PATHOGENESIS OF EXPERIMENTAL SHOCK : II. ABSENCE OF ENDOTOXIC ACTIVITY IN BLOOD OF RABBITS SUBJECTED TO GRADED HEMORRHAGE

A series of biological test reactions was used in order to establish the presence of bacterial endotoxins in the blood of rabbits during the progression of hemorrhagic shock. 1. When the shocked animal was used as the test object, it was not possible to induce either the generalized Shwartzman reaction or the dermal Shwartzman phenomenon with exogenous endotoxin (S. enteritidis or E. coli) as one of the two provocative factors. 2. Epinephrine instilled into the skin of rabbits either before, during, or after an episode of hemorrhagic shock did not result in the hemorrhagic skin reaction which occurs in the presence of as little as 1 µg of endotoxin intravenously. 3. Passive transfer from a donor in the irreversible phase of shock of 20 to 25 ml of blood into a primed recipient (B.P. at 40 mm Hg for 1 hour) was uniformly lethal. 4. Similar amounts of blood from such shocked donor failed upon intravenous injection to elicit a protective hemorrhagic reaction in skin sites which were infiltrated with 100 µg of epinephrine. In the same animals 1 µg of endotoxin added to the blood samples caused a positive dermal response. 5. Blood was taken from rabbits which had been pretreated with S. enteritidis endotoxin and then subjected to hemorrhagic shock (35 mm Hg for 2 hours). Such samples upon passive transfer produced positive skin reactions in epinephrine sites but were not lethal to the primed test recipient used in these studies. It is concluded that the contribution of bacterial endotoxemia to the genesis of hemorrhagic shock remains to be determined.     

INVOLVEMENT OF ADRENERGIC FACTORS IN THE EFFECTS OF BACTERIAL ENDOTOXIN

The possible involvement of adrenergic mechanisms in the effects of Escherichia coli endotoxin was investigated in several preparations. Appropriate pretreatment of rabbits with E. coli endotoxin significantly increased pressor responses to epinephrine and norepinephrine as compared to untreated controls. Exposure of isolated rabbit aorta strips to E. coli endotoxin in a medium containing whole blood or cellular constituents of blood significantly increased the response to epinephrine. Endotoxin had no effect on responses to epinephrine in ritro when plain Krebs-Ringer solution was used. Pretreatment with reserpine or phenoxybenzamine (dibenzyline) protected rabbits and mice against the acute lethal effects of E. coli endotoxin. The time period intervening between reserpine or dibenzyline administration and challenge by endotoxin precluded a direct antiendotoxic action of these agents. In addition, incubation of dibenzyline with endotoxin in vitro, under conditions which would favor reaction, did not decrease the toxicity of the latter. These results indicate that peripheral adrenergic mechanisms are intimately involved in the effects of E. coli endotoxin and support the concept that deleterious effects of endotoxin in shock probably are due to exaggeration of existing vasoconstriction in an already compromised organism.     

