Failure of antisera to J5 and R595 rough mutants to reduce endotoxemic lethality.

Rabbit antisera to J5 (Rc) Escherichia coli and R595 (Re) Salmonella minnesota rough mutants were selected for the highest content of hemagglutinating antibodies to their respective core glycolipids. Despite titers of 1:2560 vs. J5 and 1:640 vs. R595 core glycolipids, the antisera failed to passively protect ICR or CF-1 mice against lethality induced by endotoxins from a variety of wild-type, smooth enterobacteria: E. coli O111:B4, E. coli O127:B8, Salmonella typhimurium, S. minnesota, and Citrobacter freundii. J5 antisera, however, reduced lethality from J5 core glycolipid. In contrast, O-specific rabbit antisera were consistently protective against the lethal activity of wild-type, smooth enterobacterial endotoxins, but such protection was limited to the homologous endotoxin. These findings are consistent with in vitro demonstrations of a highly restricted ability of antibodies to J5 and R595 core glycolipids to bind to endotoxins from wild-type, smooth enterobacteria.

Endotoxemia induced by antibiotic therapy: a mechanism for adrenal corticosteroid protection in gram-negative sepsis.

The following have been demonstrated: With a standardized model of P. mirabilis peritonitis in ICR mice, when optimal doses of an aminoglycoside antibiotic (kanamycin) are given so as to reduce mortality maximally, abrupt and significant increments occur in endotoxemia, which cannot be attributed to impairment of clearance of endotoxin. Within the antibiotic-treated groups, these increments in endotoxemia correlate with mortality. The level of endotoxemia in the antibiotic-treated survivors is significantly greater than in animals dying without antibiotic therapy. MP, although unable to reduce mortality from P. mirabilis peritonitis in the absence of antibiotic therapy, does so when endotoxin is added to the challenge inoculum. It is concluded from these observations that aminoglycoside antibiotic treatment can shift the lethal mechanisms during P. mirabilis peritonitis from those involving bacterial proliferation and low levels of endotoxemia to those involving bacterial death and release of large amounts of endotoxin. It is postulated that this shift in lethal mechanisms could account for the ability of MP to reduce mortality when used in conjunction with antibiotics. Finally, since animals surviving after antibiotic treatment have significantly greater endotoxemia than those dying without such treatment and since MP does not reduce mortality in the absence of antibiotic therapy but does so if endotoxin is added to the challenge inoculum, it appears that endotoxin may not play a critical role in the pathogenesis of mortality from untreated P. mirabilis peritonitis. Whether the present correlation observed between the significant rises in endotoxemia produced by antibiotics and the protection afforded by MP extends to other models of gram-negative sepsis, or to human gram-negative sepsis, is under study.

Resolution of pulmonary cyst after steroid therapy of asthma.

Emphysematous lung cysts in rare instances are associated with bronchial asthma, as evidenced by our patient and possibly another. Particularly in young patients, a trial period of steroid therapy should be instituted to confirm this possible association, to avoid unwarranted surgery and its attendant morbidity.

Induction of endotoxin tolerance with nonpyrogenic O-antigenic oligosaccharide-protein conjugates.

We prepared a dodecasaccharide, specific for the O-antigenic polysaccharide chain of Salmonella typhimurium (O-antigens 4 and 12), by the partial hydrolysis of the O-polysaccharide chain, utilizing bacteriophage 28B endo-alpha-L-rhamnosidase. The dodecasaccharide was shown by chemical and spectroscopical analyses to be totally devoid of lipid A and core oligosaccharide. By coupling this dodecasaccharide to human serum albumin, a glycoconjugate (DODECA-4809-ITC-HSA) was prepared and found to be (i) nonpyrogenic, (ii) unable to gelate a Limulus amoebocyte lysate, and (iii) unable to induce early-phase pyrogenic tolerance to endotoxin. Rabbits immunized either intravenously (with the glycoconjugate suspended in saline) or intrapopliteally (with the glycoconjugate suspended in Freund complete adjuvant) developed a significant although modest pyrogenic tolerance against challenge with the O-antigenic homologous S. typhimurium lipopolysaccharide (P less than 0.025 and P less than 0.01 for immunized and control rabbits, respectively). The evoked tolerance was O-antigen specific since no pyrogenic tolerance against challenge with lipopolysaccharide from S. thompson (possessing identical lipid A and core oligosaccharide structures but differing in the O-antigen polysaccharide chain) could be seen (P greater than 0.1). These results demonstrate that a nonpyrogenic O-antigenic polysaccharide hapten, when coupled to an immunogenic carrier protein, evokes immune responses which mediate significant, although modest, late-phase tolerance and is capable of partly reducing the pyrogenic activity of the O-antigenic homologous lipopolysaccharide.

Experimental gram-negative bacterial sepsis: prevention of mortality not preventable by antibiotics alone.

