The pyrogenic effect of scarlet fever toxin

Scarlet fever toxin was found to liberate leukocytic pyrogen from granulocytesin vitro. In comparative experiments withSalmonella paratyphi B endotoxin and scarlet fever toxin it was tested whether leukocytes from rabbits tolerant to one of these toxins are able to synthetize and liberate endogenous pyrogen. Leukocytes from rabbits tolerant to endotoxin liberated leukoeytic pyrogen following challenge with endotoxin or with scarlet fever toxin. Leukocytes from animals tolerant to scarlet fever toxin liberated leukocytic pyrogen in the presence of endotoxin, but were insensitive to homologous, i.e. scarlet fever toxin. Similarly, leukocytes from cortisone-treated animals did not liberate leukocytic pyrogen if they were incubated with scarlet fever toxin, but liberation of leukocytic pyrogen did take place under challenge with endotoxin. Leukocytes from normal animals incubated in Hanks solution without toxin did not synthetize endogenous pyrogen.     

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.     

Macrophage procoagulant factors--mediators of inflammatory and neoplastic tissue lesions

Mononuclear phagocytes, a specialized cell lineage comprising bone-marrow precursors, blood monocytes and tissue macrophages, can interact with blood coagulation mechanisms with resulting thrombus formation or extravascular fibrin accumulation. Such procoagulant activity is usually activation dependent and requires interaction of the cells with immune or nonimmune stimuli. In the former case (e.g., alloantigens, soluble protein antigens) collaboration of mononuclear phagocytes with T lymphocytes is necessary and is mediated by cell-to-cell contact or lymphokines. Prototype of a direct acting stimulus is bacterial lipopolysaccharide. Mononuclear phagocyte procoagulant activity is expressed in the form of cell membrane-bound or released factors which display molecular heterogeneity. They include the initiator of the extrinsic clotting pathway, tissue factor, known clotting proteases such as factors V and VII, and novel proteolytic enzymes including prothrombinase and a factor X activator. Mononuclear phagocyte procoagulants are pathogenetically involved in generalized disorders with intravascular coagulation and thromboembolic phenomena. These disorders, exemplified by the Shwartzman reaction and possibly by paraneoplastic thromboembolism, are initiated by blood monocytes. Extravascular fibrin deposition can be initiated by tissue-infiltrating monocytes and macrophages in disease states such as acute renal allograft failure and solid tumours.     

Tolerance and Other Biological Properties of Vibrio parahaemolyticus Endotoxins

Biological properties of endotoxins prepared from three strains of Vibrio parahaemolyticus were compared with reference to commercially prepared Salmonella typhi endotoxin. Endotoxin assays performed in rabbits included dermal Shwartzman reactivity, pyrogenicity, heat stability, and ability to induce tolerance as well as cross-tolerance. Mice were used for endotoxin LD₅₀ determinations. Results showed V. parahaemolyticus endotoxins were similar to that of S. typhi strain O901. Induction of tolerance to V. parahaemolyticus strain 11590 endotoxin resulted in complete cross-tolerance to S. typhi endotoxin, and vice versa. Partial cross-tolerance to S. typhi endotoxin was demonstrated with rabbits rendered tolerant to endotoxin from V. parahaemolyticus strains Sak-3 and FC1011. Absorption spectra, nitrogen, phosphorus and carbohydrate analyses revealed additional similarities between endotoxins from V. parahaemolyticus and endotoxin from a member of the Enterobacteriaceae.     

Endotoxic substance of cryptococcus neoformans

In this study an endotoxic substance was extracted from the cells ofCryptococcus neoformans and the physicochemical and biological properties of this substance (Cr-ET) were investigated. In comparison with endotoxin of gram-negative bacteria, the lethality of Cr-ET for mice and chick embryos was low and such biological activities were weak as the pyrogenic effect on rabbits and effects on the leucocyte count and blood sugar level in rabbits. Skin reactions (both primary and Shwartzman reactions) were elicited in rabbits by relatively large dose of Cr-ET. Unlike bacterial endotoxin, hyperreactivity to Cr-ET was not induced in mice by prior infection with BCG.     

Endotoxin and Acute Renal Failure Associated with Obstructive Jaundice

A single dose of endotoxin given to rats with obstructive jaundice produced death with intravascular coagulation. This action was apparently due to delayed clearance of endotoxin from the circulation. The finding is relevant to “hepatorenal failure,” which can be caused by bacteraemia after biliary tract operations.     

Biological Activities of Endotoxins from Yersinia enterocolitica

The chemical properties and the general biological activities of lipopolysaccharide (LPS) and Boivin-type endotoxin obtained respectively by phenol-water and trichloroacetic acid extraction from Yersinia enterocolitica serotypes 03 and 09 were studied. The yield of LPS from the O9 strain was about 10% of the O3 strain possibly because of the lower solubility of O9-LPS in aqueous phase. However, the chemical composition of O9-LPS was similar to that of O3-LPS in the proportions of reducing sugar, glucosamine, heptose, KDO, and lipid A. In pyrogenicity and local Shwartzman reactivity in rabbits and lethality for mice, there was also no difference between O3 and O9-LPS. The anthrone-positive carbohydrate and lipid A contents of Boivin-type endotoxin from O3 were higher than those of the endotoxin from O9. The biological activities of Boivin-type endotoxin from O3 were also remarkably higher than those of the endotoxin from O9. It seems that endotoxin of Y. enterocolitica serotype O3 may play an important role in infection by this organism.     