THE ROLE OF THE RETICULO-ENDOTHELIAL SYSTEM IN HEMORRHAGIC SHOCK

"Blockade" of the RES by thorotrast so lowered the tolerance of hemorrhagic shock in rabbits and dogs that a reversible degree of hemorrhagic shock became irreversible. This was true not only in normal rabbits, but in rabbits made resistant to hemorrhagic shock by producing resistance to endotoxins. Rabbits which had been pretreated with thorotrast and then subjected to hemorrhagic shock displayed at death the hemorrhagic lesions and the renal cortical necrosis characteristic of the Shwartzman reaction, in addition to the intramural hemorrhages in the gut which are characteristic of animals dying of hemorrhagic or of endotoxic shock. Elimination of the Shwartzman reaction by the prior administration of nitrogen mustard did not prevent the endotoxemia or the death in shock. Rabbits made more resistant to thorotrast than normal rabbits by prior repeated administration of this substance were also more resistant than normal rabbits to endotoxin, and survived an ordinarily lethal exposure to hemorrhagic shock. During the first few hours after its administration thorotrast induced excessive vulnerability not only to endotoxin and to hemorrhagic shock, but also to an additional small dose of thorotrast. Moreover, a non-absorbable antibiotic given by gavage shortly after thorotrast produced the same lesions as these other agents; i.e. endotoxic shock, the Shwartzman reaction, and death. These data indicate that the lesions induced by thorotrast are produced by endotoxins which the injured or blockaded RES cannot inactivate. The presence of endotoxins in the blood of these rabbits was indicated by the lethal effect of this blood in test recipients. The foregoing observations did not apply to rabbits with an intestinal flora free of coliform bacteria. Over 80 per cent of such rabbits were resistant to an ordinarily lethal exposure to hemorrhagic shock, and they escaped the damage caused by the usual doses of thorotrast. They did, however, develop endotoxic shock and die if given a large dose of thorotrast. These data were taken to indicate that coliform-free rabbits are not entirely free of endotoxins. (In the ordinary environment animals cannot avoid swallowing endotoxin and coliform bacteria.) The absence of the Shwartzman reaction in the coliform-free rabbits is taken to signify that this reaction requires the participation of the endotoxins derived from the intraintestinal bacteria. The absence of endotoxic shock in the coliform-free rabbits is taken to signify that the endotoxins of the coliform bacteria are involved in the shock and death of the coliform-bearing rabbits. Finally the prevention by dibenamine of both the Shwartzman reaction and endotoxic shock and death in rabbits with a normal flora demonstrates that adrenergic activity plays an indispensable role in both phenomena. The foregoing data provide strong support for the thesis that when the RES is severely disabled by any agent, endotoxins which normally and continuously enter the circulation from the gut will produce endotoxic shock and death.     

ON THE ABSORPTION OF BACTERIAL ENDOTOXIN FROM THE GASTRO-INTESTINAL TRACT OF THE NORMAL AND SHOCKED ANIMAL

Coliform-free rabbits fed P³² labeled E. coli 0111B₄ prior to the induction of experimental hemorrhagic shock were shown to have a substantial amount of the type-specific 0111B₄ antigen in the circulating blood, liver, and spleen, whereas normal rabbits fed the same amount of these bacteria and held under identical conditions, but not exposed to shock, have the antigen within the liver, and occasionally in the kidney, but not in the blood. That the antigen recovered from the blood and tissues was derived from this specific strain of bacteria was demonstrated by the use of the hemagglutination inhibition reaction, by the absence of cross-reacting antigens in appropriate control animals, and by agreement in the amount of antigen as estimated by two different technics. Transport of bacterial endotoxin across the intestinal membrane appears to be achieved primarily by passive diffusion. The accumulation of biologically active endotoxin in the blood and tissues of the shocked animal appears to be due to a reduction in the detoxifying potential of the reticulo-endothelial system, and not to a greater than normal absorption of endotoxin from the intestine. The absence of toxicity in the specific antigen extracted from normal liver demonstrates that the degradation of endotoxic potency can be achieved without altering the chemical integrity of the polysaccharide moiety of the molecule. The implications of the hypothesis that there is a continuous but fluctuating absorption of bacterial endotoxin from the intestine are briefly discussed, and the contribution of free circulating bacterial endotoxin of intestinal origin to the fate of the shocked animal is noted.     

ON THE RELATION OF THE SIZE OF THE INTRAINTESTINAL POOL OF ENDOTOXIN TO THE DEVELOPMENT OF IRREVERSIBILITY IN HEMORRHAGIC SHOCK

Additional evidence is presented affirming the role of the intestinal pool of endotoxin in producing irreversibility in prolonged hemorrhagic shock. The fact that coliform-free rabbits tolerate exposure to a degree and duration of hemorrhagic shock which is lethal for rabbits that possess the normal flora, and that these tolerant rabbits lose their tolerance when E. coli are introduced into the gut several hours before inducing shock, demonstrate the critical importance of the size of the pool of endotoxin. That there is a proportionality between the size of the pool of endotoxin and the tolerance of hemorrhagic shock is suggested by the survival rate of several series of coliform-free rabbits fed E. coli by gavage. The rate was less the more firmly the E. coli were reestablished in the flora. The presence of the usual number of coliform bacteria in the intestinal flora does not mean the presence of the usual amount of endotoxin in these bacteria. The amount of endotoxin depends not only on the size of the population, but also, as our own experience demonstrates, on the particular ecological factors extant at any particular time which govern the amount of endotoxin elaborated by any given strain or strains of coliform bacteria.     

