with impaired host resistance. Nonspecific stimulators of host defense mechanisms, i.e., immunomodulators, may be of benefit in such situations. A small animal model (guinea pigs) was developed to study the efficacy of immunomodulators in burns. Anesthetized animals received a 20% total body surface area, full-thickness, scald burn. There was no mortality associated with this injury, but these animals were highly susceptible to challenge with Pseudomonas aeruginosa strain 1244 by direct injection into the burn wound within 24 hours of injury. This susceptibility persisted about 7 days. The standard model adopted was to injure animals, then challenge with 1 median lethal dose (LD50) of P. aeruginosa 96 hours after injury. Using this model, six synthetic immunomodulators were tested: CP-20,961, CP-46,665, muramyl dipeptide, thymopoietin pentapeptide (TP-5), levamisole, and lithium. Drug administration began 24 hours after injury and ended prior to challenge with P. aeruginosa at 96 hours. CP-20,961, muramyl dipeptide, levamisole, and lithium all had no beneficial effect on survival. A single dosage (0.3 mg/kg, I.V.) of CP-46,665, administered 24 hours postinjury, increased the survival rate from 50% to 85% and mean survival time (MST) from 8.2 days to 12.4 days. TP-5, given in four doses (0.1 mg/kg, I.V. each) every 24 hours, increased the survival rate from 40% to 80% and MST from 6.9 days to 11.6 days. These data show that immunomodulators could be of benefit in burns, but also that not all agents are effective in this particular situation.     

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.     

A single dose of endotoxin increases intestinal permeability in healthy humans

To investigate the effects of endotoxin on gut barrier function, we performed paired studies of intestinal permeability in healthy humans (N = 12) receiving intravenous Escherichia coli endotoxin (4 ng/kg) or 0.9% saline solution. Two nonmetabolizable sugars, lactulose and mannitol, which are standard permeability markers, were administered orally, 30 minutes before and 120 minutes after the test injection. The 12-hour urinary excretion of these substances after endotoxin/saline solution administration was used to quantitate intestinal permeability. After endotoxin administration systemic absorption and excretion of lactulose increased almost two-fold (mean +/- SEM, 263 +/- 36 mumol per 12 hours vs 145 +/- 19 mumol per 12 hours during saline studies). Similar but less marked alterations in mannitol absorption and excretion occurred after endotoxin injection (5.7 +/- 0.3 mmol per 12 hours vs 4.9 +/- 0.3 mmol per 12 hours). When individual 12-hour lactulose excretion after endotoxin administration was related to the magnitude of systemic responses, a significant relationship occurred between lactulose excretion and elaboration of norepinephrine and between lactulose excretion and minimum white blood cell count. These data suggest that a brief exposure to circulating endotoxin increases the permeability of the normal gut. These observations are consistent with the hypothesis that during critical illness, prolonged or repeated exposure to systemic endotoxins or associated cytokines may significantly compromise the integrity of the gastrointestinal mucosal barrier.     

The effect of insulin-like growth factor I on postburn hypermetabolism

These studies were undertaken to examine the effects of insulin-like growth factor I (IGF-I) on the hypermetabolic state that follows major thermal injury. Male Sprague-Dawley rats weighing 275 to 325 gm were subjected to a 50% total body surface area, full-thickness scald burn. At the time of injury, osmotic pumps were surgically implanted and used to deliver IGF-I, 1000 micrograms/day, or an equivalent volume of placebo solution by constant infusion of 5 microliters/hr for 14 days. Metabolic rates were studied with oxygen-consumption measurements performed on days 3, 7, 10, and 14 after injury. Animal weight measurements were also performed at the above intervals, with serum total IFG-I levels measured at death. Total serum IGF-I levels were decreased significantly 14 days after injury compared with uninjured animals (p less than 0.05). Treatment with IGF-I resulted in a significant decrease in oxygen consumption and a significant increase in body weight compared with burned animals and those treated with placebo 10 and 14 days after injury (p less than 0.05). These data suggest that IGF-I causes a significant change in the metabolic response that follows severe thermal injury and point to a possible role for IGF-I in the treatment of patients after severe thermal injury.     

