Evaluation of leukocyte chemotaxis in vitro in thermally injured patients.

Leukocyte chemotaxis in vitro was studied by a modification of the Boyden technic in 46 thermally injured patients. All patients demonstrated a decrease in leukocyte migration through a Nuclepore filter toward a standard casein-serum chemotactic agent. Leukocyte chemotaxis was inversely correlated with burn size during the first 72 h after injury. After 72 h, leukocyte chemotaxis directly correlated with clinical status and was highly predictive for ultimate mortality. Since mortality was largely due to infection, these findings suggest that suppression of leukocyte chemotaxis may explain the susceptibility to opportunistic infection in thermally injured patients.

Effects of heme proteins on nitric oxide levels and cell viability in isolated PMNs: a mechanism of toxicity.

Isolated human PMNs served as a model to determine oxyhemoglobin (oxyHb) binding and the effects of oxymyoglobin (oxyMb) or oxyHb on production of both nitric oxide (NO*) and superoxide (O2*-) and the resulting cytotoxicity. Physiologically relevant concentrations of NO* and H2O2 oxidized, to a similar extent, 2,7-dichlorodihydrofluorescein (DCFH) loaded into polymorphonuclear neutrophils (PMNs). Activation of PMNs with phorbol 12-myristate 13-acetate (PMA) markedly increased the internalization of extracellular oxyHb (10-250 microg/mL). OxyMb (10-300 microg/mL) or oxyHb (30-300 microg/mL) enhanced DCFH oxidation by a concentration-dependent mechanism in unstimulated, lipopolysaccharide (LPS) and tumor necrosis factor alpha (TNF-alpha)-, and PMA-stimulated PMNs. This increased DCFH oxidation was eliminated by NO* synthase inhibitors, glutathione and ascorbate, and was reduced by albumin. Nitrite accumulation in PMN filtrates mirrored NO*-induced DCF fluorescence. OxyMb-induced increases in NO* levels paralleled alterations in DNA and cell membrane damage and ATP levels in PMNs and co-cultured lymphocytes, and were attenuated by NO* synthase inhibitors. OxyMb eliminated extracellular O2*- at protein concentrations 100- to 1000-fold above those of superoxide dismutase. These results suggest that heme proteins bind and internalize into PMNs and increase NO*-induced damage in neighboring cells by inhibiting O2*(-)-scavenging of NO*. We propose a mechanism whereby heme protein-induced NO* levels may contribute to immunosuppression and increased infection rates associated with transfusions and cellular damage during inflammation.

Depressed reflex vasomotor control of the burn wound.

Total leg blood flow was measured by venous occlusion plethysmography in five normals and 14 burned patients before and after 30 min of external heating. Leg surface temperatures were held constant, but rectal temperatures increased on the average of 0.4 to 0.5 degrees C in all subjects following this heat load. Leg blood flow increased by 56.0% in the controls, 63.2% in five patients with essentially no leg burn (mean burn size = 1.5% leg surface), and 9.6% in nine patients with major leg injuries (mean burn size 55% leg surface). Failure of reflex vasodilatation in the burned leg was evident up to 107 days postinjury even when the wound was well-healed. All subjects sweated freely from the unburned skin. In two patients, where arm and leg blood flows were measured simultaneously, flow to the uninjured arm increased while that to the injured leg remained unchanged. This lack of reflex vasodilatation in the burned limbs suggests either that wound vessels are denervated or that they are so dilated in the basal state that further dilatation is limited. The bulk of this and other data would support the denervation concept. This physical or chemical denervation could occur at the time of injury, be localised to the area of the wound, and result in loss of both neurogenic vasoconstrictor tone and active reflex vasodilatation.

Hormonal changes in burned hamsters.

Burned male Syrian hamsters (burn size 23% of body surface) exhibited reduced total (T4) and free (FT4) serum concentrations, a defect in T4 binding to serum proteins manifested by the T4 dialyzable fraction but not the in vitro T3 charcoal uptake, and reduced serum testosterone concentration. These changes are similar to those noted previously in burned humans. Unlike such patients, burned hamsters did not exhibit reduced serum T3 nor elevated rT3 concentrations in a reproducible manner. Pinealectomy performed before burning in hamsters did not prevent the burn-induced depression in serum T4 and testosterone.