The role of epinephrine in the reactions produced by the endotoxins of gram-negative bacteria. I. Hemorrhagic necrosis produced by epinephrine in the skin of endotoxin-treated rabbits

Extensive lesions of dermal hemorrhagic necrosis occurred in rabbits when epinephrine (or norepinephrine) was injected into the skin within 4 hours after an intravenous injection of endotoxin. As little as 5 µg. of intradermal epinephrine, and 1 µg. of intravenous endotoxin, were sufficient to produce lesions. Similar lesions, but smaller in size and surrounded by a zone of acute inflammation, were produced by intradermal injection of a mixture of comparable amounts of endotoxin and epinephrine. No lesions were produced by combinations of endotoxin with serotonin, pitressin, or ephedrine. Both types of epinephrine-endotoxin lesion were prevented by pretreatment with cortisone, dibenzyline, and chlorpromazine. They were not prevented by heparin or nitrogen mustard. The lesions produced by intradermal mixtures of epinephrine and endotoxin were greatly enhanced in size and severity in animals treated with nitrogen mustard. Both types of lesion were prevented in rabbits rendered "tolerant" by repeated injections of sublethal amounts of endotoxin. It is concluded that endotoxin has the property of altering the reactivity of blood vessels to epinephrine in such a way that this hormone becomes a potent necrotizing agent. The possibility that this effect may represent a basic mechanism in the various intoxicating actions of endotoxin, and certain implications of this hypothesis, are discussed.     

INACTIVATION OF BACTERIAL EXOTOXINS AND ENDOTOXIN BY IRON : IN VITRO STUDIES

After incubation with various agents in vitro, the lethal effects of aqueous solutions of Cl. perfringens alpha toxin, C. diphtheriae toxin, and E. coli endotoxin were tested in mice, guinea pigs, and rabbits, respectively. Iron, copper, cysteine, ascorbic acid, and versene counteracted the lethal effects of alpha toxin in mice, while magnesium, manganese, zinc, and citrate did not. Iron also counteracted the lethal effects of diphtheria toxin in guinea pigs. After incubation of endotoxin with iron, its lethal effects and tissue-necrotizing actions in rabbits were counteracted. However, the pyrogenic properties of the toxin were not affected. The solubilities of perfringens alpha toxin and diphtheria toxin were markedly reduced after incubation with detoxifying metals, and resolubilization of these toxins with chelators resulted in partial restoration of toxicity. Addition of versene to detoxified endotoxin also resulted in partial recovery of lethal effectiveness. The inactivation of bacterial toxins by iron under in vitro conditions is not specific to this metal, is a reversible process, and may be due to desolubilization, reduction, or to competition by the metal for sites on the toxin normally bound by other cations in vivo. Although no evidence is presented in this paper to support the view that there is a relationship between the inactivation of endotoxin and the storage iron in the reticuloendothelial system of shocked animals, the observation of an in vitro inactivation of endotoxin by inorganic iron warrants consideration of such a mechanism.     

