Immunosuppressive action of the lethal toxin of Bacillus anthracis in mice

In experiments on inbred mice infected with B. anthracis capsular strain 71/12 of Tsenkovsky's second vaccine B. anthracis lethal toxin introduced in mixture with spores has been shown to aggravate anthrax infection in CBA mice susceptible to anthrax, while producing a faint effect on the infectious process in BALB mice with hereditary resistance to anthrax. B. anthracis purified edema toxin has been found to produce a weaker aggravating effect with respect to anthrax infection than the lethal toxin. As revealed in these experiments, the capacity of the lethal toxin to suppress the activity of peritoneal macrophages in vitro is the more pronounced, the more resistant to anthrax are the mice used as the donors of these macrophages. The mechanism of hereditary immunity which may ensure resistance to infection in the presence of immunosuppression is discussed.     

Morphological abnormalities, neonatal mortality, and reproductive abnormalities in mice transgenic for diphtheria toxin genes that are driven by the promoter for adipocyte lipid binding protein.

Transgenic mice were used in an experiment that was designed to serve as a model of a possible approach to reducing the amount of carcass fat in meat animals. The objective was to reduce the number of adipocytes in transgenic mice thereby restricting the capacity to accumulate lipid. Our approach employed the technique of genetic ablation. The promoter for the adipocyte lipid binding protein gene was used in an attempt to direct expression of diphtheria toxin genes specifically to adipocytes. Three diphtheria toxin genes were used; they encode, respectively, an extremely cytotoxic wild type toxin, a less toxic attenuated toxin, and a nonfunctional toxin. While it was not possible to accurately assess effects of the transgenes on lipid accumulation, several informative observations were noted. A large percentage of transgenic founder mice that harbor either wild type or attenuated toxin genes are morphologically abnormal, die as neonates, or exhibit reproductive abnormalities including sterility or failure to transmit the transgene to offspring. In contrast, mice that harbor the nonfunctional toxin gene or are nontransgenic rarely have these same abnormalities. These results suggest that the transgenic mice are expressing the transgenes in cells other than adipocytes and that the aberrant production of functional toxin is responsible for the congenital abnormalities. The production of morphological and reproductive abnormalities in transgenic animals should be useful for investigating normal developmental processes.     

Morphological abnormalities,neonatal mortality,and reproductive abnormalities in mice transgenic for diphtheria toxin genes that are driven by the promoter for adipocyte lipid binding protein

Transgenic mice were used in an experiment that was designed to serve as a model of a possible approach to reducing the amount of carcass fat in meat animals. The objective was to reduce the number of adipocytes in transgenic mice thereby restricting the capacity to accumulate lipid. Our approach employed the technique of genetic ablation. The promoter for the adipocyte lipid binding protein gene was used in an attempt to direct expression of diphtheria toxin genes specifically to adipocytes. Three diphtheria toxin genes were used; they encode, respectively, an extremely cytotoxic wild type toxin, a less toxic attenuated toxin, and a nonfunctional toxin. While it was not possible to accurately assess effects of the transgenes on lipid accumulation, several informative observations were noted. A large percentage of transgenic founder mice that harbor either wild type or attenuated toxin genes are morphologically abnormal, die as neonates, or exhibit reproductive abnormalities including sterility or failure to transmit the transgene to offspring. In contrast, mice that harbor the nonfunctional toxin gene or are nontransgenic rarely have these same abnormalities. These results suggest that the trans-genic mice are expressing the transgenes in cells other than adipocytes and that the aberrant production of functional toxin is responsible for the congenital abnormalities. The production of morphological and reproductive abnormalities in transgenic animals should be useful for investigating normal developmental processes.     

Induction of an immune response in athymic nude mice to thymus-dependent antigens by Pseudomonas aeruginosa exotoxin A

Exposure of spleen cells from athymic nude mice to Pseudomonas aeruginosa exotoxin A induces these cells to respond to the thymus-dependent (TD) antigen sheep erythrocytes (SRBC). The response induced by toxin is dose dependent, antigen specific, and not due to polyclonal B-cell activation. Enhancement of the anti-SRBC response can be observed when toxin addition precedes antigen stimulation by 24-48 hr, which decreases when toxin administration follows antigen stimulation. A significant response is also observed when toxin and antigen are added simultaneously. A significant anti-SRBC response can be observed out to Day 10 postantigen and toxin stimulation after attaining a peak response at Day 5. Cultures exposed to toxin in the presence or absence of antigen exhibited a higher cell number and relative number of B cells as compared to control cultures. Exposure of T-cell depleted B cells from euthymic +/nu mice to toxin plus antigen does not result in an anti-SRBC response indicating that exotoxin A alone is not sufficient to induce B-cell responsiveness to T-dependent antigens and that other cells and/or factors are involved in the toxin-induced responsiveness of nude mice to T-dependent antigens.     