Alterations of mental status and thyroid hormones after thermal injury.

In 16 burn patients, mean values for serum T4 and T3, their T3 uptake-derived free indices (FT4I and FT3I) and dialysis-derived free concentrations (FT4 and FT3) were depressed (all P less than 0.001) compared to respective means in 13 normal subjects. In the patients, the free hormone indices were relatively more depressed below control values than were the free hormone concentrations. However, within the group of burn patients, variation in FT4I reflected that of FT4 (r = 0.91), and variation in FT3I reflected that of FT3 (r = 0.93). We then studied serum T4, T3, and their free indices in 134 patients (burn size, 6-96% of the skin area), including 45 nonsurvivors, none of whom received steroid, dopamine, or iodine treatment. At each sampling, the level of obtundation (LO) was determined on a 6-point scale from normal to deep coma. Whereas initially low mean FT4I values rose in survivors, they remained lower in nonsurvivors than in survivors until death in the nonsurvivors. In nonsurvivors, mean LO worsened in the first week and remained worse than that in survivors until death. Multiple regression analyses showed that for a given age or burn size, nonsurvival was better correlated with lower T4 or FT4I than with T3 or FT3I, but was even more closely correlated with worse LO (P less than 0.001). Exclusion of data obtained within 24 h of narcotic or tranquilizer doses did not weaken the relationship of nonsurvival with LO and FT4I. Nonsurvival after burn injury was associated with reduced T4, FT4I, and mental status for up to weeks before death, this association being independent of treatment with drugs acting on mental status or thyroid function.

Opportunistic infections in severely burned patients.

The risk of infection in burn patients, which is proportional to the extent of burn, reflects the combined effect of impairment of all aspects of the host defense system and microbial factors. The microbial flora colonizing the burn wound changes with time following injury and provides the organisms causing infections in burn patients. The temporal pattern of the predominant gram-negative organisms causing infections in a burn unit resembles that of a succession of mini-epidemics necessitating an active program of microbial surveillance to guide treatment of infections. Topical chemotherapy has significantly reduced the occurrence of invasive burn wound infections, but microbial control is imperfect and the burn wound, as well as the patient as a whole, must be closely monitored (using wound biopsies as indicated) to diagnose and treat infection in a timely manner. The treatment of burn wound infections is guided by extent and depth of microbial invasion, density of microorganisms, and systemic changes. As a manifestation of immunologic impairment, infection in sites other than the burn wound remains the most frequent cause of death in burn patients. The use of broad spectrum serologic agents to enhance immuno-competence in extensively burned patients may reduce the occurrence of life threatening opportunistic infections.

Replacement therapy with modified immunoglobulin G in burn patients: preliminary kinetic studies.

Suppression of serum immunoglobulin G for periods ranging from days to weeks following thermal injury may enhance the risk of infection in burn patients. In an initial trial, we attempted to determine whether intravenous pulses of Immunoglobulin G (IgG) will establish and maintain normal serum IgG concentrations in this interval. The levels of endogeneous serum IgG in eight control patients, mean total burn size 45 percent body surface area (no IgG infusions), were measured by radial immunodiffusion on various postburn days. Commercially available reduced alkylated IgG (5 percent Gamimune, Cutter Biological, Berkeley, California) was infused in doses of 500 mg/kg twice per week in four patients (total burn size 32 percent) and once per week in five patients (total burn size 47 percent), beginning during the first postburn week. Circulating IgG was measured prior to each infusion and at three postinfusion times: (1) 15 minutes (peak), (2) one day, and (3) either day 3, 4, or 6. Surgery or blood transfusions prior to one of these time points invalidated kinetic analysis of some infusions. Exponential two-point decay constants for total serum IgG after each of 24 infusions were calculated separately for early (day 0-1) and later (day 1-3 or 1-4) postinfusion intervals and assessed by stepwise regression analysis to determine sources of variation in decay. Early decay was seen to be faster with larger burn size after accounting for variation of decay with preinfusion and peak IgG values. Later decay was not related to burn size. Maltose, a constituent of the IgG preparation, was detectable in serum for only four to eight hours after each infusion and may have contributed to a 20 percent increase in total serum glucose between four and eight hours postinfusion. Mean serum IgG in patients given infusions twice weekly was in the normal range after one infusion, about a week earlier than in untreated patients. Such infusions maintained normal IgG levels.