PATHOGENESIS OF EXPERIMENTAL SHOCK : IV. STUDIES ON LYSOSOMES IN NORMAL AND TOLERANT ANIMALS SUBJECTED TO LETHAL TRAUMA AND ENDOTOXEMIA

Fatal shock was produced in animals by drum trauma, temporary occlusion of the superior mesenteric artery, and bacterial endotoxin. Measurements were made of release of beta glucuronidase and cathepsins from the large granule fractions of livers, and of levels of circulating beta glucuronidase and acid phosphatase in these animals. Experiments were also carried out with animals rendered tolerant by previous exposure to sublethal amounts of trauma or by pretreatment with cortisone. The results show that release of beta glucuronidase and cathepsins from the large granule fraction of liver was increased during traumatic and endotoxin shock in the rat. Similarly, circulating levels of acid phosphatase and beta glucuronidase were increased during traumatic shock in rats and rabbits, and during endotoxin shock in rats. The data also indicate that tolerance to traumatic injury, induced by prior conditioning, prevented the increase in levels of circulating acid phosphatase normally observed after stress, and may have been associated with an increased stability of hepatic lysosomal particles. In addition, cortisone, which appears to "stabilize" hepatic lysosomes in vivo, also reduced the increase in plasma acid phosphatase brought about by endotoxin and trauma. From the foregoing observations, it is suggested that: (a) Disruption of lysosomes and release of their contained enzymes in free, active form may occur in liver and intestine of shocked animals. (b) The activation of lysosomal hydrolases within cells and their release into the circulation may play an important role in exacerbating tissue injury and accelerating the development of irreversibility during shock. (c) The increased stability of lysosomes of tolerant and of cortisone-treated animals may constitute an important component of the resistance of these animals to shock.     

ON THE COLLAPSE OF BACTERIAL ENDOTOXIN RESISTANCE FOLLOWING HEMORRHAGE

Unanesthetized immature albino rabbits exposed to 2 hours of severe but reversible hemorrhagic shock induced with aseptic precautions by multiple cardiac bleedings exhibited no increase in susceptibility to single intravenous injections of 200 µg./kg. E. coli endotoxin administered 4 hours post retransfusion, a quantity of endotoxin that was found to be the largest dose uniformly non-lethal to normal rabbits. Paired and randomly selected rabbits treated identically except for the additional procedure of a femoral arterial cutdown and ligation (without aseptic precautions) exhibited increases in susceptibility to the same endotoxin of several hundredfold. This effect could not be attributed to the femoral cutdown and arterial ligation alone since such trauma when coupled with sham cardiac bleedings failed to increase susceptibility to 200 µg./kg. of endotoxin. These data appear valid since sham cardiac bleedings produced no detectable impairment of myocardial contractility, while 2 hours of hemorrhagic shock at 50 mm. Hg with the Lamson reservoir technique caused an increase in endotoxin susceptibility comparable to that seen when cardiac bleedings were combined with a non-sterile femoral arterial cutdown and ligation. The mechanisms increasing the susceptibility to E. coli endotoxin were investigated. It was found that (a) rabbit femoral skeletal muscle is normally contaminated with clostridia, (b) the use of aseptic femoral wounding precautions exerted some suggestive protective influence, (c) the use of aseptic wounding precautions combined with immediate topical sulfanilamide and wound closure exerted a significant protective influence, and (d) prophylactic polyvalent gas gangrene antitoxin protected in a manner not demonstrable when diphtheria antitoxin was employed as a control. These observations suggest that clostridial wound infection is one mechanism whereby a femoral arterial cutdown lowers endotoxin resistance of the rabbit following hemorrhagic shock. It is, however, not the only mechanism since the ligation of a femoral artery during hemorrhagic shock led to edema following retransfusion equivalent to a mean of approximately 30 per cent of the original circulating plasma volume. The intensification of shock caused by this transudation presumably intensified reticulo-endothelial injury, and thus further lowered the resistance of the rabbit to an intravenous injection of endotoxin 4 hours following retransfusion.     

