Effect of a human immunoglobulin preparation for intravenous use in a rabbit model of meningococcal endotoxin-induced shock.

BACKGROUND AND METHODS: Endotoxin shock is mediated by various cytokines, including tumor necrosis factor. Treatment of patients with i.v. immunoglobulin has been shown to reduce the concentration of circulating cytokines. The purpose of this study was to determine the protective effects of immunoglobulin for i.v. use on meningococcal endotoxin-induced shock in a rabbit model. Experimental animals were challenged with i.v. meningococcal endotoxin (lipo-oligosaccharide) 10 micrograms/kg, and treated with either a 2-hr i.v. immunoglobulin infusion (400 mg/kg) or a similar saline infusion that was initiated 30 mins before endotoxin challenge. Control animals were challenged with saline alone. RESULTS: Compared with untreated control animals, pulse rate increased (p less than .007) and mean arterial pressure and serum bicarbonate concentrations decreased (p less than .02) in both experimental groups, but did not differ between immunoglobulin-treated and saline-treated animals (p greater than .05) at any time after the endotoxin challenge. Geometric mean serum endotoxin concentrations were significantly (p less than .03) lower in the immunoglobulin-treated animals at 60, 120, 180, 240, 300, and 360 mins after the endotoxin challenge. The geometric mean serum tumor necrosis factor level at 1 hr after the endotoxin challenge in the immunoglobulin-treated experimental animals was lower than in saline-treated animals (5.53 vs. 8.47 tumor necrosis factor enzyme-linked immunosorbent assay U/mL), but not significantly so (p greater than .05). Mortality rate was similar in both experimental groups; eight (67%) of 12 saline-treated experimental rabbits and seven (70%) of ten immunoglobulin-treated rabbits died. All untreated control animals survived 24 hrs. CONCLUSIONS: In this model of circulatory shock in rabbits, i.v. immunoglobulin: a) does not significantly alter the physiologic responses to endotoxin challenge; b) significantly reduces endotoxin concentrations; c) reduces tumor necrosis factor concentrations, but not significantly; and d) does not improve survival rate.

Limulus antilipopolysaccharide factor protects rabbits from meningococcal endotoxin shock.

Limulus antilipopolysaccharide factor (LALF), an 11.8-kDa peptide isolated from amebocytes of Limulus polyphemus, neutralizes meningococcal lipooligosaccharide (LOS)-induced gelation of limulus amebocyte lysate. Rabbits challenged with an LD90 of LOS (10 micrograms/kg) premixed with LALF in vitro (n = 10) had significantly higher mean arterial pressure, arterial pH, serum bicarbonate concentrations, and survival (90% vs. 8%, P = .005) than did rabbits challenged with LOS alone. Relative to untreated controls, rabbits pretreated with LALF intravenously (iv) at 1.2 mg/kg (n = 21) also had significant improvements in physiologic measurements and higher survival (52% vs. 8%, P = .003). Even when LALF (1.2 mg/kg iv) was given 1/2 h after LOS challenge, animals showed significant improvements in physiologic measurements and survival (33% vs. 8% in untreated controls P = .028). LALF-treated animals also had significantly lower circulating endotoxin activity and tumor necrosis factor concentrations. Thus, LALF attenuates the toxic effects of meningococcal LOS in rabbits even when administered after LOS challenge and deserves further evaluation as a potential therapeutic agent for treating gram-negative septic shock.

Effect of meningococcal endotoxin in a rabbit model of shock.

