Mechanisms by which intracellular calcium induces susceptibility to secretory phospholipase A2 in human erythrocytes.

Exposure of human erythrocytes to the calcium ionophore ionomycin rendered them susceptible to the action of secretory phospholipase A(2) (sPLA(2)). Analysis of erythrocyte phospholipid metabolism by thin-layer chromatography revealed significant hydrolysis of both phosphatidylcholine and phosphatidylethanolamine during incubation with ionomycin and sPLA(2). Several possible mechanisms for the effect of ionomycin were considered. Involvement of intracellular phospholipases A(2) was excluded since inhibitors of these enzymes had no effect. Assessment of membrane oxidation by cis-parinaric acid fluorescence and comparison to the oxidants diamide and phenylhydrazine revealed that oxidation does not participate in the effect of ionomycin. Incubation with ionomycin caused classical physical changes to the erythrocyte membrane such as morphological alterations (spherocytosis), translocation of aminophospholipids to the outer leaflet of the membrane, and release of microvesicles. Experiments with phenylhydrazine, KCl, quinine, merocyanine 540, the calpain inhibitor E-64d, and the scramblase inhibitor R5421 revealed that neither phospholipid translocation nor vesicle release was required to induce susceptibility. Results from fluorescence spectroscopy and two-photon excitation scanning microscopy using the membrane probe laurdan argued that susceptibility to sPLA(2) is a consequence of increased order of membrane lipids.

Diagnosis and management of acute aortic valvular disruption secondary to rapid-deceleration trauma.

Acute aortic valve rupture with resultant aortic insufficiency is a rare complication of blunt trauma. We describe a case in which a patient fell 70 feet, sustaining avulsion of two leaflets of the aortic valve along with multiple other injuries, primarily orthopedic. Our case demonstrates that patients with acute aortic regurgitation can be managed nonoperatively if necessary in the acute setting, enabling management of other significant trauma. Subsequent semielective valvular replacement may be undertaken if other injuries must take precedence.

Transverse rectus abdominis musculocutaneous flap breast reconstruction in sickle cell disease.

Patients with homozygous sickle cell disease pose an enormous challenge to the reconstructive surgeon. Historically, the risk of attempting flap reconstruction was considered prohibitive. A successful case of immediate breast reconstruction with a "supercharged" bipedicled transverse rectus abdominis musculocutaneous flap is presented. Perioperative transfusions that maintained the sickle hemoglobin S level below 30% were crucial in preventing erythrocyte sickling in the microcirculation of the flap during the period of relative ischemia.

Chronic pathophysiologic elevation of corticosterone after thermal injury or thermal injury and burn wound infection adversely affects body mass, lymphocyte numbers, and outcome.

The purpose of the present studies was to investigate the effect of glucocorticoids on catabolism and lymphocyte numbers in a rat model of thermal injury or thermal injury plus burn wound infection. Thermal injury alone caused only an acute increase in plasma corticosterone concentrations. Furthermore, body weight declined moderately (5%), and lymphocyte numbers in lymph nodes draining the burn wound and blood increased markedly, whereas splenic lymphocyte numbers declined by about 60%. By contrast uninjured rats subjected to chronic elevation of corticosterone by corticosterone pellet implantation showed large decreases in body weight and lymphocyte numbers in all tissues examined. The combination of injury and chronic corticosterone elevation resulted in body weight and lymphocyte changes intermediate between injury alone and corticosterone treatment alone. Chronic elevation of corticosterone for 4 days before burn wound infection significantly decreased survival time and survival. Burn wound infection immediately after injury caused chronic elevation of endogenous plasma corticosterone and body weight and numeric lymphocyte changes that were remarkably similar to those of uninjured rats treated with corticosterone. Finally, the glucocorticoid receptor antagonist RU 486 significantly increased survival time in thermally injured, burn wound-infected rats. These results lend support to a hypothesis that chronic elevation of plasma cortisol concentrations as observed in patients with burns may be deleterious.