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.     

Ultrastructural manifestations of pulmonary thrombohemorrhagic syndrome in endotoxic shock

In electron microscopic investigation in the initial period of endotoxin shock in rats, rabbits and dogs there are haemorrhages and microthrombi with precipitation of fibrin. In intermediate period of shock signs of intravascular coagulation predominate and on the stage of late endotoxemia qualitatively new sign of destruction is interstitial fibrosis. Involving of coagulative link completes and enhances microcirculatory changes in the lungs and promotes formation of acute lung insufficiency.     

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.     

Hypotension and disseminated intravascular coagulation following intralesional bacillus Calmette-Guérin therapy for locally metastatic melanoma

Summary Four patients developed serious hypotension and signs of disseminated intravascular coagulation shortly after a second round of Tice bacillus Calmette-Guérin (BCG) injections into locally recurrent cutaneous melanoma satellite nodules. Each of these patients survived following intensive therapy with isoniazid, pyridoxine, steroids, pressors, antibiotics, and cardio-renal support including, in one case, three acute hemodialyses. Plasma specimens from two of the four patients caused gelation of lysate from the amebocytes ofLimulus polyphemus, indicating the presence of endotoxin or an endotoxin-like substance. In vitro studies on the BCG preparations led us to conclude that this endotoxin activity in the plasma is not the result of direct injection of endotoxin with the BCG preparation, but rather from release of endotoxin from endogenous sources, such as the intestinal tract during a period of relative hypotension following an allergic reaction. Prior immunity appeared to be the consistent factor in the toxic reactions reported herein. Finally, we present recommendations for serial monitoring of these patients and discuss the use of an alternative agent for intralesional therapy.     

Effect of Thyrotropin-Releasing Hormone and Its Synthetic Analogue Digipramine on Certain Indices of Hemostasis <Emphasis Type="Italic">in vitro</Emphasis> and <Emphasis Type="Italic">in vivo</Emphasis>

The effect of neuropeptide thyrotropin-releasing hormone (TRH) and its synthetic analogue digipramine on certain indices of blood coagulation and fibrinolysis were studied in vitro and in vivo. The peptides added to the pool of normal rat plasma at 10⁻¹⁰ to 10⁻³ M increased the procoagulant activity but had virtually no effect on fibrinolysis. Intravenous administration of TRH and digipramine increased the procoagulant activity of the blood and platelet aggregation but decreased fibrinolysis; digipramine had a more pronounced effect on the coagulation potential of the blood and a less pronounced effect on fibrinolytic indices as compared to TRH. Intranasal administration of the peptides did not change the pattern of their effect on indices of hemostasis although the effects became less pronounced.__________Translated from Izvestiya Akademii Nauk, Seriya Biologicheskaya, No. 3, 2005, pp. 311–315.Original Russian Text Copyright © 2005 by Grigorjeva, Golubeva.     

Mechanistic insight into the procoagulant activity of tumor-derived apoptotic vesicles

BackgroundChemotherapy induces the release of apoptotic vesicles (ApoV) from the tumor plasma membrane. Tumor ApoV may enhance the risk of thrombotic events in cancer patients undergoing chemotherapy. However, the relative contribution of ApoV to coagulation and the pathways involved remain poorly characterized. In addition, this study sets out to compare the procoagulant activity of chemotherapy-induced ApoV with their cell of origin and to determine the mechanisms of ApoV-induced coagulation.MethodsWe utilized human and murine cancer cell lines and chemotherapeutic agents to determine the requirement for the coagulation factors (tissue factor; TF, FII, FV, FVII, FVIII, FIX and phosphatidylserine) in the procoagulant activity of ApoV. The role of previously identified ApoV-associated FV was determined in a FV functional assay.ResultsApoV were significantly more procoagulant per microgram of protein compared to parental living or dying tumor cells. In the phase to peak fibrin generation, procoagulant activity was dependent on phosphatidylserine, TF expression, FVII and the prothrombinase complex. However, the intrinsic coagulation factors FIX and FVIII were dispensable. ApoV-associated FV could not support coagulation in the absence of supplied, exogenous FV.ConclusionsApoV are significantly more procoagulant than their parental tumor cells. ApoV require the extrinsic tenase and prothrombinase complex to activate the early phase of coagulation. Endogenous FV identified on tumor ApoV is serum-derived and functional, but is non-essential for ApoV-mediated fibrin generation.General significanceThis study clarifies the mechanisms of procoagulant activity of vesicles released from dying tumor cells.     