Update on current therapeutic approaches in burns.

Burn injury results in a rapid loss of intravascular volume as wound edema forms, which reduces the circulating blood volume and generates the need for fluid therapy to combat hypovolemia. Fluid resuscitation of a burn patient is usually carried out with isotonic, sodium- and chloride-containing fluids, such as lactated Ringer's solution. The initial 24 h resuscitation volume is based on the burn size and body weight of the patient. Following a successful resuscitation, the burn patient develops stereotypic neurohormonal and metabolic responses that, depending on the extent of injury, last for several weeks or months. Breathing of incomplete products of combustion by the fire victim produces inhalation injury, the incidence of which rises with increasing burn size and the severity of which is proportional to the duration of exposure. Systemic hypoxia from carbon monoxide toxicity causes early death; chemical airway injury increases mortality and predisposes to subsequent pneumonia that further reduces survival. The diagnosis of inhalation injury is made by bronchoscopy and/or xenon scan and therapy involves support of ventilation. Thermal destruction of the cutaneous mechanical barrier and the presence of nonviable avascular burn eschar as well as impairment of other host defenses render the burn patient susceptible to local as well as systemic infections. Care following resuscitation is focused on topical antimicrobial therapy, burn wound excision, and wound closure by grafting. Nutritional support and the prevention and control of infection are constant themes in burn patient management. A progressive improvement in general care of the acutely injured patient, prevention of shock, effective means of maintaining organ function, prevention and control of burn wound and other infections, and physiologically based metabolic support have significantly increased burn patient survival in recent decades.

Underestimation of thermal lung water volume in patients with high cardiac output.

When utilizing the intravascular double-indicator dilution technique to measure extravascular lung water, blood flow may be so high that diffusion equilibrium of the diffusible indicator fails to occur and the water distribution space is underestimated during the first 7 days after thermal injury. We serially measured cardiac index and lung water in five severely burned patients (mean age 24 years, range 18 to 33 years; mean burn size 56% total body surface, range 43% to 80%) by a rebreathing method utilizing two gases of differing solubility and by the thermal-indocyanine green dye (ICG) double-indicator dilution technique. Rebreathing lung water, determined by a time- and blood flow-insensitive method, increased significantly over the study period, from 6.6 ml/kg on admission to the hospital 11.3 ml/kg on postburn day 6 (+70%, P less than 0.01). Thermal-ICG lung water decreased slightly as blood flow rose. Rebreathing lung water correlated with clinical data in a patient with pulmonary edema, while thermal-ICG lung water changed in the opposite direction. Our data suggest that the thermal-ICG technique may be diffusion limited by short transit times at the high flows characteristic of burned and other critically ill patients with hyperdynamic circulations. Additionally, segmented redistribution of pulmonary blood flow known to occur in burn patients may contribute to underestimation of lung water.

Decreased wound neutrophils and indiscrete margination in the pathogenesis of wound infection.

To assess the pathogenesis of increased susceptibility to infection and septic death in a rat model, neutrophils (PMNs) in the wound, circulating PMNs, and their in vivo activity were evaluated after 30% and 60% burns. Eight hours after injury there were twice as many PMNs in the wounds of rats that sustained 30% compared with 60% burns. There was no difference between these two groups in the number of circulating PMNs at 2, 4, 6, and 8 hours after injury. In vivo evaluation of PMN response to infusion of F-Met-Leu-Phe revealed that circulating PMNs were more sensitive 4 hours after 60% burns compared with sham burns. At this time PMNs were found to be less sensitive to zymosan-activated serum infusion after 30% burns compared with sham burns. However, the PMNs in rats with 30% burns were more sensitive to this stimulus than were PMNs in rats with 60% injuries. Thus rats with greater injury, known to be more susceptible to wound infection, have fewer PMNs in their wounds 8 hours after injury. This is preceded by an increased sensitivity of PMNs in vivo to bacterial chemotactic factor and a relative increase in sensitivity to wound factors. This unusual finding implicates indiscrete margination as a factor in the pathogenesis of infection.

The effect of exogenous substrate on hepatic metabolism and membrand transport during endotoxemia.