Recurrent and chronic appendicitis: the other inflammatory conditions of the appendix.

Episodic abdominal pain, a common clinical problem, can be a diagnostic and therapeutic conundrum when the surgeon encounters it acutely in the emergency department. Appendicitis is often excluded from the differential diagnosis because the natural history of appendicitis is usually appreciated as acute, progressing to some degree of peritonitis quite rapidly and inevitably. However, recurrent and chronic forms of appendicitis occur also and can mislead the clinician. Herein, we describe two patients with recurrent appendicitis that were misinterpreted as other abdominal conditions, and we review the literature implicating recurrent and chronic appendicitis as disease processes, distinct from acute appendicitis, that occur with an incidence of approximately 10 per cent and 1 per cent, respectively.

Platelet-activating factor antagonism attenuates platelet and neutrophil activation and reduces myocardial injury during coronary reperfusion.

Platelet-activating factor (PAF) is known to be synthesized during tissue reperfusion and to be involved in the activation of platelets and neutrophils in inflammatory processes. The hypothesis of the present study is that PAF is central in the pathophysiology of myocardial reperfusion and that specific PAF receptor antagonism may reduce myocardial reperfusion injury. Utilizing an intact sheep model that involved a 90-min occlusion of the mid-left anterior descending coronary artery followed by 6 hr of reperfusion, a study group that received a specific PAF receptor antagonist (L-659,989, 5 mg/kg) 10 min before reperfusion was compared to a control group that received a saline placebo (n = 8 in each group). Coronary sinus platelet aggregating activity and neutrophil oxidative burst were studied by standard platelet aggregometry and the 2',7'-dichlorofluorescein flow cytometric assay, respectively. Left coronary flow and left ventricular functions measured as peak +/- dp/dt and stroke work were analyzed. The extent of myocardial infarction at the end of 6 hr of reperfusion was measured by standard histochemical stainings. The results demonstrated that platelets were hyperaggregable and that neutrophil oxidative burst was increased in the myocardial compartment during the first 3 hr of coronary reperfusion after 90 min of ischemia. The administration of the PAF antagonist immediately before reflow effectively prevented the activation of platelets and neutrophils. This was associated with significantly improved coronary reflow and ventricular function during the observed reperfusion period and with reduced myocardial infarct measured at 6 hr of reperfusion. We conclude that the use of a specific PAF receptor antagonist, L-659,989, immediately before controlled coronary reflow attenuated the activation of platelets and neutrophils that occurred during reperfusion. These anti-platelet and anti-neutrophil effects together with the inhibition of the known direct deleterious effects of PAF on the myocardium translated into improved ventricular function and reduced myocardial infarct.

Human keratinocyte growth factor effects in a porcine model of epidermal wound healing.

Keratinocyte growth factor (KGF) is a member of the fibroblast growth factor (FGF) family (hence the alternative designation FGF-7). It is produced by stromal cells, but acts as a mitogen for epithelial cells. We examined the effects of topically applied KGF on healing of wounds in a porcine model. In partial-thickness wounds, KGF stimulated the rate of reepithelialization (p < 0.0002), associated with a thickening of the epidermis (p < 0.0001). Epidermis from KGF-treated full-thickness wound sites was significantly thicker (0.31 +/- 0.22 mm) compared with mirror image control sites (0.18 +/- 0.12 mm) (p < 0.0001). Moreover, the majority (77%) of KGF-treated wounds exhibited epidermis with a deep rete ridge pattern as compared with control sites. These effects were observed as early as 14 d and persisted for at least 4 wk. KGF treatment also increased the number of serrated basal cells associated with increased deposition of collagen fibers in the superficial dermis adjacent to the acanthotic epidermis. Electron microscopy revealed better developed hemidesmosomes associated with thicker bundles of tonofilaments in the serrated cells. The pattern of epidermal thickening observed in KGF-treated wounds resembled psoriasis. Psoriasis is a disease associated with epidermal thickening, parakeratosis as well as hyperproliferation that extends beyond the basal layer. In striking contrast to psoriasis, KGF-treated wounds exhibited normal orthokeratotic maturation, and proliferation was localized to the basal cells. Our present findings have significant implications concerning the role of KGF as a paracrine modulator of epidermal proliferation and differentiation.

