Influence of hypothermia on the generalized Shwartzman reaction.

In normothermic rabbits the generalized Shwartzman reaction can usually be elicited by two intravenous injections of endotoxin spaced 24 hr apart. The reaction could be prevented by cooling the rabbits. Hypothermia is effective only at the time of the second injection. The protective effect can clearly be demonstrated by gross and histologic examination. The possible mechanism of the protective action of hypothermia is discussed.     

Interleukin 1: a common endogenous mediator of inflammation and the local Shwartzman reaction

In this study we investigated the role of interleukin 1 (IL 1) in the induction of inflammatory lesions and in the preparation and provocation of the local Shwartzman reaction. Both of these phenomena can be induced with a variety of agents. This suggested to us that a common endogenous mediator may be crucial to the development of these two lesions. When IL 1 was injected intradermally into shaved rabbit backs, 51Cr-labeled neutrophils accumulated at the injection site. Neutrophils began to accumulate less than 1 hr after injection, and the maximum rate of accumulation was observed by 4 hr. This activity was dose dependent. It was calculated that in all animals, 10(-14) mol of IL 1 induced significant neutrophil accumulation, whereas in many animals, as little as 10(-15) mol of IL 1 sufficed. When 4.2 X 10(-9) mol of E. coli 0111:B4 lipopolysaccharide W was injected i.v. 24 hr after an intradermal injection of IL 1 (2.9 X 10(-13) mol), a local Shwartzman reaction was seen 4 hr later at the intradermal injection site. IL 1 injected i.v. 24 hr after an intradermal injection of either IL 1 or lipopolysaccharide also produced a local Shwartzman reaction. These data indicate that IL 1 may be the common endogenous mediator of the inflammatory response, and IL 1 may serve in the same role for the preparation and provocation of the local Shwartzman reaction.     

Effect of lead acetate on the susceptibility of rats to bacterial endotoxins

Selye, H. (Université de Montréal, Montreal, Canada), B. Tuchweber, and L. Bertók. Effect of lead acetate on susceptibility of rats to bacterial endotoxins. J. Bacteriol. 91:884-890. 1966.-A single, normally well-tolerated, intravenous injection of lead acetate increases the sensitivity of the rat to the endotoxins of various gram-negative bacteria about 100,000 times above normal. Under the conditions of these experiments, the mortality and organ changes normally produced by the intravenous injection of 100 mug of Escherichia coli endotoxin were essentially the same as those obtained by use of 1 nanogram in lead-sensitized rats. The sensitizing effect of lead acetate for E. coli endotoxin is greatest when the two agents are given simultaneously. However, considerable sensitization is still detectable when endotoxin is injected up to 1 hr before or 7 hr after sensitization with lead. No sensitization was noted when the endotoxin was administered 24 hr before or after lead acetate. Under our experimental conditions, the minimal dose of lead acetate which could still induce significant sensitization to E. coli endotoxin was 1 mg per 100 g of body weight. Although lead acetate induces a high degree of susceptibility to various endotoxins, other reticuloendothelial blocking agents did not acquire unusual toxicity after pretreatment with lead. Finally, none of the other metals or reticuloendothelial blocking agents tested could duplicate the pronounced decrease in endotoxin resistance induced by lead acetate.     

牙髓紫卟啉菌内毒素对炎症性细胞因子的介导作用

牙髓紫叶琳菌ＡＴＣＣ３５４０６是近年来新发现的重要致病性专性厌氧菌,采用改良酚－氯仿－石油醚法提取牙髓紫卟啉菌ＡＴＣＣ３５４０６内毒素脂多糖,通过Ｋｒａｍｅｒ测定法、软琼脂细胞培养法以及胸腺细胞增殖法测定脂多糖的细胞生物学活性。结果显示：纯化脂多糖可不同程度地诱导小鼠模型生成肿瘤坏死因子（ＴＮＦ）、集落刺激因子（ＣＳＦ）和白介素－１（ＩＬ－１）,并在一定范围内呈剂量依赖型,提示牙髓紫卟啉菌内毒素在动物模型和细胞模型中具有显著的细胞生物学和免疫学活性．     

Modification of Selected Host-reactive Properties of Endotoxin by Treatment with Sodium Deoxycholate

Endotoxin dissociated into subunits by sodium deoxycholate treatment exhibited diminished capacity to kill chick embryos, protect mice against the lethal effects of infection with Salmonella typhi, evoke hemorrhagic necrosis in skin inoculated with epinephrine, prepare for and provoke the dermal Shwartzman reaction, and induce pyrogenic tolerance. Surfactant-treated material which had been allowed to reaggregate displayed activity equivalent to that of untreated material. These findings were consistent with the working hypothesis that a macromolecular complex of critical size is required in order for endotoxin to elicit its characteristic effects in the host.     