Endotoxin in the form of a lipooligosaccharide (LOS) plays a key role in the development of shock in meningococcal sepsis. To examine hemodynamic and biochemical alterations during meningococcal endotoxic shock, we established a rabbit model. Thirty-nine rabbits, weighing 2.5-4.4 kg, were studied. After anesthesia with intramuscular ketamine (20 mg/kg) and xylazine (4 mg/kg), femoral venous and arterial catheters were inserted. Control animals received only saline, while rabbits in each of four additional groups were given LOS in 10-fold increments from 0.1 microgram/kg to 100 microgram/kg. Mean arterial pressure (MAP), heart rate (HR), respirations (RR), temperature (T), urine output, and arterial blood gases (pH, PCO2, PO2, and bicarbonate) were determined at baseline and hourly. Endotoxin levels and TNF levels were measured at 30, 60, 120, 180, 240, 300, and 360 min post-LOS. Survival was recorded. One-way analysis of variance (ANOVA) and the Scheffe procedure, paired samples t-test, two-tailed t-test, and Fisher's exact test were used. Pearson's coefficients were calculated. Animals receiving meningococcal LOS developed tachycardia and compensated metabolic acidosis with an initially normal pH and MAP. With progression of the shock state, the pH decreased and hypotension ensued. Maximal levels of endotoxin were measured 30 min after LOS injection and declined during the ensuing 6 hr. TNF rose from undetectable to markedly elevated levels and peaked at 60-120 min post-LOS. Increasing the amount of injected endotoxin produced more profound degrees of shock until a dose of 10.0 micrograms/kg was reached. There was no correlation between serum TNF at 60 min and survival at 6 hr or 24 hr.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of polymyxin B on experimental shock from meningococcal and Escherichia coli endotoxins.

Meningococcemia is the most frequent cause of septic shock in healthy children. To determine whether polymyxin B (PMB) might improve mortality from meningococcal shock, its protective activity was evaluated in rabbits challenged with an LD90 of meningococcal lipooligosaccharide (LOS) and compared with an LD80 of Escherichia coli O111:B4 lipopolysaccharide (LPS). PMB (5 mg/kg) administered intravenously 30 min before meningococcal LOS challenge had no significant effect on heart rate, mean arterial pressure, serum bicarbonate, serum tumor necrosis factor (TNF) levels, or survival relative to controls. However, PMB premixed with LOS in vitro increased serum bicarbonate levels (P less than .05) and improved 24-h survival (P less than .05). In contrast, PMB given before E. coli LPS challenge increased serum bicarbonate levels, decreased TNF levels, and improved 24-h survival (all, P less than .05). In vitro studies confirmed that PMB at 10 micrograms/ml neutralized E. coli LPS but not meningococcal LOS activity. Thus, pretreatment with PMB apparently protects rabbits against shock induced by E. coli LPS but not by meningococcal LOS.

Production of tumor necrosis factor by human monocytes in response to toxic-shock-syndrome toxin-1.

We studied the effect of toxic-shock-syndrome toxin-1 (TSST-1) on production of tumor necrosis factor (TNF) by human monocytes. Adherent mononuclear cells were stimulated with TSST-1 and their supernatants assayed for TNF by using L929 cells in a cytotoxicity assay. TSST-1 stimulated production of TNF over a wide range of concentrations. The cytotoxicity of monocyte supernatants was neutralized by antibody to TNF but not by antibody to interleukin-1 or by normal rabbit serum. TSST-1 and lipopolysaccharide (LPS) had a synergistic effect on monokine production. Monocytes "primed" with TSST-1 produced more interleukin-1 and TNF in response to LPS than did unprimed cells. Treating monocytes with LPS before TSST-1 and co-incubating the two agents with cells for 24 h also enhanced monokine production under some circumstances. These studies suggest a role for TNF in the pathogenesis of toxic shock syndrome, as a consequence of induction by TSST-1 alone or the synergistic effects of several bacterial products.

Neutralization of toxic shock syndrome toxin-1 by monoclonal antibodies in vitro and in vivo.

Sixteen monoclonal antibodies (MAbs) directed against toxic shock syndrome toxin-1 (TSST-1) were generated by immunization of mice with purified TSST-1 and subsequent fusion of spleen cells with myeloma cells. Antibody-producing clones, identified by an enzyme-linked immunosorbent assay, were maintained as ascites tumors, and MAbs were purified by protein A chromatography. High-titered clones were further characterized and tested for the ability to neutralize several biological activities of TSST-1. The MAbs, which are of several immunoglobulin subtypes, reacted specifically with purified TSST-1 and TSST-1 present in Staphylococcus aureus culture supernatants. Three MAbs neutralized TSST-1-induced mitogenesis in a dose-dependent manner. Three of eight MAbs tested were able to neutralize induction by TSST-1 of interleukin-1 production by human monocytes. One neutralizing MAb, 8-5-7, was tested for the ability to protect rabbits from a constant infusion of TSST-1. Rabbits given the MAb had an attenuated clinical illness and were protected from the hypocalcemia, lipemia, and hepatic and renal insufficiency seen in control rabbits. Six of seven control rabbits died, compared with only one of seven rabbits treated with MAb 8-5-7. These experiments suggest that MAb 8-5-7 is directed against an antigenic determinant critical to the toxicity of TSST-1 and that the MAbs should be useful as probes in structure-function analyses of the TSST-1 molecule.