Breakdown of hepatic tight junctions during reoxygenation injury.

We investigated whether reoxygenation following anoxia increased biliary permeability and whether or not allopurinol had a protective effect. Isolated rat livers were perfused for 30 min in a one-pass system with buffer equilibrated with 100% nitrogen after stabilization, and then for 60 min with the oxygenated buffer. Hepatic tight junction permeability was assessed by quantifying the early appearance in the bile of horseradish peroxidase (HRP) injected with the perfusate. This early peak represents paracellular passage of HRP, whereas a later second peak results from transcellular passage. In the control livers, 7% of the total HRP passage (93 +/- 50 pg/g liver) was paracellular and 93% was transcellular. After 30 min of reoxygenation following anoxia, however, 516 +/- 20 pg/g liver of HRP passed paracellularly. Addition of allopurinol (5 micrograms/ml) to the perfusate from the start of perfusion reduced paracellular passage of HRP to 219 +/- 49 pg/g liver after anoxia and reperfusion (P less than 0.01). Allopurinol also reduced the cumulative lactate dehydrogenase (LDH) release during the first 30 min of reoxygenation from 2.1 +/- 0.3 x 10(4) to 1.4 +/- 0.4 x 10(4) units/g liver (P less than 0.01). Reduction of the anoxic period from 30 min to 25 min significantly reduced the change in tight junction permeability and the extent of cellular injury: Paracellular passage of HRP was 336 +/- 20 pg/g and LDH release was 0.7 +/- 0.1 x 10(4) units/g liver, both significantly lower than those at 30 min (P less than 0.01). No significant difference in hepatic ATP levels after 60 min of reoxygenation was noted among the experimental groups, but all had lower levels than the control group. The protective effect of allopurinol suggests that the mechanism of biliary reoxygenation injury involves free radical generation. Susceptibility of tight junctions suggests a pattern of injury similar to that involved in anoxic damage of the vascular endothelium.     

Immunosuppressive effects of burn injury and nonspecific blood transfusion

Burn injuries and blood transfusions both have been implicated as causes of suppressed immune responses. Skin allograft survival in a burned mouse model was studied to determine the relationships among burn injury, blood transfusion, and phlebotomy before transfusion as they affected immunocompetence. At 7 days after 20% TBSA full-thickness burn injury, allograft skin survival was prolonged compared to nonburned control. When increasing volumes of blood were transfused, allograft survival times decreased accordingly. Phlebotomy before transfusion tended to enhance this response. Similar results were seen at 14 days after burn injury, although phlebotomy before transfusion did not further decrease allograft survival time at 14 days. This study demonstrated that blood transfusions were not additively immunosuppressive over burn injury alone. Increased amounts of transfused blood restore allogeneic responsiveness. Phlebotomy may enhance this response when performed early after burn injury.     

Prebiotic ingestion does not improve gastrointestinal barrier function in burn patients

Prebiotics increase intestinal levels of health-promoting bacteria implicated in decreasing pathogen colonization, stimulating immune functions and stabilizing gut barrier functions, parameters which are altered in burn patients. We propose that regular intake of a prebiotic, oligofructose (OF), might help to improve the altered gastrointestinal (GI) permeability observed in burn patients. A randomized, double-blind, controlled clinical trial was carried out in 41 burn patients (mean burn surface area=17.1+/-8.2%) who ingested daily 6 g of oligofructose (OF group) or sucrose as placebo (Control group) during 15 days. Gastrointestinal permeability to sucrose and lactulose/mannitol (L/M) was evaluated on days 1 (before treatment) 3, 7, 14 and 21. A permeability test was also performed in 18 healthy subjects as controls. Thirty-one patients completed the protocol (dropout rate=24.4%). Healthy subjects had a basal sucrose excretion of 21.3 mg (14.0-32.5 mg) and a basal L/M ratio of 0.017% (0.009-0.022%). Sucrose excretion increased 5-fold and L/M ratio 4.4-fold in burn patients on day 1 and these high levels of marker excretion decreased significantly throughout the study (p=0.016 and 0.000001, respectively). No differences between the OF and Control groups were observed for sucrose excretion or L/M ratio. In conclusion, the normalization of gastrointestinal permeability is not accelerated by prebiotic intake.     