Synthesis and characterization of gentiobiose heptaacetate conjugate vaccines that produce endotoxin-neutralizing antibodies

We have prepared aminoethyl (AE), aminopropyl (AP), and aminopentyl (APT) derivatives of gentiobiose heptaacetate (GH). These spacer compounds (AEGH, APGH, APTGH) have been coupled to succinylated diphtheria toxoid (Suc.DT) to produce conjugate vaccines. These conjugates all bind to the anti-lipid A human monoclonal antibody A6(H4C5) in an ELISA binding assay. Rabbits immunized with the APGH conjugate vaccine in either Freund's complete adjuvant or aluminum hydroxide gel produced antibody levels of 5120 and 3600 ELISA units, respectively, compared to an antibody level of less than 20 ELISA units for the prebleed sera. Sera from mice immunized with either the aminopropyl or the aminopentyl conjugate had antibody levels of 5120 and 2560 ELISA antibody units, respectively. These antibodies neutralized endotoxin in a Limulus lysate neutralization assay. Protection against the local Shwartzman reaction was demonstrated (p less than 0.05) in eight out of nine rabbits immunized with the Suc-DT-APGH conjugate vaccine compared to three out of 10 rabbits immunized with the carrier protein Suc-DT. Passive transfer experiments demonstrated that four out of five rabbits receiving immune serum were protected from Shwartzman reaction compared to one out of five rabbits receiving normal serum (p less than 0.1). These results indicated that epitopes contained in gentiobiose heptaacetate when properly presented as conjugate vaccines were capable of inducing neutralizing antibodies against endotoxin.     

Role of Leukocytes in Blood Coagulation and the Generalized Shwartzman Reaction

IN spite of intensive investigation, many of the factors which initiate blood coagulation and thrombosis remain obscure. The generalized Shwartzman phenomenon, which is due at least in part to intravascular coagulation, is classically obtained by two appropriately spaced, sub-lethal, intravenous doses of endotoxin given to rabbits and is characterized by disseminated thrombi in lungs, kidney and spleen; the characteristic lesion in the kidneys is renal cortical necrosis. Although the Shwartzman phenomenon can be prevented by anticoagulation^{1, 2}, its mechanism remains obscure. Leukocytes have been implicated as the mediators but only indirect evidence is available¹. Leukocytes also possess procoagulant and anticoagulant activity^3–5, the former, however, has always been considered too weak to be physiologically significant or able to cause intensive intravascular clotting with defibrination. We now have evidence that endotoxin given to rabbits may endow their leukocytes with considerable procoagulant activity in vitro, sufficient to produce intravascular clots in various organs when infused to untreated normal rabbits.     

Haematologic effect and Shwartzman reactivity of radiodetoxified endotoxin.

Comparative experiments were made in rabbits with Escherichia coli O89 endotoxin and endotoxin detoxified by ionizing radiation (60Co-gamma, 5 Mrad). Radiation significantly weakened the leukopenia and thrombocytopenia provoking effect of endotoxin. Radiodetoxified endotoxin decreased the fibrinogen level only slightly and caused insignificant changes in reptilase time. The complement level was decreased less by the detoxified than by the parent endotoxin. Even the local Shwartzman phenomenon inducing capacity of radiodetoxified endotoxin decreased significantly, particularly when it was used for preparation and provocation, too.     

Tests for studying invasive properties of selected Proteus mirabilis strains]

Proteus mirabilis is an important pathogen of the urinary tract infections (UTI). Lipopolysaccharide (LPS, endotoxin) is one of the pathogenic factors of pathogenicity of these bacteria. In this paper we described the invasion of L929 mouse fibroblasts by P. mirabilis strains, classified into the O10, O23, O30, O43 serogroups. The maximal invasiveness was observed between 4-6 hours of incubation of the tested cells with bacteria. The cytotoxic effect slightly increased with the incubation time, probably as a result of the production of HpmA hemolysin. Incubation of L929 fibroblasts with LPS led to decrease of bacterial invasiveness. We observed that with the time of incubation of L929 cells with LPS (2-22 h), the invasiveness decreased (longer incubation time with LPS--weaker penetration).     

