Infection and subsequent multiple organ failure continue to be a significant cause of morbidity and mortality after major burn trauma (1-6). The need for intubation and mechanical ventilation after burn injury frequently contributes to the development of pneumonia and infectious complications (7). Recent studies suggest that signaling through the Toll receptor family is an important factor in priming for an exaggerated immune response to a second delayed stimulus, such as post burn pneumonia or infection (8, 9). The synergistic effect of burn injury and postburn infectious complications likely contribute to the pathogenesis of downstream organ injury and dysfunction and has been termed the 2-hit hypothesis (10). The CD14 transmembrane/TLR4 protein complex, together with other proteins within plasma membrane lipid rafts bind lipopolysaccharide, activating a complex signaling cascade that activates the innate immune system (11-13).
We previously described a model of burn injury complicated by aspiration-related pneumonia in mice. That study confirmed that either Gram-positive or Gram-negative bacterial challenge after burn injury increased the morbidity and the mortality compared with those observed in either burn alone or sepsis alone (14). Inasmuch as the severity of organ dysfunction and mortality in injury followed by infection has been related to the time between the 2 sequential insults (15, 16), we developed a model of burn injury on 40% of the total body surface area (TBSA) in adult Sprague-Dawley rats, followed on postburn day 7 by intratracheal challenge with either Gram-positive (Streptococcus pneumoniae) or Gram-negative bacteria (Klebsiella pneumoniae). The animals were given standard fluid resuscitation, and hemodynamic function, systemic inflammatory responses (in vivo), cardiac function, and proinflammatory and anti-inflammatory cytokine responses (in vitro) were studied 24 h after the second insult (bacterial challenge) and on postburn day 8. This study was directed to determine whether sepsis, which occurs after an initial insult (such as burn injury), exacerbates the myocardial inflammatory responses, perhaps contributing to greater myocardial contraction and relaxation defects in a 2-hit model.
MATERIALS AND METHODS
Adult Sprague-Dawley rats (weight, 325-350 g) were used in this study. Animals obtained from Harlan Laboratories (Houston, Tex) were conditioned in-house for 5 to 6 days after arrival, with commercial rat chow and tap water available at will. All studies performed in this study were reviewed and approved by the University of Texas Southwestern Medical Center's institutional review board for the care and handling of laboratory animals and conformed to all guidelines for animal care as outlined by the American Physiological Society and the National Institutes of Health.
Catheter placement, burn procedure, and fluid resuscitation
The rats were anesthetized lightly with isoflurane 12 to 13 h before the burn experiment; then, the body hair on the side, back, and neck was closely clipped. The neck region was treated with a surgical scrub, the left carotid artery was exposed, and a polyethylene catheter (gauge, PE-50) inserted into the artery was advanced retrogradely to the level of the aortic arch. In addition, a polyethylene catheter (gauge, PE-50) placed in the right external jugular vein was used to administer fluids and drugs. All catheters were filled with heparinized saline, exteriorized, and secured. Twelve hours after catheter placement, the animals were deeply anesthetized with isoflurane and secured in a constructed template device. Then, the surface of the skin exposed through the aperture in the template was immersed in 100°C water for 10 s on the back and upper sides. The use of the template produced a well-circumscribed burned area, avoided injury to the abdominal organs, and accomplished full-thickness dermal burns on 40% TBSA. Exposure to this water temperature in adult rats has been shown previously by our laboratory to destroy all underlying nerves and to avoid injury to underlying organs. Sham burn rats were subjected to identical preparation, except that they were immersed in room temperature water and served as controls. Immediately after immersion, the rats were dried and returned to individual cages. Then, each external jugular catheter was connected to a swivel device (BSP99 Syringe Pump; Braintree Scientific, Inc, Braintree, Mass) for fluid administration (lactated Ringer's solution; volume, 4 mL/kg per percentage of burn, with one half of the calculated volume administered during the first 8 h after burn and the remaining volume administered during the next 16 h after burn). Buprenorphine (dose, 0.5 mg/kg) was administered every 12 h during the postburn period. Burned rats did not display discomfort or pain, moved freely about the cage, and consumed food and water at will. In the sham burn group, the external jugular vein was cannulated; then, lactated Ringer's solution (dosage, 0.2 mL/kg per hour) was administered to maintain catheter patency, and an identical regimen of analgesics (buprenorphine) was administered throughout the study period. Twenty-four hours after burn injury, systemic blood pressure was measured using a Gould-Statham pressure transducer (Model IDP23; Gould Instrument Systems, Oxnard, Calif) connected to a Grass medical recorder (Model 7D Polygraph; Grass Instruments, Quincy, Mass). A Grass tachycardiograph (Model 7P4F) was used to monitor heart rate. A Grass PolyVIEW data acquisition system was used to convert the acquired data into digital form.