A rabbit model of toxic shock syndrome that uses a constant, subcutaneous infusion of toxic shock syndrome toxin 1.

We have developed a rabbit model of toxic shock syndrome that uses a subcutaneous infusion pump to administer toxic shock syndrome toxin 1 (TSST-1). A dose of 150 micrograms, infused at a constant rate over a period of 7 days, resulted in a characteristic illness highlighted by fever, conjunctival hyperemia, cachexia, and lethargy. The illness was uniformly fatal, with a mean interval until death of 3.2 +/- 0.4 days. Serial determinations of serum chemistries confirmed the multisystem nature of this illness. Rabbits developed profound hypocalcemia, with levels falling from 15.5 +/- 0.2 to 7.6 +/- 0.4 mg/dl under the influence of TSST-1. Blood urea nitrogen and creatinine rose dramatically, in the setting of oliguria or anuria. Serum glutamicpyruvic transaminase was the most reliable indicator of hepatic dysfunction, with the mean rising from 48 U/liter before administration of TSST-1 to 546 U/liter among rabbits surviving 2 days of the infusion. Creatine phosphokinase also rose dramatically in 10 of 16 rabbits. Rabbits demonstrated relative neutrophilia and lymphopenia as well as an increase in the partial thromboplastin time. Histopathologic examination demonstrated disease of multiple organs, particularly the liver, spleen, and lymph nodes, all of which demonstrated inflammation, thrombosis, hemorrhage, and erythrophagocytosis. The concurrent administration of prednisolone with TSST-1 prevented death in four of four rabbits and greatly lessened the morbidity. Rabbits were not protected from morbidity or mortality by the concurrent administration of polymyxin B. We believe that a constant, subcutaneous infusion of TSST-1 in rabbits provides a reproducible model for studying the pathogenesis of TSS.

Induction of interleukin-1 by strains of Staphylococcus aureus from patients with nonmenstrual toxic shock syndrome.

We studied the induction of human interleukin-1 (IL-1) production in strains of Staphylococcus aureus isolated from patients with nonmenstrual toxic shock syndrome (TSS). Of the 20 TSS-associated strains studied, 11 produced and nine did not produce TSS toxin-1 (TSST-1). Human monocytes were incubated with dilute staphylococcal supernatants, and IL-1 production was measured in a lymphocyte-activating factor assay. All 20 TSS-associated strains were potent inducers of IL-1, in comparison with none of 10 vaginal isolates of S. aureus from healthy women. TSST-1-positive strains were more potent than TSST-1-negative strains. Nine TSST-1-negative TSS-associated strains were compared with 14 strains of S. aureus from other clinical settings and were found to be significantly more potent inducers of IL-1 (P less than .01). Eight of these nine TSS-associated strains produced at least one staphylococcal enterotoxin. Stimulation of monocytes by products of S. aureus may play a role in the pathogenesis of TSS.

Competitive, enzyme-linked immunosorbent assay for toxic shock syndrome toxin 1.

We developed a competitive, enzyme-linked immunosorbent assay for the quantitation of toxic shock syndrome toxin 1 (TSST-1). Polyvalent immunoglobulin G from immunized rabbits was used as the capture antibody, and alkaline phosphatase conjugated to purified toxin served as the indicator enzyme. A standard curve was generated with each experiment, from which the concentration of toxin in culture supernatants was extrapolated. The assay was useful for determining toxin concentrations of 0.03 to 0.5 micrograms/ml, which is a substantial, practical improvement over immunodiffusion methods. Staphylococcal enterotoxins A through E were not significantly cross-reactive in the assay, and staphylococcal protein A did not interfere with quantitation of TSST-1. By testing a variety of staphylococcal strains, we found 100% concordance between toxin determinations made with our assay and those made by the investigators from whom the strains were obtained. The competitive, enzyme-linked immunosorbent assay is a highly reproducible, inexpensive means of determining TSST-1 concentrations and may have broad applicability in the field of toxic shock research.