Failure of bacterial endotoxin to produce the generalized Shwartzman reaction in the Syrian hamster

The ability of bacterial endotoxin to produce the generalized Shwartzman reaction (GSR) in pregnant and nonpregnant hamsters was investigated. Endotoxins prepared from Escherichia coli O127:B8, Salmonella enteritidis, and S. typhosa 0-901 did not produce the GSR in nonpregnant hamsters. Injection of lead acetate did not make the hamsters susceptible to the GSR producing effects of endotoxin. Endotoxin administered to hamsters on either or both the 14th and 15th day of the 16-day gestation period caused fetal death, but did not provoke the GSR. The immunization of hamsters with boiled suspensions of gram-negative bacteria isolated from hamster feces did not protect against the GSR produced in pregnant hamsters by the injection of the antimitotic drug colchicine late in the gestation period. It appeared that colchicine was acting to produce the GSR by a mechanism other than the release of endogenous endotoxin through the damaged intestinal wall. Ascitic fluid, amniotic fluid, and serum obtained from pregnant hamsters developing the GSR after the administration of colchicine did not provoke the GSR in other pregnant hamsters.     

DERMATITIS-PRODUCING ALGA LYNGBY A MAJUSCULA GOMONT IN HAWAII. II. BIOLOGICAL PROPERTIES OF THE TOXIC FACTOR1,2

The course of the reaction produced by intracutaneous injection of the toxin of Lyngbya majuscula Gomont is that of a severe acute inflammatory reaction. No protective or sensitizing effect is induced by a previous exposure. The response from intravenous injection into a rat indicates that the toxin acts as a general cell toxin. The protozoan Tetrahymena pyriformis and rabbit erythrocytes are lysed by the toxic principle, which also possesses antibacterial activity. Of the organisms tested, Mycobacterium species are markedly inhibited, while Bacillus cereus, Gaffkya tetragena, and Sarcina lutea are slightly to moderately inhibited. Other components of the alga, which are steam distillable, have been found to have antibacterial activity but are not involved in the skin reaction.     

Detection and study of the labile toxic factor of Proteus mirabilis]

The author studied the dynamics of toxin formation of the high- and low-virulent Pr. mirabilis strains. The high-virulent strain produced toxic substances of the exotoxin type detectable in 1-2-day broth cultures. Later the activity of the culture medium of both cultures under study was due to substances of the endotoxin type. By physico-chemical and immunobiological properties, and also by chemical composition of Pr. mirabilis "early toxin" was similar to the exoenterotoxins of enterobacteria. A principal possibility of concentration and purification of Pr. mirabilis "early toxin" with the use of ultrafiltration and gel-chromatography was demonstrated.     

Fever in rats during normal and dehydrated conditions

The effect of endogenous pyrogen (EP, from rabbit) and endotoxin (Salmonella typhosa) on rectal temperature (Tre) was investigated in normal and dehydrated rats of both sexes. Intraperitoneal injection of either EP or endotoxin did not affect body temperature. In addition, no changes in Tre were observed when endotoxin was injected intravenously in normally hydrated male rats, but significant falls in Tre occurred in normal female rats. However, intravenous injection of EP produced fever in both sexes, but females generally showed smaller responses. A second intravenous injection of endotoxin, given 3 days after the first injection, always produced fever in normally hydrated rats. The pattern of this febrile response was monophasic. In contrast to the response in normal rats, intravenous endotoxin produced significant fevers with a biphasic pattern in dehydrated rats of either sex, but the febrile responses of male rats were greater than those of female rats. On the other hand, there were no significant differences between febrile responses to intravenous EP exhibited by normal and dehydrated animals. These results show that rats of both sexes possess physiological mechanisms capable of producing a fever following intravenous injections of EP.     

Endotoxin-like activities inMyxococcus xanthus

Vegetative cells as well myxospores ofMyxococcus xanthus have shown anticomplementary activity and the capacity to be used as active agents in the skin preparation of the Shwartzman reaction and in its intravenous induction. These endotoxin-like properties were not extractable by the hot phenol-water methods. Our results suggest the presence of a lipid A analog in both vegetative cells and myxospores, and emphasize the difficulty of lipopolysaccharide detection; this is perhaps a consequence of a developing associated change in polysaccharide moiety of the myxobacterial lipopolysaccharides; this may be the basis of the special immunomodulation pattern shown byM. xanthus myxospores.     

A possible effect on the Shwartzman reaction in the rabbit caused by fentanyl-fluanisone tranquillization

Shwartzman's reaction was elicited in four Chinchilla rabbits. Two of the animals were tranquillized with fentanyl-fluanisone (Hypnorm: Janssen Pharmaceuticals). There was a reduced response in the tranquillized rabbits while the untranquillized animals developed the typical dose-responsive skin reaction. Fentanyl-fluanisone tranquillization may interfere with the development of the Shwartzman reaction in Chinchilla rabbits.     