Endotoxemia in dogs reduced hepatic uptake and biliary excretion of indocyanine green dye. This diminished active membrane transport was associated with reduced hepatocyte membrane potential difference. Studies of arteriovenous concentration differences and flow across the liver demonstrated that endotoxemia increased hepatic glucose and lactate production and decreased oxygen consumption. Correction of this energy deficit occurred following infusion of glucose and insulin, but not after administration of isocaloric quantities of intravenous amino acids. The glucose-insulin infusion during endotoxemia shifted the liver back to an organ of glucose uptake, improved oxygen consumption, and provided the necessary energy for normal dye transport and maintenance of the normal membrane potential difference.

Granulocyte oxidative activity after thermal injury.

BACKGROUND: Alterations in granulocyte function after thermal injury have been described. We have serially studied the level of granulocyte cytosolic peroxidase activity in 23 thermally injured patients during the first 6 weeks after injury. The patients' mean age and burn size were 35.1 +/- 15.7 years and 41.6% +/- 16.8% (range, 18% to 88%), respectively. Fourteen patients had concomitant inhalation injury, and the overall mortality rate was 4.3%. METHODS: Purified granulocytes were obtained from peripheral blood after red cell lysis and Ficoll-Hypaque (Pharmacia Inc., Piscataway, N.J.) gradient separation. Cells were loaded with dichlorofluorescin diacetate, and baseline fluorescence was measured by flow cytometry. After phorbol myristate acetate stimulation, fluorescence was measured again. Cells from unburned normal subjects were used as daily controls. RESULTS: The data are expressed as percent of stimulated control granulocyte fluorescence. Unstimulated patient granulocytes demonstrated a significantly higher baseline activity than did unstimulated controls (22.9% vs 15.4%; p < 0.05). Mean fluorescence from stimulated granulocytes was 114% of the control values (p < 0.05). CONCLUSIONS: Granulocytes from thermally injured patients exhibited a baseline increase in cytosolic oxidase activity, suggesting in vivo activation and a greater than normal oxidase activity after in vitro stimulation.

Cimetidine prevents reduction in gastric mucosal blood flow during shock.

This study evaluated Cimetidine's possible role in regulating gastric mucosal blood flow in the anesthetized, stressed miniature swine. Stress consisted of hemorrhagic shock to a mean arterial pressure of 50 mm Hg. Twenty-one animals were divided into three experimental groups: untreated controls, preshock Cimetidine treatment group, and postshock Cimetidine treatment group. Gastric mucosal blood flows were determined (microsphere method) during a stabilization period and after 90 minutes of shock. Central hemodynamic indices were monitored throughout each experiment. In the fundus, mucosal blood flow decreased 59% in the controls, 11% in the preshock, and 28% in the postshock Cimetidine groups. Antral mucosal blood flow decreased 57% in controls, 19% in the preshock, and 33% in the postshock Cimetidine groups. In the corpal mucosa, blood flow decreased 53% in controls, 11% in the preshock group, and 41% in the postshock Cimetidine group. Cimetidine administration, both before and after shock, conferred significant protection on mucosal blood flow changes related to shock. Preshock drug administration had a significantly greater protective effect than postshock treatment on blood flow in the corpal mucosa.

Effect of inhaled nitric oxide on pulmonary function after sepsis in a swine model.

BACKGROUND: Inhaled nitric oxide (NO) has been shown to improve sepsis induced pulmonary dysfunction. This study evaluated the mechanism by which inhaled NO improves pulmonary function in a porcine sepsis model. METHODS: After an infusion of Escherichia coli lipopolysaccharide (LPS, 200 micrograms/kg), animals were resuscitated with saline solution (1 ml/kg/min) and observed for 3 hours while mechanically ventilated (fraction of inspired oxygen, 0.6; tidal volume, 12 ml/kg; positive end-expiratory pressure, 5 cm H2O). Group 1 (LPS, n = 6) received no additional treatment. Group 2 (NO, n = 6) received inhaled NO (40 ppm) for the last 2 hours. Group 3 (control, n = 5) received only saline solution without LPS. Cardiopulmonary variables and blood gases were measured serially. Multiple inert gas elimination technique was performed at 3 hours. Wet to dry lung weight ratio was measured after necropsy. RESULTS: Lipopolysaccharide resulted in pulmonary arterial hypertension, pulmonary edema, and hypoxemia. Multiple inert gas elimination technique analysis indicated a significant increase in blood flow to true shunt and high ventilation perfusion distribution (VA/Q) areas with an increased dispersion of VA/Q distribution. All of these changes were significantly attenuated by NO. CONCLUSIONS: Inhaled NO significantly improved LPS induced VA/Q mismatching by decreasing both true shunt and high VA/Q areas, by decreasing pulmonary edema, and by redistributing blood flow from true shunt to ventilated areas.