The rats were randomly divided into sham and burn groups. In the rats designated for the burn group, a full-thickness burn was accomplished on 40% TBSA; then, lactated Ringer's solution was initiated as described previously. On day 7 after burn, the rats (both burn and sham) were subdivided to receive either intratracheal Streptococcus pneumoniae or Klebsiella pneumoniae. Additional sham burn and burn rats were administered intratracheal vehicle to produce sham septic-challenged groups. Thus, 6 experimental groups were produced, as shown in Table 1. The experimental groups were group 1 (sham burn + sham sepsis), group 2 (sham burn + Streptococcus pneumoniae challenge to produce Gram-positive sepsis alone), group 3 (sham burn + Klebsiella pneumoniae challenge to produce Gram-negative sepsis in the absence of burn injury); group 4 (burn + sham sepsis to produce burn alone); group 5 (burn + Streptococcus pneumoniae-related sepsis), and group 6 (burn + Klebsiella pneumoniae-related sepsis). All animals were studied 24 h after bacterial challenge (on postburn day 8).
Preparation of inoculum
Streptococcus pneumoniae type 3 and Klebsiella pneumoniae were obtained from the American Type Culture Collection (ATCC 6303; Rockville, Md) in lyophilized form. Each bacterial strain was reconstituted and then injected into the lungs of a rat to increase virulence. Lung lavage was plated and purified; then, aliquots of each bacterial strain were prepared and stored at −80°C. Before each experiment, individual aliquots were thawed, inoculated onto trypticase soy agar blood agar plates, and incubated overnight at 37°C. The plate was washed with sterile endotoxin-free phosphate-buffered saline solution; then, a concentration of 1 × 107 colony-forming units (CFUs) per milliliter was determined by absorbance at 540 nm. The bacteria was agitated and drawn up into sterile tuberculin syringes in 0.4-mL aliquots, producing a final inoculum of 4 × 106 CFUs in 0.4 mL. The CFUs were determined by plating 10 μL of the 1 × 104 CFUs/mL bacterial suspension onto blood agar plates and incubating the plates overnight at 37°C. The number of viable bacteria inoculated into the animals in either the pneumonia alone groups or in the burn + pneumonia groups was approximately 4 × 106 CFUs.
To produce sepsis, the animals were anesthetized with isoflurane and placed in a supine position; then, the area over the trachea was prepared with a surgical scrub (povidone iodine, Betadine). A midline incision was made over the trachea; the trachea was identified and isolated via blunt dissection. An aliquot of bacterial suspension (concentration, 4 × 106 CFUs in 0.4 mL) of either Streptococcus pneumoniae or Klebsiella pneumoniae or sterile, endotoxin-free phosphate-buffered saline solution was injected directly into the trachea using a 30-gauge needle; the wound was then closed with surgical staples. The animals were placed on a 30-degree inclination, with the head up, to ensure that the injected fluid entered the lungs. All rats were administered i.p. injection of 10 mL lactated Ringer's solution while anesthetized to ensure hydration. All animals administered with either intratracheal pneumoniae had a blood sample collected 24 h after septic challenge. The hearts were then collected for in vitro perfusion (cardiac function or myocyte secretion of cytokines) and the lungs were collected to determine pulmonary inflammation and morphology.