The relationship between the vascular manifestations of shock produced by endotoxin,trauma, and hemorrhage. I. Certain similarities between the reactions in normal and endotoxin-tolerant rats

The vascular effects of lethal doses of E. coli endotoxin, as observed in the mesentery of the rat, resemble the reactions of traumatic and hemorrhagic shock in the following respects: a profound inhibition of arteriolar and precapillary reactivity to topical epinephrine occurs after an initial stage of hyperreactivity; the small veins show failure to relax completely following constrictor doses of epinephrine; and the terminal vessels develop an unusual sensitivity to fluctuations in temperature of the fluid irrigating the tissue. Rats in which tolerance to bacterial endotoxin is induced, by repeated doses given daily, become highly resistant to the lethal effects of both drum trauma and hemorrhagic shock. However, rats in which the adaptation to traumatic shock is produced by repeated exposure to drum trauma, do not develop a significant degree of tolerance to lethal doses of endotoxin. The injection of small non-lethal doses of bacterial endotoxin during non-lethal episodes of trauma or hemorrhage, leads to the development of irreversible shock and death. The bearing of these findings on the problem of the relationship between endotoxin and traumatic shock is discussed.     

EFFECT OF NUTRITION ON THE RESISTANCE OF MICE TO ENDOTOXIN AND ON THE BACTERICIDAL POWER OF THEIR TISSUES

Mice on inadequate nutritional regimens were found to be more susceptible to various bacterial diseases than mice fed a complete diet containing 15 to 20 per cent casein. The tests of susceptibility included: (a) infections with virulent bacteria; (b) injection of large doses of avirulent coagulase-negative staphylococci; and (c) lethal effects of bacterial endotoxins. The infection-enhancing effect of nutritional deficiencies could be rendered even more striking by administering the infective inoculum simultaneously with a sublethal dose of endotoxin. Despite their great susceptibility to infection, malnourished animals retained much of their ability to eliminate bacteria from the blood, liver, spleen, kidneys, and lungs, at least during the early phases of the infectious processes. This was true even when the animals had received endotoxin simultaneously with the infective dose. The results suggest that under the conditions of the present study, the nutritional state influenced the outcome of infection not primarily by affecting the fate of the pathogens in vivo, but rather by modifying the ability of the host to resist their toxic effects.     

MECHANISMS OF ENDOTOXIN TOLERANCE : III. THE REFRACTORY STATE DURING CONTINUOUS INTRAVENOUS INFUSIONS OF ENDOTOXIN

Bacterial endotoxins were administered by continuous intravenous infusions at constant rates to normal man and rabbits. An initial progressive febrile reaction was followed by progressive defervescence to baseline. The resulting pyrogenic refractory state was characterized as follows: (a) reticuloendothelial blockade with thorotrast neither prevented nor reversed its course; (b) passive transfer was unsuccessful with refractory phase plasma; (c) infusions of normal plasma or fresh whole blood failed to restore responsiveness; (d) a minimum of 4 hours of continuous endotoxin infusion was required for full development of unresponsiveness; (e) circulating antibody titers to endotoxin remained unaltered; (f) peripheral leukocytosis appeared; (g) infusion of febrile phase plasma reevoked an immediate, monophasic fever; (h) endotoxinemia could be demonstrated by pyrogen bioassay; (i) 10-fold increases in endotoxin infusion rates reevoked fever; (j) impaired responsiveness extended to heterologous endotoxins; (k) dermal inflammatory responses to endotoxin were suppressed in man while tuberculin reactivity remained unimpaired; dermal inflammatory responses to endotoxin were enhanced in rabbits; and (l) pyrogenic reactivity to endotoxin reappeared within 24 hours in man; refractoriness persisted in rabbits. It is concluded that the pyrogenic refractory state reflects an inability of the host to continue to mobilize endogenous pyrogen during sustained endotoxinemia. Such observations, together with previous studies, are consistent with two distinct immunologic mechanisms of resistance to endotoxin pyrogenicity: (a) desensitization at the cellular level; and (b) elaboration of circulating antibodies which assist reticuloendothelial clearance and destruction of endotoxin. Whereas both such mechanisms may contribute to pyrogenic tolerance, the characteristics of the pyrogenic refractory state suggest the participation only of the former.     