Effect of thermal injury on endotoxin-induced lung injury

We studied the effects of a burn injury on the response of the lung to endotoxin. Seventeen unanesthetized sheep with lung lymph fistulas were studied. Eight were given Escherichia coli endotoxin (1.5 micrograms/kg) alone and nine were given the same dose 72 hours after a 25% total body surface burn injury. At this time after burn, all physiologic parameters were at baseline levels. A characteristic two-phase lung injury was seen after administration of endotoxin with an initial hypertension phase, characterized by pulmonary artery hypertension, and a second or permeability phase, characterized by an increase in protein-rich lymph flow. all eight animals that underwent only endotoxin administration survived, whereas four of the nine burned animals died during the permeability phase in pulmonary edema. Major physiologic differences between the groups were noted during the permeability phase, including a more severe hypoxia, pulmonary hypertension, and increased postburn lymph flow. Major biochemical changes included significant increases in lymph thromboxane, thromboxane B2, and beta-glucuronidase activity in the burn group. We conclude that the lung is more sensitive to endotoxin after burn, probably as a result of an increased release of products of arachidonic acid metabolism and products of leukocyte activation caused by the body burn.     

The effect of interleukin 1 alpha on survival in a murine model of burn wound sepsis

We examined the effects of human recombinant interleukin 1 alpha (IL-1 alpha) in a murine model of burn wound sepsis. The BDF1 male mice received a 15% burn injury, followed by burn wound inoculation with Pseudomonas aeruginosa. Improvement in survival was noted in the mice that received a single injection of 100 or 1000 ng of IL-1 alpha in comparison with the control animals (IL-1 alpha, 100 ng vs control, 60% vs 13%; IL-1 alpha, 1000 ng vs control, 40% vs 0%). The animals that received 1 ng twice daily for 7 days had improved survival in comparison with the controls (IL-1 alpha vs control, 70.8% vs 20.8%). The animals that received a single injection of 1000 ng after a bacterial challenge with 10(4) P aeruginosa of IL-1 alpha had fewer positive blood cultures at 48 hours compared with the controls (57% vs 89%). In addition, the animals that received 100 ng of IL-1 alpha had significantly increased absolute neutrophil counts at 6, 24, and 48 hours after thermal injury and bacterial challenge with 10(3) colony-forming units of P aeruginosa. The use of cytokines to modulate the host response to injury or infection may lead to additional strategies to improve the outcome following burn injury.     