To isolate cardiac myocytes, the rats (n = 4-5 rats from each experimental group) received an i.p. injection of heparin (2,000 units) 20 to 30 min before killing. The rats were decapitated and the hearts were harvested and placed in a petri dish containing ice-cold (temperature, 4°C) heart medium (composition: NaCl, 113 mmol/L; KCl, 4.7 mmol/L; KH2PO4, 0.6 mmol/L; Na2HPO4, 0.6 mmol/L; MgSO4, 1.2 mmol/L; NaHCO3, 12 mmol/L; KHCO3, 10 mmol/L; D-glucose, 20 mmol/L; amino acids [50× Gibco/BRL 11130-051], 0.5× MEM [Eagle minimum essential medium]; HEPES, 10 mmol/L; taurine, 30 mmol/L; carnitine, 2.0 mmol/L; and creatine, 2.0 mmol/L). The hearts were cannulated via the aorta and perfused with room temperature heart medium at a rate of 12 mL/min for a total of 5 min in a nonrecirculating mode. Enzymatic digestion was initiated by perfusing the heart with digestion solution that contained 24.5 mL of heart medium described previously and 50 mg of collagenase II (Worthington 4177; Lot MOB3771), 50 mg of bovine serum albumin (BSA), Fraction V (Gibco/BRL 11018-025), 0.5 mL of trypsin (concentration, 2.5%; 10× Gibco/BRL 15090-046), and 15 μmol/L of CaCl2. Enzymatic digestion was accomplished by recirculating this solution through the heart at a flow rate of 12 mL/min for 20 min. All solutions perfusing the heart were maintained at a constant temperature of 37°C. At the end of the enzymatic digestion, the ventricles were removed and were mechanically disassociated in 6 mL of enzymatic digestion solution containing a 6-mL aliquot of 2× BSA solution (composition: 2 gm of BSA, Fraction V, to 100 mL of heart media). After mechanical disassociation with fine forceps, the tissue homogenate was filtered through a mesh filter into a conical tube. The cells adhering to the filter were collected by washing with an additional 10-mL aliquot of 1× BSA solution (composition: 100 mL of heart medium described previously and 2 mg of BSA, Fraction V). The cells were then allowed to pellet in the conical tube for 10 min. The supernatant was removed and the pellet was resuspended in 10 mL of 1× BSA. The cells were washed and pelleted further in BSA buffer with increasing increments of calcium level (100 μmol/L, 200 μmol/L, 500 μmol/L, to a final concentration of 1,000 μmol/L). After the final pelleting step, the supernatant was removed and the pellet was resuspended in MEM (prepared by adding 10.8 gm 1× MEM, Sigma M-1018, 11.9 mmol/L NaHCO3, 10 mM HEPES, and 10 mL penicillin-streptomycin, 100× Gibco/BRL 1540-122 with 950 mL MilliQ water); the total volume was adjusted to 1 L. At the time of MEM preparation, the medium was bubbled with 95% O2-5% CO2 for 15 min and the pH adjusted to 7.1 with 1 mol/L NaOH. The solution was then filter sterilized and stored at a temperature of 4°C until use. At the final concentration of calcium, the cell viability was measured and the cell suspensions with viability greater than 85% were used for subsequent studies. Myocytes with a rodlike shape, clearly defined edges, and sharp striations were prepared with a final cell count of 5 × 104 cells/mL per well.
Measurement of cardiac myocyte cytokine secretion
Primary cardiac myocytes were pipetted into microtiter wells (5 × 104 cells/well), and cells were incubated in a CO2 incubator at 37°C for 18 h. At the end of the incubation period, supernatants were collected to measure secreted inflammatory cytokines (enzyme-linked immunosorbent assay). Cell viability and morphology were examined. Additional aliquots of myocytes were loaded with either Fura-2AM or sodium-binding benzofurzan isophthalate to measure myocyte calcium and sodium, respectively.
Cardiac myocyte calcium and sodium measurement
Myocyte loading with Fura-2AM was accomplished in 45 min, whereas myocyte loading with benzofurzan isophthalate was accomplished in 1 h at room temperature in the dark. The myocytes were then suspended in 1.0 mmol/L calcium containing MEM, washed to remove extracellular dye, and placed on a glass slide on the stage of a Nikon inverted microscope. The microscopy was interfaced with Grooney optics for epi-illumination, a triocular head, phase optics, a ×30 phase-contrast objective, and a mechanical stage. The excitation illumination source (300 W compact Xenon arc illuminator) was equipped with a power supply. In addition, the InCyt Im2 fluorescence imaging system (Intracellular Imaging Inc, Cincinnati, Ohio) included an imaging workstation and Intel Pentium Pro 200 MHz-based PC. The computer-controlled filter changer allowed for alternation between the 340- and the 380-nm excitation wavelengths. Images were captured using monochrome charge-coupled device camera equipped with a TV relay lens. The InCyt Im2 image software allowed for the measurement of intracellular calcium and sodium concentrations from the ratio of the 2 fluorescent signals generated from the 2 excitation wavelengths (340 and 380 nm); background was removed using the InCyt Im2 software. The calibration procedure included the measurement of fluorescence ratio with buffers containing different concentrations of either calcium or sodium. At each wavelength, the fluorescence emissions were collected for 1-min intervals; the time between data collection was 1 to 2 min. Inasmuch as quiescent or noncontracting myocytes were used in these studies, the calcium levels measured reflect diastolic levels.