Comparison of peripheral blood leukocyte kinetics after live Escherichia coli, endotoxin, or interleukin-1 alpha administration. Studies using a novel interleukin-1 receptor antagonist.

OBJECTIVE: This study was undertaken to evaluate whether hematologic and immunologic effects observed after bacteremia and endotoxemia in the host could be replicated by administration of recombinant human interleukin-1 alpha (IL-1 alpha) in a primate model. Furthermore, to determine whether endogenously produced interleukin-1 (IL-1) contributes to the changes observed during endotoxemia or gram-negative septic shock, a specific IL-1 receptor antagonist (IL-1 ra) was administered. SUMMARY BACKGROUND DATA: The lipopolysaccharide (LPS) component of the outer membrane of gram-negative bacteria initiates a constellation of metabolic and immunologic host responses. IL-1, a macrophage-derived cytokine, acts as a key mediator in the host response to infection and inflammation. METHODS: Baboons were randomly assigned to receive either recombinant human IL-1 alpha, LPS, or live Escherichia coli both with or without concomitant administration of IL-1ra. Blood was collected hourly and analyzed using flow cytometric techniques. RESULTS: Both endotoxemia and live E. coli bacteremia induced an acute granulocytopenia; however, the granulocytopenia gradually resolved in the endotoxemic group, but was sustained in the bacteremic group. An early lymphopenia and monocytopenia was elicited by LPS or E. coli and persisted throughout the experiment. Recombinant human IL-1 alpha induced the following: (1) an early, transient decline in granulocytes followed by a sustained granulocytosis; (2) a lymphopenia; and (3) a transient monocytopenia followed by a gradual return to baseline. Although IL-1ra had no effect on leukocyte kinetics with either live E. coli or LPS, the IL-1ra significantly abrogated the monocytopenia seen with recombinant human IL-1 alpha administration alone. CONCLUSIONS: These results suggest that administration of recombinant human IL-1 alpha can replicate some of the characteristic patterns of hematologic change associated with bacteremia and endotoxemia. However, an endogenous IL-1 response is not required for these changes to occur. Rather, the data suggest that other inflammatory mediators induced by endotoxemia or gram-negative bacteremia, such as tumor necrosis factor-alpha (TNF alpha), may be involved.

Influence of hypercortisolemia on the acute-phase protein response to endotoxin in humans.

BACKGROUND: The response to systemic infection includes the coordinated appearance of hepatic acute-phase proteins, the production of which may be influenced by a counterregulatory hormonal background. This study sought to assess the potential for hypercortisolemic conditions to influence fibrinogen kinetics and other acute-phase protein responses in humans with endotoxemia. METHODS: Eleven hospitalized healthy male volunteers underwent two separate determinations of fibrinogen kinetics, one baseline and one after administration of endotoxin (2 ng/kg intravenously; lot EC-5). Seven volunteers were studied without hormonal manipulation and four in the presence of a hypercortisolemic background (hydrocortisone infusion, 3 micrograms/kg/min). Fibrinogen fractional synthetic rates were estimated from the incorporation of orally administered 15N-glycine, and fibrinogen, C-reactive protein, cortisol, tumor necrosis factor-alpha, and interleukin-6 levels were also determined. RESULTS: The presence of an antecedent hypercortisolemic background resulted in an attenuated interleukin-6 response, as well as decreased fibrinogen synthesis and C-reactive protein appearance. CONCLUSIONS: The current data suggest that glucocorticoid hormonal influences are of importance in the regulation of endotoxin-induced cytokine and acute-phase protein responses.

In vivo effects of the antiglucocorticoid RU 486 on glucocorticoid and cytokine responses to Escherichia coli endotoxin.