Removal of necrotic tissue with an ananain-based enzyme-debriding preparation

An enzymatic debriding preparation was formulated with purified enzyme derived from a crude pineapple stem extract. The primary component of this preparation was the sulfhydryl protease ananain which represented >/=85% of the proteolytic activity. The remaining proteolytic activity in the preparation was contributed by a co-purifying homologous cysteine protease comosain. Taken together these two proteases provided a protein purity of greater than 95% as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis. This ananain-based enzyme preparation exhibited both gelatinolytic and fibrinolytic activity in vitro. Ananain-based enzyme preparation was formulated in a hydrophilic cream vehicle at concentrations ranging from 115 to 260 U/gm vehicle. Ananain-based enzyme preparation formulated in this fashion is referred to as Vianain debriding agent. Vianain was applied to partial-thickness cutaneous burn wounds produced in the skin of domestic pigs. A maximum of two 4-hour applications of Vianain provided complete debridement of eschar from the partial-thickness burn wounds as judged by light and electron microscopic analyses of biopsy specimens harvested before and after debridement. Wounds debrided with Vianain exhibited more rapid reepithelialization as compared with wounds that were not debrided. Wounds on pigs that were hyperimmunized to ananain-based enzyme preparation before burning and debridement with Vianain exhibited a similar enhancement in reepithelialization as compared with wounds treated with vehicle alone. The capacity of Vianain to debride necrotic tissue was also evaluated in a guinea pig ischemic ulcer model. Full-thickness ischemic lesions were created on the back of guinea pigs. Vianain was applied to the hardened necrotic tissue for 6 hours per day for up to a maximum of 5 days. Complete debridement of these wounds was accomplished within 4 to 5 days. Treatment of ischemic cutaneous ulcerations in this animal model with two commercially available enzyme-debriding agents provided little or no debridement of the necrotic tissue. In vitro, Vianain treatment of surgically debrided human tissue samples, obtained from patients with burn injury or cutaneous ulcers, showed that the protease preparation was effective in rapidly digesting these necrotic tissues.     

Cutaneous burn increases apoptosis in the gut epithelium of mice

Background: Gut mucosal homeostasis depends on a balance between cell proliferation and cell death. After cutaneous burn injury, gut mucosal weight has been shown to decrease. This decrease in weight was paradoxically associated with an increase in gut proliferative factors. For mucosal weight to decrease in the presence of increased proliferation, there must be an even greater increase in cell death. We postulate that cutaneous burn injury causes an increase in gut epithelial cell death primarily by apoptosis.Study Design: We produced a 30% full-thickness scald burn in the dorsum of anesthetized male C57BL6 mice and collected the proximal small bowel at 12, 24, 36, 48, and 60 hours after injury. Sham burned animals served as controls. Apoptosis and proliferation were measured by immunohistochemical assays (terminal deoxyuridine nick-end labeling for apoptosis and proliferative cell nuclear antigen assay for proliferation). Apoptosis was also measured by ELISA for cytoplasmic histone-associated DNA fragments. Mucosal height was determined on histologic sections. The two groups were compared at each time point using Wilcoxon two-sample test and t-test with Bonferroni’s correction where appropriate.Results: The percentage of apoptotic cells (number of cells stained by terminal deoxyuridine nick-end labeling per 100 villus cells) was significantly higher at 12, 24, and 48 hours after injury. This increase was corroborated by an increase in the ELISA at 12 hours. Proliferation as measured by immunostaining for proliferative cell nuclear antigen significantly increased at 12, 24, 48, and 60 hours. Mucosal height as a gross measure of mucosal atrophy was not different between the groups.Conclusions: We have shown an increase in apoptosis coupled with an increase in proliferation after a burn injury. These results imply an increase in cell turnover in the gut epithelial cells after a burn. Impaired bowel function has been demonstrated repeatedly after burn injury, and this increase in cell turnover may be related.     

The importance of lipid type in the diet after burn injury.

The effects of different types of dietary lipids were tested in burned guinea pigs. All diets were identical except for the type of lipid, with total energy intake from lipids equaling 10%. All animals received a 30% total body surface area (TBSA) flame burn and were fed identically by pump-controlled gastrostomy feedings for 14 days. When compared to safflower oil (74% linoleic acid) as well as linoleic acid alone, fish oil (18% eicosapentaenoic acid or EPA) administration resulted in less weight loss, better skeletal muscle mass, lower resting metabolic expenditure, better cell mediated immune responses, better opsonic indices, higher splenic weight, lower adrenal weight, higher serum transferrin, and lower serum C3 levels. With the exception of better cell mediated immune responses in the animals fed linoleic acid, the administration of indomethacin made little difference. These findings can be explained by a reduction in the synthesis of the dienoic prostaglandins that are derived from the omega 6 series of fatty acids, some of which are significantly immunosuppressive. Regulation of dietary lipids may be an important therapeutic advance in nutritional support after burn injury, and controlled trials should be considered.