SOME EFFECTS OF PROTEOLYTIC INHIBITORS ON TISSUE INJURY AND SYSTEMIC ANAPHYLAXIS

A study was made of the development of various forms of local and systemic injury in animals treated with inhibitors of proteolytic activity. The agents used were tosylarginine methyl ester (TAME), epsilon aminocaproic acid (EACA), and soybean trypsin inhibitor (SBTI). 1. Hemorrhagic necrosis in the skin of the rabbit following intradermal epinephrine in combination with bacterial endotoxin (either intravenous or local) was clearly suppressed by EACA, TAME, and SBTI, given systemically. Tosylarginine (TA) was ineffective. 2. No effect was observed on the classical Shwartzman reaction, the local Arthus phenomenon, or inflammation induced by xylene. 3. The lethal effects of systemic anaphylaxis in the mouse, acute endotoxemia in the rat and mouse, and drum shock in the rat are suppressed by EACA and TAME. 4. There was no effect on the generalized Shwartzman phenomenon in the rabbit and on anaphylaxis in the guinea pig. 5. The effects of EACA or TAME on the injury reactions under investigation were not due to a pharmacological or chemical action on vascular behavior per se. 6. The data provide corroborative evidence for a proteolytic step in injury phenomena which may be mediated through some common activation system. 7. The working hypothesis is advanced that local or systemic stress through the release of epinephrine may result in an increase of a circulating activator of proteolysis and that this in turn may give rise to the release of vasoactive substances,—possibly histamine, serotonin, or a polypeptide.     

MECHANISMS OF ENDOTOXIN TOLERANCE : I. RELATIONSHIP BETWEEN TOLERANCE AND RETICULOENDOTHELIAL SYSTEM PHAGOCYTIC ACTIVITY IN THE RABBIT

Pyrogenic tolerance following 7 daily intravenous injections of 2.0 µg/kg E. coli endotoxin in albino rabbits was associated with significant increases in RES phagocytic activity as measured with colloidal carbon. Nevertheless, 4 hours after RES blockade with thorotrast (3 ml/kg), the tolerant rabbits exhibited significantly lower fever indices following intravenous endotoxin challenge than did non-tolerant control animals despite comparably depressed capacities to clear carbon from the blood. Moreover, plasma from rabbits tolerant to endotoxin induced significant tolerance in normal rabbits prepared by thorotrast blockade without enhancing the depressed carbon clearance. This passive protection extended to heterologous endotoxins. Analysis of the data indicates that RES blockade does not abolish tolerance; rather blockade resets the reactivity to endotoxin in the normal and tolerant animal, rendering both exquisitely reactive, but permitting retention of the major portion of tolerance. Apparently the tolerant animal possesses a dual endotoxin defense system. One system is abolished by thorotrast; the other is in part humoral, accounts for the greater portion of tolerance, and is thorotrast-resistant. The nature of the humoral component is not defined but is consistent with that of an opsonin with high endotoxin specificity.     

The role of epinephrine in the reactions produced by the endotoxins of gram-negative bacteria. II. The changes produced by endotoxin in the vascular reactivity to epinephrine,in the rat mesoappendix and the isolated,perfused rabbit ear

The effects of endotoxin on the epinephrine reactivity of blood vessels in the rat mesoappendix have been studied. Following intravenous injection of a relatively small, sublethal dose of endotoxin, the terminal arterioles and venules exhibited greatly augmented and prolonged vasoconstrictor responses to epinephrine and norepinephrine. Hyperreactivity became evident within 30 minutes after injection of endotoxin, and persisted for as long as 6 hours. After larger doses of endotoxin, sufficient to cause illness or death, the vascular hyperreactivity to epinephrine was of briefer duration, and was followed by a stage of increasing hyporeactivity reaching levels much below normal. With lethal doses, the terminal arterioles and venules became completely refractory to epinephrine, while heightened reactivity persisted in the larger arteries and veins. The end result was pooling of stagnant blood in distended capillaries and venules, accompanied by the appearance of petechiae. Topical applications of epinephrine during this stage were followed promptly by an increase in petechial hemorrhage at the site of testing. Rats which were rendered tolerant to the lethal effect of endotoxin, by repeated daily injections of small doses, developed resistance to the effects of endotoxin on epinephrine reactivity. Neither hyperreactivity nor hyporeactivity to epinephrine were demonstrable in these animals, nor were spontaneous abnormalities of blood flow or petechial hemorrhages observed in the mesoappendix. Analogous results were obtained in perfusion studies of the vessels of the isolated rabbit ear. Perfusion of small amounts of endotoxin was followed within a few minutes by potentiation of epinephrine reactivity. Larger doses caused complete reversal of this effect, to such an extent that epinephrine now produced marked degrees of vasodilation. The possible meaning of these observations in the interpretation of the endotoxin-epinephrine skin lesions described in the preceding paper is discussed. It is suggested that abnormal reactions to epinephrine or norepinephrine in the tissues of intact animals may represent a basic mechanism in the intoxicating and tissue-damaging properties of endotoxin.     