Inhibition by pentoxifylline of procoagulant activity produced by endotoxin-active monocytes]

When appropriately stimulated, monocytes are able to initiate blood coagulation through the membrane expression of tissue factor. This procoagulant activity is thought to play a role in activating coagulation in response to inflammatory stimuli in vivo. We found that pentoxifylline, a methylxanthine derivative already reported to regulate some monocyte functions, inhibits the procoagulant activity developed by monocytes in vitro in response to endotoxin. This effect was accompanied by an early increase in intracellular levels of cyclic AMP and was mimicked by compounds that induce an increase in cyclic AMP levels. These results suggest that the suppressive effect of pentoxifylline occurs at least in part via an increase in intracellular cyclic AMP levels.     

Production of the Shwartzman Reaction with Microbial L Forms

Study of potential pathogenicity of microbial L forms was done by the localized Shwartzman reaction. Stable L forms of Proteus mirabilis served as skin preparation in rabbits for induction of Shwartzman reaction by subsequent intravenous injection of either P. mirabilis L forms or Escherichia coli endotoxin. The intensity of the reaction was positively correlated to numbers of L forms in the skin. L forms also served as the intravenous challenge. In vivo multiplication of L forms was not a prerequisite for the reaction, as it could be produced with nonviable, osmotically lysed L forms. The reaction produced with L forms in the skin was more intense than that produced with the parent bacterial form. These latter observations, coupled with the demonstration that L forms disappeared from the skin (lysed?) after 4 hr, in contrast to bacteria which were recoverable for 72 hr (duration of study), suggest release of endotoxin by L forms as a pathogenic mechanism.     

Local Schwartzman phenomenon in axenic rabbits]

Local Schwartzman phenomenon was produced by coli-endotoxin in all the germfree rabbits tested (11 in all) at the age of 102 to 135 days. Any kinds of natural antibodies were not detected in sera of the rabbits, which in fact were found to be agammaglobulinaemic in most cases, as revealed by immunoelectrophoresis. These facts suggested that the germfree rabbits utilized here had not been sensitized to bacterial endotoxins. From the results obtained here, it may be concluded that the existence of hypersensitivity to endotoxin is not necessary to the production of local Schwartzman phenomenon by bacterial endotoxin.     

Platelet Requirement in the Interaction of the Complement and Clotting Systems

THE study of immunological reactions in rabbits genetically deficient in the sixth component of complement (C6) can provide information about the biological role of the complement system. Since their discovery¹, these rabbits have been found to produce no C6 protein and they can form antibody against it (ref. 2 and review in ref. 3). Furthermore, although the plasma and serum of these rabbits contain the normal coagulation factors, the whole blood clotting time is longer than normal⁴.     

Adjuvant Action of Capsular Polysaccharide of Klebsiella pneumoniae on Antibody Response

The neutral fraction (neutral CPS-K) of Klebsiella pneumoniae capsular polysaccharide (CPS-K) from type 1, Kasuya strain, has already been reported as the active substance responsible for the strong adjuvant effect of CPS-K. The present results demonstrate that neutral CPS-K exhibits further common biological activities with lipopolysaccharide (LPS) isolated from Salmonella enteritidis. The intensity of the lethality in mice of neutral CPS-K by the intraperitoneal route is very similar to that of LPS. Its lethality for mice by the intravenous (i.v.) route is significantly stronger than that of LPS, because the degree of increase in the sensitivity to their lethality by i.v. challenge is smaller for LPS than for neutral CPS-K. The intensity of the pyrogenicity of neutral CPS-K in rabbits is approximately one-tenth of that of LPS as judged by the minimal pyrogenic doses and fever indices. The skin-preparatory potency of neutral CPS-K for the dermal Shwartzman phenomenon in rabbits is also approximately one-tenth of that of LPS compared on the basis of the minimal skin-preparatory doses. When injected i.v., neutral CPS-K exhibits a provocative effect on hemorrhagic reactions in skin sites prepared with neutral CPS-K or LPS.     

Rheological study on coagulation of blood with special reference to the triggering mechanism of venous thrombus formation

A markedly reduced blood flow, an elevation of hematocrit and an increased aggregability of erythrocytes [red blood cells (RBCs)] are risk factors for venous thrombus formation (intravascular blood coagulation). However, these risk factors alone seem to be insufficient to stimulate the coagulation cascade in the absence of a primary triggering mechanism. In this paper, our rheological and biochemical studies on blood coagulation, especially focusing on procoagulant activity of RBCs, are summarized. It is shown that the intrinsic coagulation pathway is triggered by the activation of factor IX (F-IX) by RBCs. The F-IX-activating enzyme in normal human erythrocyte (RBC) membranes was purified, identified and characterized. The activation of F-IX by RBCs was enhanced by a decrease in flow shear rate and an elevation in hematocrit. The procoagulant ability of RBCs and coagulation of blood obtained from individuals with a relatively high level of hypercoagulability were enhanced compared with those for normals. The studies demonstrated a new triggering mechanism for coagulation or thrombus formation that may occur under stagnant flow conditions.