Isolated coronary perfused heart
Additional rats from each experimental groups (n = 9-10 rats/group) were heparinized, and the hearts were removed and placed in ice-cold (temperature, 4°C) Krebs-Henseleit bicarbonate-buffered solution (composition in mmol/L: NaCl, 118; KCl, 4.7; NaHCO3, 21; CaCl2, 1.25; MgSO4, 1.2; KH2PO4, 1.2; glucose, 11). All solutions were prepared each day with demineralized, deionized water and bubbled with 95% O2-5% CO2 (pH, 7.4; Po2, 550 mmHg; Pco2, 38 mmHg). A 17-gauge cannula, placed in the ascending aorta, was connected via glass tubing to a buffer-filled reservoir (Ismatec, model 7335-30; Cole-Parmer Instrument Co, Chicago, Ill) and was used to maintain perfusion of the coronary arteries by retrograde perfusion of the aortic stump cannula. Coronary perfusion pressure was measured and effluent was collected to confirm the coronary flow rate. Contractile function was assessed by measuring intraventricular pressure with a water-filled latex balloon attached to a polyethylene tube and threaded through the apex of the left ventricular (LV) chamber. Peak LV systolic pressure and LV end-diastolic pressure were measured with a Statham pressure transducer (Model P23ID; Gould Instrument Systems) attached to the balloon cannula, and the rates of LV pressure (LVP) rise (+dP/dt) and fall (−dP/dt) were obtained suing an electronic differentiator [Model 7P20C; Grass Instruments] and recorded [Grass Model 7DWL8P]. Left ventricular developed pressure was calculated by subtracting the end-diastolic from the peak systolic pressure. A Grass PolyVIEW data acquisition system was used to convert acquired data into digital form.
All values are expressed as mean ± SEM. Analysis of variance was used to assess an overall difference among the groups for each of the variables. The Levene test for equality of variance was used to suggest the multiple-comparison procedure to be used. If equality of variance among the groups was suggested, multiple comparison procedures were performed (Bonferroni test); if inequality of variance was suggested by the Levene test, the Tamhane multiple comparisons (which do not assume equal variance in each group) were performed. Probability values less than 0.05 were considered statistically significant (analysis was performed using SPSS for Windows, Version 7.5.1).
All rats moved freely about the cage after recovery from anesthesia and consumed food and water at will. All rats maintained their body weight during the 8-day postburn period. There were no deaths in either the sham burn, sham sepsis rats (group 1) or in the rats given burn injury and sham sepsis to produce burn alone (group 4). The mortality rate in rats injected with Streptococcus pneumoniae in the absence of previous burn injury (group 2) 24 h after bacterial challenge was 10.5%, whereas the mortality rate in the Klebsiella pneumoniae-challenged rats in the absence of burn injury (group 3) was 7%. The presence of a previous burn injury aggravated the mortality rate in the Klebsiella pneumoniae- challenged rats (19%, group 6), but not in the Streptococcus pneumoniae-challenged rats (mortality rate at 24 h, 11%). Blood cultures were positive 24 h after either Gram-positive or Gram-negative bacterial pneumonia challenge, regardless of the presence or absence of a previous burn injury (Table 2). The cultures of bronchoalveolar lavage (BAL) fluid were positive 24 h after intratracheal bacterial challenge, and the bacterial counts in BAL samples were similar in groups 2, 3, 5, and 6 (Table 2). Blood and BAL cultures were negative for bacteria in sham burn, sham sepsis rats (group 1) and in rats given burn injury alone (group 4).
Mean arterial blood pressure was lower 24 h after intratracheal bacterial challenge, regardless of presence or absence of a previous burn injury and despite fluid administration. In all rats given septic challenge, acidosis was apparent from the rise in whole blood lactate and base excess changes (Table 3).