Effect of blood transfusions on immune function. Part VI. Effect on immunologic response to tumor.

Transfusions are reported to increase the incidence of tumor metastasis in clinical studies and primary tumor growth in animal studies. We evaluated the effect of transfusions on immunologic response to primary and metastatic tumors in multiple rat models. One half of the animals were administered lactated Ringer's solution and one half ACI rat blood at the time of tumor challenge. In 80 rats a slow-growing colon tumor was implanted subcutaneously. At 4 months there were no significant differences in tumor size or leukocyte infiltration of the tumor. Similar results were obtained with a rapidly growing colon cancer. Analysis of T-lymphocyte subpopulations in both groups showed no differences. Rats transfused at the time of intravenous challenge with a suspension of 1 x 10(6) tumor cells had a mean survival time of 38.3 +/- 0.8 days and the control group had a mean survival time of 41.1 +/- 0.8 days (p = 0.016). One week after transfusion, natural killer cell lysis of tumor cells at a 100:1 effector/target cell ratio was 18.0% +/- 1.8% in the transfusion group and 23.0% +/- 1.3% in the control group (p = 0.034). In conclusion, transfusions in multiple rat cancer models did not affect primary tumor growth or the host's immunologic response to it but did significantly impair natural killer cell function and survival with tumor metastases.

Evaluation of (1-sarcosine, 8-isoleucine) angiotensin II as a therapeutic agent for oleic acid-induced pulmonary edema.

(1-Sarcosine, 8-isoleucine) angiotensin II was assessed as a therapeutic agent for acute respiratory distress syndrome with oleic acid pulmonary edema in sheep used as an experimental model. Under general anesthesia with controlled mechanical ventilation with 100% oxygen, 32 sheep received oleic acid (0.075 ml/kg) intravenously. After oleic acid infusion, 20 animals were treated with continuous intravenous infusion of the angiotensin II analogue; nine received 300 ng/kg/min, six received 600 ng/kg/min, and five received 2000 ng/kg/min. Cardiopulmonary measurements were repeated every 30 minutes for 270 minutes. According to time-integrated PaO2, six of 15 animals of the groups given 300 and 600 ng/kg/min (43%) did not respond to the treatment. All animals responded in the group given 2000 ng/kg/min. Animals in the latter group had lower Qs/Qt, PaCO2, and airway resistance than had the control animals. Elevation of pulmonary vascular resistance was limited and mean arterial blood pressure was well maintained. These results reveal that (1-Sar, 8-Ile) angiotensin II is effective in the treatment of oleic acid-induced pulmonary edema.

Altered muscle metabolism in rats after thermal injury.

Burn injury is associated with an elevation in total body oxygen consumption, increased hepatic alanine uptake and conversion to glucose, and a negative nitrogen balance. The primary source of the alanine used for gluconeogenesis by the liver and of the nitrogen lost as urea is believed to be from skeletal muscle. Selected muscle regulatory enzymes and pyruvate and oleate oxidation rates were assayed for maximal activity during the postburn period. Male Sprague-Dawley rats that received 50% total body surface scald burns on the dorsum and abdomen were examined for citrate synthase (CS), phosphofructokinase (PFK), and glutamate-pyruvate transaminase (GPT) activity in uninjured muscle at 3, 7, 13, and 20 days postburn, and the ability of muscle to oxidize pyruvate and oleate was measured at 3 and 13 days after injury. Cs, PFK, and GPT activities increased significantly (p less than 0.05) by 13-20 days after injury in the soleus and diaphragm. The epitrochlearis showed no change in CS, but PFK and GPT were elevated within this time frame. The gastrocnemius muscle showed an elevated oleate oxidation rate at 13 days after injury, but no change at 3 days postburn. Pyruvate oxidation rates were unaltered. The results of this study indicate that during the postburn period several metabolic alterations occur in muscle. These adaptations include: (1) elevated CS activity which may be associated with increased oxidative capacity,, (2) increased PFK activity which implies that more substrate is being shuttled through the glycolytic pathway, (3) increased GPT activity which may reflect increased pyruvate conversion to alanine, and (4) increased oleate oxidation rates which demonstrate that muscle is utilizing more fatty acid substrates during the postburn period.