Outbred Swiss mice were inoculated intraperitoneally or intravenously with one 90 to 100% lethal dose of Escherichia coli O:18, Proteus mirabilis, or Klebsiella pneumoniae. After carefully timed intervals, aminoglycoside antibiotics were begun at dosages nnd intervals predetermined to constitute optimal therapy. With progressive increases in delay of antibiotic therapy, mortality rates increased progressively from 0% to 90 to 100%. Standardized models of infection were developed by selecting delay periods before initiating antibiotic therapy such that 50 to 70% mortalities resulted. Utilizing these models, agents with reputed anti-endotoxin activity were administered concomitantly with the delayed antibiotic therapy to determine if any could prevent gram-negative septic mortality no longer preventable by the antibiotics alone. The following were observed: (i) adrenal corticosteroids prevented mortality that was no longer preventable by optimal aminoglycoside antibiotics alone. The following were preventable by optimal aminoglycoside antibiotic therapy alone; (ii) specific antisera also did so, provided anaphylaxis was circumvented; (iii) in one model (P. mirabilis), such protection by adrenal corticosteroids and specific antiserum could be additive; (iv) adrenal corticosteroids and specific antiserum acted synergistically with the aminoglycoside antibiotics--no protection was achieved by delayed administration of the steroids or antiserum alone; (v) timing was crucial--the synergistic protective activity of adrenal corticosteroids and of specific antiserum with aminoglycosides declined rapidly as infection progressed; (vi) cyclophosphamide pretreatment markedly impaired the synergistic protective activity of specific antiserum and of adrenal corticosteroids with aminoglycosides; (vii) no reputed anti-endotoxin agents other than adrenal corticosteroids and specific antiserum proved capable of preventing mortality not preventable by aminoglycoside antibiotics alone. These included antisera to rough mutant Enterobacteriaceae of Rc, Rd, and Re chemotypes, anticoagulants (heparin), ascorbic acid, antiproteolytic agents (aprotinin), alpha adrenergic blockers (phenoxybenzamine), prostaglandin synthetase inhibitors (acetylsalicylic acid, sodium salicylate, indomethacin), nicotinamide, glucose, and insulin-glucose-potassium mixtures.

Mechanisms of endotoxin tolerance. The role of the spleen.

Splenectomy markedly impaired the production of circulating anti-endotoxin antibodies during the initial 10 days after .v. administration of a Boivin preparation of Escherichia coli endotoxin (ET) in both rabbit and man. Increase in antibodies with secondary (flocculating and bactericidal) activities were virtually abolished, whereas increases in antibodies with primary (binding) activity were significantly reduced. On the basis of these findings, splenectomized rabbit and man were employed to test the hypothesis that the early phase (less than 72 h) of pyrogenic tolerance to endotoxin is independent of anti-endotoxin antibody but that such antibody contributes significantly to the later phase (less than or equal to 72 h) of tolerance. In the splenectomized rabbit, the initial pyrogenic reponses to ET and the subsequent tolerant responses at 24 and 48 h were comparable to sham-operated controls...

Mechanisms of endotoxin tolerance. IX. Effect of exchange transfusion.

Healthy New Zealand rabbits were injected iv with an LD-80 dose of E. coli endotoxin. Twenty minutes later, after removal of over 50% of the endotoxin by the RES, exchange transfusion was performed, accomplishing a rapid and sustained reduction in the level of endotoxemia simulating that seen in animals rendered highly tolerant by seven prior sublethal injections of toxin. Depite such reduction in endotoxemia, 96-hr mortality was only slightly, and not significantly reduced compared to sham exchanged controls (70 vs 83% respectively). Additional control studies indicated that exchange tranfusion per se did not enhance endotoxin mortality. The findings directly support the concept that endotoxin tolerance is based primarily upon enhanced RES resistance to endotoxin toxicity rather than upon enhanced RES clearance of circulating endotoxin.

The nature of endotoxin tolerance.

Certain of the mechanisms by which man develops pyrogenic tolerance to bacterial endotoxins have been considered. After an initial intravenous injection of toxin, two temporally distinct phases of tolerance can be discerned, early and late, each with very different characteristics. Early tolerance appears to be mediated by a non-antibody mechanism entailing a transiently occurring refractory state, apparently involving to a major degree decreased production of endogenous pyrogen by the macrophage system, particularly the hepatic macrophages. Late tolerance appears to be mediated by anti-endotoxin antibodies directed against both "O" and common core antigens which blunt the release of endogenous pyrogen from macrophages. The common core antigens are masked in the presence of the "O" antigenic side chains and become effective immunogens only when these "O" side chains are lacking. Accelerated reticuloendothelial system clearance of circulating endotoxin provides an ancillary protective mechanism in that it brings the toxin more efficiently into the macrophages that are refractory or protected by antibody. When endotoxin is administered repeatedly at closely spaced intervals, both the early phase (non-immune) and late phase (immune) mechanisms may become superimposed. In addition, a third mechanisms, enhanced detoxification capabilities of macrophages, also now appears to come into play. At any given time, it is the relative contribution of each mechanism, which in turn is dependent upon the immunization schedule, antigenicity of the endotoxin, dosage, and immunological competency of the host, that determines the expression of the endotoxin tolerant state.