Effects of thalidomide on the local Shwartzman reaction in mice and rabbits

Abstract The Shwartzman reaction is an animal model displaying histopathological vasculitis phenomena. Extravasation and swelling due to increased vascular permeability and cellular infiltration, which are hallmarks of the Shwartzman reaction, were evaluated as leakage of i.v.-injected Evans Blue dye and by histological and immunohistological characteristics in rabbits and mice. (±)-Thalidomide, (−)-thalidomide, (+)-thalidomide and dexamethasone inhibited the increase of vascular permeability in the local Shwartzman reaction. Histologically, the intensity of the Shwartzman reaction was reduced. In mice thrombus formation and leukocytoclastic vasculitis was inhibited by (±)-thalidomide and (+)-thalidomide. ICAM-1 expression was markedly reduced after (+)-thalidomide injection. Thalidomide and dexamethasone pretreatment reduced Mac-1 expression on perivascular infiltrated granulocytes. The inhibitory effect of thalidomide on vasculitis of the Shwartzman reaction may thus be related to reduction of adhesion molecule expression.     

Acute alcohol intoxication and endotoxemia desensitize HIV-1 gp120-induced CC-chemokine production by Kupffer cells

Chemokines are involved in the inhibition of HIV-1 infection and in the pathogenesis of tissue injury in a number of conditions, including endotoxemia and alcoholic liver disease. CC chemotactic peptides (MIP-1alpha, MCP-1 and RANTES) are produced by a wide variety of cell types in response to immunological stimuli, bacterial endotoxin and gp120 from HIV-1 and HIV-2. This work tests the hypothesis that prior exposure to endotoxin and/or ethanol in vivo inhibits the production of CC-chemokines following a secondary challenge with HIV-1 gp120 in vitro. Male Sprague-Dawley rats received in intravenous infusion of ethanol to maintain blood ethanol level at 170 mg/dl for 3 hr. Escherichia coli LPS (1 mg/Kg) was given intravenously 5 min after the ethanol bolus was injected. Control groups received similar volumes of saline. Three hr after LPS treatment, Kupffer cells were obtained and treated with HIV-1 gp120 (5 microg/10(6) cells/24 hr). At the end of the incubation period, cells were obtained for RT-PCR analysis of CC-chemokine mRNA expression. Chemokine release in culture supernatants was measured by ELISA. Results show that in vivo ethanol was associated with downregulation of MIP-1alpha and MCP-1 mRNA expression and protein release in primary cultures of Kupffer cells. However, ethanol alone primed isolated Kupffer cells for enhanced RANTES mRNA and protein release in the presence or absence of HIV-1 gp120. These results demonstrate that acute ethanol intoxication and endotoxemia may selectively act as a desensitizing agent in response to a secondary challenge with bacterial or viral products.     

Identification of Staphylococcal Hemolysins by an Electrophoretic Localization Technique

A technique for identifying and characterizing staphylococcal hemolysins by first separating them electrophoretically in barbital-buffered agar gel (pH 8.4) at 5 ma/cm for 2 hr and then determining their hemolytic activities by exposing them to human, horse, rabbit, and sheep erythrocytes is described. The alpha-hemolysin produced by a White variant of the Wood 46 strain of Staphylococcus aureus migrated 18 mm towards the cathode, and it lysed horse, rabbit, and sheep erythrocytes, whereas a Clear variant of the Wood 46 strain of S. aureus produced a lysin which migrated similarly to the alpha-hemolysin but lysed only rabbit cells. This latter lysin was tentatively named alpha(1)-lysin. This strain of S. aureus also produced beta-hemolysin which migrated 36 mm towards the cathode and lysed sheep cells. beta-Hemolysin produced by some strains of S. aureus showed considerable tailing during electrophoresis, whereas beta-hemolysin produced by other strains of S. aureus migrated as a well-defined peak. A lysin migrating 11 mm towards the anode was probably delta-lysin. It was, however, not produced in sufficient concentration when the cultures were grown in semisolid medium.     

Biological activity of synthetic heptaacyl lipid A representing a component of Salmonella minnesota R595 lipid A

A synthetic lipid A (preparation 516), containing seven acyl groups and representing one component of natural free lipid A of Salmonella minnesota R595, has been investigated for biological activity in a number of endotoxin test systems. It was found that the synthetic preparation was, in typical in vivo endotoxin tests (lethality, pyrogenicity, Shwartzman reactivity) as well as in its antigenicity and macrophage activation capacity, significantly less active than natural Salmonella lipid A. However, in other in vitro assay systems (B-cell mitogenicity, complement activation, Limulus amoebocyte lysate gelation) it expressed similar activity as Salmonella lipid A.