Systemic and myocardial cytokines (pg/mL) were measured 24 h after bacterial challenge. There was a significant rise in proinflammatory and anti-inflammatory cytokine levels measured in plasma (Table 4) 24 h after intratracheal bacterial challenge, regardless of Gram-positive or Gram-negative bacterial challenge. Similarly, myocardial inflammation was evident at this time in all rats given intratracheal bacterial challenge, as indicated by the rise in cardiac myocyte secretion of TNF-α (Fig. 1A), IL-1β (Fig. 1B), and IL-6 (Fig. 1C). This proinflammatory cytokine response was paralleled by a compensatory anti-inflammatory response, as indicated by the rise in myocyte IL-10 secretion (Fig. 1D). The proinflammatory and anti-inflammatory responses that occurred with sepsis in the absence of burn injury (groups 2 and 3) were exacerbated by a previous burn injury (groups 5 and 6).
Either burn injury or sepsis has been shown to alter myocyte calcium and sodium homeostasis, perhaps contributing to myocardial contraction and relaxation defects. In this study, sepsis, in the absence of burn injury (groups 2 and 3), promoted myocyte calcium (Fig. 2, top) and sodium loading (Fig. 2, bottom) compared with ion concentrations measured in myocytes from the sham burn, sham sepsis rats (group 1; P < 0.05). Gram-positive and Gram-negative sepsis, which occurred 8 days after a previous burn injury (groups 5 and 6, respectively), exaggerated myocyte calcium and sodium accumulation over that observed with sepsis alone (groups 2 and 3; P < 0.05).
Cardiac contraction and relaxation were first assessed as hearts were perfused in vitro (Langendorff method) at a constant LV preload (LV volume), constant heart rate, and constant coronary flow rate. As shown in Table 5, septic challenge in the absence of burn injury caused LVP and ±dP/dt to fall compared with values measured in sham burn, sham septic rats (group 1; P < 0.05). The LVP and ±dP/dtmax were reduced to a similar extent in Gram-positive and Gram-negative bacteria-challenged rats in the presence of previous burn injury. The LV function was further examined as LV volume was incrementally increased in all hearts. As shown in Figure 3, LVP and ±dP/dt responses to increasing preload was significantly reduced in rats given Gram-positive (Fig. 3A) and Gram-negative (Fig. 3B) bacterial challenge, as indicated by the downward shift of LV function curves calculated for groups 2 and 3 and compared with function curves calculated for the sham burn, sham sepsis rats (group 1). As shown in Figure 3, A and B, the LV dysfunction was exacerbated in groups given bacterial challenge after an initial burn injury (groups 5 and 6).
Inasmuch as infectious complications occur frequently after major burn injury, contributing to postburn mortality (1-6), we developed a rat model of burn injury on 40% TBSA, followed by either Gram-positive or Gram-negative bacterial challenge on postburn day 7. We elected to implement this "second hit" (bacterial challenge) on postburn day 7 because both experimental and clinical studies confirm significant immune depression at this time after major injury (14-17).
In our model, sepsis was apparent 24 h after intratracheal bacterial challenge from positive blood cultures and a marked inflammatory response as indicated by a rise in plasma and myocardial cytokine levels. Inflammatory responses to either Gram-positive or Gram-negative-related sepsis were paralleled by significant myocardial contraction and relaxation deficits. Bacterial challenge, which occurred after a previous burn injury, exacerbated the systemic and myocardial proinflammatory cytokine responses and exaggerated the myocardial contractile dysfunction. These data are consistent with previous clinical and experimental reports that a septic insult, which occurs after an initial injury, worsens multiple organ failure and increases mortality rate (14, 18-21).