STUDIES ON LYSOSOMES : I. THE EFFECTS OF ENDOTOXIN,ENDOTOXIN TOLERANCE,AND CORTISONE ON THE RELEASE OF ACID HYDROLASES FROM A GRANULAR FRACTION OF RABBIT LIVER

Granular fractions sedimenting between 800 g and 15,000 g have been prepared in 0.25 M sucrose from the livers of normal young rabbits and from the livers of rabbits injected with A. aerogenes endotoxin. As early as 5 minutes after endotoxin, there was an augmented release of two enzymes, beta glucuronidase and cathepsin, into the supernatant of fractions which had been incubated or incubated and irradiated with a mercury vapor lamp at 37°C. These effects were maximum at 30 minutes after endotoxin and were associated with a depletion of the total activities of the two enzymes within the granules. Concurrently there was a rise in the activity of the two enzymes in fractions of the homogenate which were unsedimentable at 15,000 g. Fractions prepared from animals made tolerant to endotoxin no longer responded to incubation and irradiation by an augmented release of these two hydrolases, nor did fractions prepared from animals pretreated with glucocorticoids. Pretreatment with DOCA did not prevent release of enzymes after endotoxin. The results have been interpreted to indicate that one consequence of the injection of endotoxin is an effect upon the stability of lysosomes, with subsequent release of acid hydrolases into the cell sap or surrounding tissue. Glucocorticoids may exert their pharmacologic effects through the protection of these subcellular particles against a variety of injurious agents.     

A LABILE SERUM FACTOR IN EXPERIMENTAL ENDOTOXIN SHOCK: CROSS-TRANSFUSION STUDIES IN DOGS

Peripheral vascular failure caused by endotoxin in the dog has an initial stage of vasoconstriction. Preliminary studies in vitro demonstrated that the constriction was due to the interaction of endotoxin with a heat-labile serum or plasma factor and platelets, resulting in the liberation of histamine. Further studies on the intact dog support and extend this concept. A standardized dose of Escherichia coli endotoxin produced fatal shock in control adult mongrel dogs within 28 hours. The characteristic pattern of changes included progressive hypotension, oliguria and anuria, hemoconcentration, and acidosis. Normal dogs were protected against endotoxin by transfusions of blood in which the essential serum factor was depleted in one of two ways. First, plasma separated from the blood of normal animals was heated at 56°C for 30 minutes, and the infused reconstituted whole blood protected normal dogs. Protection was not afforded by unheated reconstituted blood. Second, blood from immune dogs obtained within 24 hours after a second lethal dose of endotoxin protected recipient dogs. However, protection was not demonstrated with blood collected 72 hours after a second injection of endotoxin. The nature of the serum factor essential for endotoxin activity is not known. It is postulated that an enzyme or enzyme system is involved, and the possible role of complement is discussed.     