The endogenous adrenocortical response to sepsis is critical for host survival. The in vivo interactions among the endogenous glucocorticoid response, the induction of cytokines, and host survival during endotoxemia were explored in this study by use of the glucocorticoid receptor antagonist RU 486. Male Lewis rats underwent sterile insertion of a right jugular venous catheter. After a 72-h recovery period, animals received a 50% lethal dose of Escherichia coli endotoxin (2.5 mg/kg) via the catheter after pretreatment for 30 min prior to lipopolysaccharide (LPS) treatment with (i) vehicle alone intravenously (i.v.) (-corticosterone [-Cort]/-RU 486/+LPS) (n = 10), (ii) the antiglucocorticoid RU 486 (10 mg/kg) i.v. (-Cort/+RU 486/+LPS) (n = 11), or (iii) RU 486 (10 mg/kg) i.v. in animals that had undergone subcutaneous implantation of a corticosterone pellet at the time of catheter insertion (+Cort/+RU 486/+LPS) (n = 10). Except in animals receiving corticosterone pretreatment, baseline plasma corticosterone levels were low in all groups. Plasma corticosterone levels increased significantly (P less than 0.001) above the baseline following LPS administration. Animals in the -Cort/+RU 486/+LPS-treated group exhibited significantly increased mortality (P less than 0.001), with only 9% of the animals surviving at 72 h, as well as significantly increased plasma interleukin-6 levels, compared with animals receiving the vehicle alone (-Cort/-RU 486/+LPS), which showed 50% mortality. Pretreatment with corticosterone and RU 486 (+Cort/+RU 486/+LPS) significantly (P less than 0.001) reversed the mortality observed with RU 486 pretreatment alone (-Cort/+RU 486/+LPS), with 70% of the animals surviving at 72 h, and significantly attenuated the peak plasma tumor necrosis factor and interleukin-6 responses to LPS, compared with those in the animals treated with vehicle alone. These data demonstrate that the blockade of glucocorticoid binding by RU 486 increases LPS-induced mortality. The reversal of this effect by the induction of hypercorticosteronemia prior to RU 486 and LPS exposure (+Cort/+RU 486/+LPS) improves survival and is further associated with significant attenuation of cytokine production. Therefore, these data suggest that the protective effect of the endogenous glucocorticoid response to acute endotoxemia may result from the down-regulation of a potentially lethal cytokine response.

Interleukin-1 receptor blockade improves survival and hemodynamic performance in Escherichia coli septic shock, but fails to alter host responses to sublethal endotoxemia.

The present study was undertaken to evaluate the extent to which an endogenous interleukin-1 (IL-1) response contributes to the hemodynamic and metabolic consequences of sublethal endotoxemia or lethal Gram-negative septic shock. Young, healthy baboons received either a sublethal dose of lipopolysaccharide (LPS) or an LD100 of live Escherichia coli bacteria, and one half of the animals in each group were continuously infused with IL-1 receptor antagonist (IL-1ra). Plasma IL-1 beta was not detected in this model of endotoxemia. Administration of IL-1ra had only minimal effects on the modest hemodynamic and metabolic responses to sublethal endotoxemia, and did not attenuate the plasma cytokine response. In contrast, high circulating levels of IL-1 beta (range 300-800 pg/ml) were seen during lethal E. coli septic shock. IL-1ra treatment significantly attenuated the decrease in mean arterial blood pressure (MAP) (from -72 +/- 8 to -43 +/- 6 mm Hg; P less than 0.05) and cardiac output (from -0.81 +/- 0.17 to -0.48 +/- 0.15 liter/min; P less than 0.05), and significantly improved survival from 43 to 100% at 24 h (P less than 0.05). The plasma IL-1 beta and IL-6 responses to lethal E. coli septic shock were also significantly diminished by IL-1ra treatment (P less than 0.05), whereas tumor necrosis factor-alpha (TNF alpha) concentrations were unaffected. We conclude that an exaggerated systemic IL-1 beta response is characteristic of lethal E. coli septic shock, and contributes significantly to the hemodynamic and metabolic consequences of E. coli septic shock. IL-1ra can significantly attenuate the cytokine cascade and improve survival.