The mechanisms by which an initial injury primes the subject, increasing susceptibility to subsequent infection, remain unclear. O'Sullivann et al. (22) describe that an initial injury contributes to predominance of T-helper 2 lymphocyte phenotype paralleled by diminished IL-12 production, which partly contributed to decreased resistance to infection. O'Suilleabhain et al. (23) confirmed the contribution of diminished IL-12 to postburn susceptibility to infection by treating burned mice with 25 ng IL-12 daily for 5 days after burn; IL-12 therapy improved the outcome in burn complicated by cecal ligation and puncture. Other investigators have implicated the inflammatory cytokines as the primary mechanism by which injury predisposes a subject to infectious complications (14, 24, 25). The finding that inflammatory cytokines impair cardiovascular function has been confirmed in heart failure, myocardial infarction, sepsis, ischemia reperfusion, hemorrhage, and burn trauma (26-30). Inthis study, either Streptococcus pneumoniae or Klebsiella pneumoniae alone produced significant myocardial contractile dysfunction, as indicated by impaired LVP and ±dP/dt responses to incremental increases in preload. This contractile dysfunction was paralleled by a rise in both systemic and compartmental (myocardial) cytokine levels. Furthermore, cardiac myocytes had a 4-fold increase in TNF-α levels compared with TNF-α levels measured in the systemic circulation, suggesting that high levels of the proinflammatory cytokine TNF-α within the myocardium likely contribute to the impairment of the contractile elements. Sepsis, which occurred 24 h after intratracheal bacterial challenge and on postburn day 8, exacerbated inflammatory cytokine secretion and worsened myocardial contractile defects. The hearts harvested from the animals given burn and either Gram-positive or Gram-negative bacterial challenge had significantly lower LVP and ±dP/dt responses to increases in preload or LV volume compared with responses measured in hearts from animals given sepsis alone. In our model, we attributed the rise in anti-inflammatory cytokine IL-10 levels in burn alone, sepsis alone, or burn + sepsis to the compensation for the significant proinflammatory response.
In our burn complicated by sepsis model, all rats given burn injury alone survived the 8-day experimental period. In contrast, the mortality rate after Streptococcus pneumoniae alone challenge in the absence of burn injury was 10%, whereas Klebsiella pneumoniae challenge alone produced a mortality rate of 7% at 24 h. When Gram-positive sepsis was complicated by previous burn injury, the mortality rate (11%) was similar to that observed in Streptococcus pneumoniae challenge in the absence of burn injury (10%). In contrast, the mortality rate at 24 h after Gram-negative bacterial challenge in rats given a previous burn injury was 19%, significantly higher than that observed after Klebsiella pneumoniae challenge in the absence of burn (7%). All rats given bacterial challenge had positive blood cultures at 24 h after challenge, but there were no significant differences in the number of CFUs per milliliter of blood regardless of Gram-positive or Gram-negative challenge or regardless of the presence or absence of burn injury. Thus, the differences in mortality rate between burn + Gram-positive sepsis and burn + Gram-negative sepsis could not be attributed to the differences in CFUs in peripheral blood. Furthermore, all rats given septic challenge had evidence of lung inflammation that was similar in all groups, regardless of Gram-positive or Gram-negative sepsis. In our model, the greater mortality rate in Klebsiella pneumoniae-challenged rats given an initial burn injury (group 6) may have been related to the more robust myocardial proinflammatory cytokine response compared with the TNF-α, IL-1β, and IL-6 levels measured in Streptococcus pneumoniae-challenged rats given a burn injury (group 5). Similarly, the plasma TNF-α and IL-6 levels with Klebsiella pneumoniae challenge after burn injury were significantly higher than the TNF-α and IL-6 plasma levels measured at 24 h after Streptococcus pneumoniae challenge alone. This greater systemic and compartmental (myocardial) inflammatory response may have contributed to the greater cardiac contractile dysfunction in the Klebsiella pneumoniae-challenged group. In this regard, LVP and ±dP/dt were reduced to a greater extent in Klebsiella pneumoniae- challenged rats given a previous burn injury compared with burn rats given Streptococcus pneumoniae challenge and burn injury. These findings suggest that further studies of in vivo and in vitro cardiac function after Gram-positive and Gram-negative bacterial challenge in the presence of previous burn injury are warranted.
In summary, we examined the myocardial performance in a model of burn injury in rats complicated by either Gram-positive (Streptococcus pneumoniae) or Gram-negative (Klebsiella pneumoniae)-related sepsis. Either burn alone or sepsis alone produced profound myocardial inflammatory responses (in creased myocyte secretion of TNF-α, IL-1β, IL-6, and IL-10) and significant myocardial contractile dysfunction (decreased LVP and ±dP/dt responses to increases in either preload or perfusate calcium); furthermore, myocardial inflammation and dysfunction were exacerbated by an earlier burn injury. The availability of these models will allow us to examine the role of signaling through the Toll family of receptors (TLR4 and TLR2) and the contribution of this signaling pathway to increased susceptibility to infectious complications after major burn injury.
The author thanks David L. Maass and D. Jean White for technical assistance.
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