Nonthyroidal control of metabolism after burn injury: possible role of glucagon.

The endocrine basis for control of metabolism in nonthyroidal illness is not yet understood. Burn injury is associated with reduced serum concentrations of thyroid hormones and with resting hypermetabolism. One index of severity is total burn size (TBS, % body surface). After overnight fasting and recumbency, resting metabolic rate (MR, O2 consumption) was measured weekly and plasma was sampled for determination of glucose, total cholesterol, triglycerides, insulin, glucagon, somatostatin, growth hormone, norepinephrine, epinephrine, and cortisol in 28 burned men, 17-23 years old, TBS 2%-85%, including 8 controls with minimal injury (TBS less than or equal to 7.5%). MR was elevated in proportion to burn size mainly in the first week then declined toward normal. Growth hormone was not changed. Two multiple regression analyses (validated by random partitioning of data) determined which plasma variables independently reflected residual variation in MR: without TBS entered as a variable, high MR was associated with elevated glucose, cortisol, and glucagon, and low cholesterol (cumulative r2 = 0.79); with TBS entered, high MR was associated with greater TBS, elevated norepinephrine, and again high glucagon and low cholesterol (r2 = 0.81). Resting metabolism after burn injury is controlled not by the thyroid but may be controlled by a set of antiinsulin hormones that does not include growth hormone, but possibly includes glucagon.

Base-line and postthermal injury plasma histamine in rats.

Although plasma histamine concentration has been reported to increase after thermal injury in the rat to as much as 100-fold over normal human plasma levels, the pathophysiological significance and relevance to human disease is questionable. Lack of confidence in the rat as a model of histamine-mediated disease is based on reports that normal rat base-line plasma histamine concentration exceeds that of human plasma by 20- to 70-fold. The present study confirms that high concentrations of histamine (20-68.9 ng/ml) are found in rat plasma obtained in an uncontrolled manner; but concentrations are lower (1.17 +/- 0.49 ng/ml) or undetectable in a sensitive radioenzymatic assay when sampling technique and plasma isolation are controlled. The primary cause for falsely elevated values for plasma histamine concentration appeared to be due to manipulation of the rat. Plasma histamine concentration increased within 1 min after thermal injury and the increase was proportional to extent of surface area injured. In contrast to the finding of a single time-related peak of plasma histamine concentration after partial-thickness burn, a biphasic elevation was found after full-thickness injury. Thus the data indicate that normal rat plasma histamine concentration is similar to that of the human and below the reported threshold for modulation of a variety of immune responses. Furthermore, the data support a role for histamine and other mast-cell mediators in the local and systemic responses to injury.

Thermoregulatory and nonthermoregulatory heat production in burned rat.

Severely burned patients are hypermetabolic within their thermoneutral zone (TNZ), where there are no thermoregulatory demands on heat production. The rat has been used as a model of postburn hypermetabolism without clear evidence that it behaves in a similar way. Male rats (400-500 g; n = 34-39) were placed as a group in a respiration chamber and metabolic rates for the average rat were determined over 3-6 h at ambient temperatures between 9 and 36 degrees C. Colonic temperatures (Tco) and body weights were measured after each run. Animals were studied sequentially as normals (N), after clipping (C) and following 50% total body surface scald burns. Clipping increased the lower critical temperature (LCT) from 27.7 to 29.1 degrees C without affecting resting heat production (N = 42.6 +/- 0.5; C = 42.0 +/- 0.8 W/m2; mean +/- S.E.) or Tco (N = 36.6 +/- 0.1; C = 36.6 +/- 0.1 degrees C) in the TNZ. Injury increased LCT to 32.8 degrees C and the burned animals were hypermetabolic (47.2 +/- 0.6 W/m2; P less than 0.05 vs. N) and febrile (36.9 +/- 0.1 degrees C; P less than 0.05 vs. N) in the elevated TNZ. These metabolic and temperature responses of burned rats are limited in magnitude but are qualitatively similar to those of patients. The extra heat production in the TNZ reflects the basic metabolic cost of injury.