Effects of intravenous IL-8 administration in nonhuman primates.

IL-8, a cytokine known for its potent and specific neutrophil activation and chemoattractant properties, has been recently detected in the circulation during septic shock, endotoxemia, and after IL-1 alpha administration. Because of its observed in vitro actions, it has been hypothesized that IL-8 may contribute to the dynamics of circulating granulocytes and to the pathologic sequelae seen in sepsis. Here, human rIL-8 is administered to healthy nonhuman primates as a single i.v. injection or as a continuous 8-h i.v. infusion. We demonstrate that both methods of i.v. administration result in a rapid but transient, severe granulocytopenia, followed by a granulocytosis that persists as long as IL-8 levels are detectable in the circulation. There were no hemodynamic changes after IL-8 administration, and animals remained clinically stable during the 24-h observation period. No detectable circulating TNF-alpha, IL-1 beta, or IL-6 response was induced by either IL-8 administration regimen. Histopathologic examination revealed mild to moderate neutrophilic margination in lung, liver, and spleen, of greater severity in baboons receiving the 8-h infusion. There was no associated neutrophilic infiltration or tissue injury. Thus, IL-8 modulates circulating granulocyte dynamics and likely directs their actions, but when administered i.v. to healthy animals, either as a bolus dose or as a continuous infusion for up to 8 h, does not induce the hemodynamic and metabolic aberrations or the acute organ damage seen during sepsis.

Effect of combined cortisol-endotoxin administration on peripheral blood leukocyte counts and phenotype in normal humans.

We studied the role of lipopolysaccharide and the associated hypercortisolemic response as mediators of leukocyte changes associated with endotoxemia. Normal human subjects were given continuous, 12-hour, intravenous infusions of cortisol. After 6 hours of cortisol infusion, lipopolysaccharide (20 U/kg) was administered in an intravenous bolus. Plasma cortisol and blood leukocyte counts and lymphocyte subset proportions were evaluated every hour throughout the 12-hour study period. After 6 hours of cortisol infusion, lymphocyte counts and proportions of CD4+ helper/inducer T cells had declined significantly. The fact that these cells did not decline further in response to lipopolysaccharide and continued cortisol infusion suggests that lipopolysaccharide-induced lymphocyte changes are cortisol dependent. In contrast, the granulocytosis normally observed after lipopolysaccharide administration was unaffected by cortisol infusion. Finally, the monocyte counts and proportions of B cells (HLA-DR+ or CD20+ cells) responded to cortisol infusion and LPS in a pattern distinct from that of lipopolysaccharide alone. These results indicate that lipopolysaccharide-induced hypercortisolemia plays a role in immune modulation during endotoxemia.

Myocardial reperfusion injury. Platelet-activating factor stimulates polymorphonuclear leukocyte hydrogen peroxide production during myocardial reperfusion.

To study the roles of platelet-activating factor, polymorphonuclear leukocytes, and oxygen free radicals in myocardial reperfusion injury, we subjected 10 sheep to 90 minutes of mid-left anterior descending coronary artery followed by 6 hours of reperfusion. Stainings with gentian violet and tetratriphenyl ammonium chloride demonstrated 20% +/- 3% of the left ventricular mass at risk for ischemia, of which 75% +/- 10% underwent infarction. Coronary sinus blood was assayed for platelet-activating factor and neutrophil hydrogen peroxide production before and during coronary occlusion and during reperfusion. Platelet-activating factor was isolated by column chromatography and lipid extraction and quantified by radioimmunoassay. Neutrophil hydrogen peroxide production was measured by a 2',7'-dichlorofluorescein flow-cytometric assay. Platelet-activating factor was elevated to 899 +/- 210 pg/ml at 15 minutes of reperfusion, compared with the preocclusion level of 271 +/- 55 pg/ml and coronary occlusion level of 359 +/- 64 pg/ml (p less than 0.05; analysis of variance). Neutrophil hydrogen peroxide production, measured on a relative fluorescence scale, was also elevated to a level of 141 +/- 27 at 1 hour of reperfusion, compared with the preocclusion level of 103 +/- 6 and the coronary occlusion level of 114 +/- 13 (p less than 0.01; analysis of variance). Both of these parameters returned toward baselines at the end of 6 hours of reperfusion. Histologic examination revealed infiltration of polymorphonuclear leukocytes into the interstitium of the reperfused myocardium. Neutrophils isolated from unoperated and healthy sheep demonstrated a graded dose response in hydrogen peroxide production when stimulated by purified platelet-activating factor in vitro. These findings suggest that platelet-activating factor is released in the coronary circulation and is a mediator of oxygen free radical production in polymorphonuclear leukocytes during myocardial reperfusion.