A SPECIFIC POISON IN THE LIVER EXTRACTS OF RABBITS INOCULATED WITH TYPHOID AND PRODIGIOSUS BACILLI INTRAVENOUSLY

1. The livers of rabbits inoculated with cultures of Bacillus typhosus or Bacillus prodigiosus under certain conditions contain a toxic substance extractable with salt solution. When the toxic extracts are injected intravenously into normal rabbits the latter animals develop symptoms resembling those of anaphylactic shock and succumb. The lethal doses of the toxic extracts are far smaller than those of normal liver extract. 2. The livers of rabbits injected with typhoid antigen also yield a toxic extract. 3. Boiling as well as filtration through a Berkefeld filter only partially detoxicates the extract. 4. Tolerance to one to two lethal doses of the poisonous extracts can be induced by cautious immunization. 5. Rabbits actively immunized to Bacillus typhosus or Bacillus prodigiosus usually resist one lethal dose of the homologous liver poison; and animals tolerant to the typhoid liver poison resist one minimum lethal dose at least of Bacillus typhosus. 6. Typhoid immune serum is not detoxicating either in vivo or in vitro for the typhoid liver poison. 7. The liver poisons are specific, since rabbits actively immunized to either Bacillus typhosus or Bacillus prodigiosus withstand at least one minimum lethal dose of the homologous but not of the heterologous-liver poisons.     

STUDIES ON THE BIOLOGIC RELATIONSHIP OF ENDOTOXIN AND OTHER TOXIC PROTEINS : I. COMPARISON OF THE PROPERTIES OF SNAKE VENOM AND ENDOTOXIN

1. Agkistrodon piscivorus venom and E. coli endotoxin were shown to be immunologically distinct, and to differ in certain biologic properties: effects on immune response, body temperature, and circulating leukocyte count, and capacity to prepare for and provoke the local and generalized Shwartzman reaction. 2. Neither a single prior injection of venom nor the existence of hyperimmunity to lethal doses of venom protected rabbits against the local and generalized Shwartzman reaction. 3. Serial intravenous injections of sublethal doses of venom produced enhanced susceptibility to venom rather than refractoriness. 4. Preparation for both the local and generalized Shwartzman reaction with endotoxin appeared to enhance susceptibility of rabbits to challenge with venom. 5. Tolerance to bacterial pyrogens established by repeated injections of endotoxin is paralleled by increased resistance to snake venom given at least 1 week later, in mice and rabbits. 6. Zymosan failed to enhance susceptibility of rabbits to venom, but thorotrast increased the number of late deaths from venom. 7. Exposure of venom to ferrous sulfate interferes with its toxicity.     

THE SUSCEPTIBILITY OF MICE TO BACTERIAL ENDOTOXINS

Albino mice (Rockefeller NCS strain) raised and maintained free of ordinary bacterial pathogens, as well as of intestinal Escherichia coli and of Proteus bacilli, were found to be highly resistant to the lethal effect of bacterial endotoxins. When newborn mice of this NCS colony were nursed by foster mothers from another colony raised under ordinary conditions (SS colony from which the NCS colony was derived), they acquired the intestinal flora of the latter animals and became susceptible to the lethal effects of endotoxins. NCS adult mice could be rendered susceptible to the lethal effect of endotoxins by vaccination with heat killed Gram-negative bacilli. The susceptibility thus induced exhibited a certain degree of specificity for the bacterial strain used in vaccination. Although untreated NCS mice were resistant to the lethal effect of endotoxins, they proved exquisitively susceptible to the infection-enhancing effect of these materials. For example, 1 µg. or less of endotoxin was found sufficient to help establish a rapidly fatal septicemia with Staphylococcus aureus. Small amounts of endotoxin (1 µg. or less), administered alone, caused a marked but transient loss of weight. Vaccination with heat-killed Gram-negative bacilli or with killed BCG increased the resistance of NCS mice to the infection-enhancing effect of small amounts of endotoxin. This protective effect exhibited a certain degree of specificity for the bacterial strain from which the toxin used in the infection-enhancing test was derived. These various findings can be explained by assuming that the pathological effects of endotoxins involve at least two unrelated mechanisms; (a) a primary toxicity illustrated in this study by the loss of weight and enhancement of infection resulting from the injection of small doses of toxin; (b) an immunological reaction with lethal consequences which became manifest only in animals sensitized to the endotoxin by prior exposure to Gram-negative bacilli.