Comparison between effects of interleukin-1 alpha administration and sublethal endotoxemia in primates.

Interleukin (IL)-1 is an early mediator of host response to inflammation, although its contribution to individual components of the acute phase reaction is still unclear. To evaluate how the hemodynamic, metabolic, and hormonal responses to sublethal endotoxemia compare with IL-1 administration, baboons received intravenously either lipopolysaccharide (LPS) or 0.1, 10, or 100 micrograms/kg IL-1 alpha. LPS induced an early tachycardia and a fall in mean arterial pressure, as well as lacticacidemia and hypoaminoacidemia. Similar hemodynamic and metabolic changes were seen with 10 or 100 micrograms/kg of IL-1 alpha. An increase in adrenocorticotropic hormone and fall in serum iron were induced by IL-1 alpha but not by LPS. Plasma tumor necrosis factor-alpha (TNF-alpha) was not measurable after IL-1 alpha administration, whereas LPS induced a monophasic TNF-alpha response. IL-6 levels were significantly greater after LPS than IL-1 alpha administration. Histopathological lesions, similar in LPS- and 100 micrograms/kg IL-1 alpha-treated groups, were present only in the adrenal cortex. We conclude that many, but not all, of the effects of sublethal endotoxemia can be replicated by IL-1 alpha administration, and these responses are dose dependent.

Pathophysiologic glucocorticoid levels and survival of translocating bacteria.

Burn wound sepsis in rats results in sustained corticosterone elevations and the prolonged presence of translocated bacteria in the mesenteric lymph nodes (MLNs). To determine if survival of bacteria in the MLNs may be influenced by pathophysiologic corticosterone levels, MLNs were quantitatively analyzed from rats randomized to the following groups: burn wound sepsis (BI); BI with adrenocortical response attenuated by cyclosporine (cyclosporine/BI); or cyclosporine/BI with corticosterone replacement (cyclosporine/BI + P). Although rates of bacterial translocation were similar, corticosterone levels were significantly different among the three groups and correlated with the number of lymphocytes and the number of enteric bacteria present per gram of MLN. Thus, pathophysiologic elevations of corticosterone levels during sepsis may exert an effect that allows survival of translocated bacteria in the MLNs of rats, perhaps due to glucocorticoid-associated alterations in regional immunity.

Biological thresholds of cold-induced phrenic nerve injury.

The effects of controlled cooling on phrenic nerve signal conduction were investigated by cooling an isolated segment of the phrenic nerve with a constant but variable temperature probe. The conduction of a standard electrical stimulus applied to the nerve proximal to the cooled section was measured by detector electrodes sutured to the diaphragm. Nerve conduction of the applied stimulus ceased between 10 degrees and 12 degrees C but returned within seconds after the probe was removed. The delay in the return of conduction increased as nerve temperature decreased until at a temperature of 4 degrees C the ability to conduct did not return after 4 hours. The amount of fat surrounding the nerve and the blood flow rate along the cooled portion of the nerve were observed to ameliorate the effects of low temperature on stimulus conduction. Total body cooling also appears to offer some protection against loss of conduction.