A comparison of toxins isolated from the cyanobacteria Oscillatoria agardhii and Microcystis aeruginosa

1. A toxin isolated from a strain of Oscillatoria agardhii var. was compared to a peptide toxin isolated from Microcystis aeruginosa. 2. The Oscillatoria toxin possessed similar hepatotoxic properties on mice as the Microcystis toxin but had a higher LD50 than the latter; 320 micrograms/kg compared to 43 micrograms/kg (i.p. mouse), respectively. 3. Ultra-violet and infra-red spectra showed that the Oscillatoria toxin is a peptide which is not identical to the Microcystis toxin. 4. The spectra also indicated some structural similarities in these toxins.     

Analysis of Pulmonary Inflammation and Function in the Mouse and Baboon after Exposure to Mycoplasma pneumoniae CARDS Toxin

Mycoplasma pneumoniae produces an ADP-ribosylating and vacuolating toxin known as the CARDS (Community Acquired Respiratory Distress Syndrome) toxin that has been shown to be cytotoxic to mammalian cells in tissue and organ culture. In this study we tested the ability of recombinant CARDS (rCARDS) toxin to elicit changes within the pulmonary compartment in both mice and baboons. Animals responded to a respiratory exposure to rCARDS toxin in a dose and activity-dependent manner by increasing the expression of the pro-inflammatory cytokines IL-1α, 1β, 6, 12, 17, TNF-α and IFN-γ. There was also a dose-dependent increase in several growth factors and chemokines following toxin exposure including KC, IL-8, RANTES, and G-CSF. Increased expression of IFN-γ was observed only in the baboon; otherwise, mice and baboons responded to CARDS toxin in a very similar manner. Introduction of rCARDS toxin to the airways of mice or baboons resulted in a cellular inflammatory response characterized by a dose-dependent early vacuolization and cytotoxicity of the bronchiolar epithelium followed by a robust peribronchial and perivascular lymphocytic infiltration. In mice, rCARDS toxin caused airway hyper-reactivity two days after toxin exposure as well as prolonged airway obstruction. The changes in airway function, cytokine expression, and cellular inflammation correlate temporally and are consistent with what has been reported for M. pneumoniae infection. Altogether, these data suggest that the CARDS toxin interacts extensively with the pulmonary compartment and that the CARDS toxin is sufficient to cause prolonged inflammatory responses and airway dysfunction.     

Anthrax toxin targeting of myeloid cells through the CMG2 receptor is essential for establishment of Bacillus anthracis infections in mice

Bacillus anthracis kills through a combination of bacterial infection and toxemia. Anthrax toxin working via the CMG2 receptor mediates lethality late in infection, but its roles early in infection remain unclear. We generated myeloid-lineage specific CMG2-deficient mice to examine the roles of macrophages, neutrophils, and other myeloid cells in anthrax pathogenesis. Macrophages and neutrophils isolated from these mice were resistant to anthrax toxin. However, the myeloid-specific CMG2-deficient mice remained fully sensitive to both anthrax lethal and edema toxins, demonstrating that targeting of myeloid cells is not responsible for anthrax toxin-induced lethality. Surprisingly, the myeloid-specific CMG2-deficient mice were completely resistant to B. anthracis infection. Neutrophil depletion experiments suggest that B. anthracis relies on anthrax toxin secretion to evade the scavenging functions of neutrophils to successfully establish infection. This work demonstrates that anthrax toxin uptake through CMG2 and the resulting impairment of myeloid cells are essential to anthrax infection.     

Essential involvement of IFN-gamma in Clostridium difficile toxin A-induced enteritis

Clostridium difficile has emerged as the important causative agent of antibiotics-associated pseudomembranous colitis; especially its toxin A is presumed to be responsible for the colitis. We examined the pathophysiological roles of IFN-gamma in toxin A-induced enteritis using IFN-gamma knockout (KO) mice. When toxin A of C. difficile was injected into the ileal loops of BALB/c wild-type (WT) mice, massive fluid secretion, disruption of intestinal epithelial structure, and massive neutrophil infiltration developed within 4 h after the injection. IFN-gamma protein was faintly detected in some CD3-positive lymphocytes in the lamina propria and submucosa of the ileum of untreated WT mice. On the contrary, at 2 and 4 h after toxin A injection, IFN-gamma protein was detected in infiltrating neutrophils and to a lesser degree in CD3-positive lymphocytes. In the ileum of WT mice, toxin A treatment markedly enhanced the gene expression of TNF-alpha, macrophage inflammatory protein-1alpha and -2, KC, and ICAM-1 >2 h after treatment. In contrast, the histopathological changes were marginal, without enhanced fluid secretion in the ileum of toxin A-treated IFN-gamma KO mice. Moreover, toxin A-induced gene expression of TNF-alpha, neutrophil chemotactic chemokines, and ICMA-1 was remarkably attenuated in IFN-gamma KO mice. Furthermore, pretreatment of WT mice with a neutralizing anti-IFN-gamma Ab prevented toxin A-induced enteritis. These observations indicate that IFN-gamma is the crucial mediator of toxin A-induced acute enteritis and suggest that IFN-gamma is an important molecular target for the control of C. difficile-associated pseudomembranous colitis.     

Transgenic mice expressing the diphtheria toxin receptor are sensitive to the toxin

Whereas diphtheria and the mechanism of action of diphtheria toxin, the bacterial molecule that induces the disease, have been studied and understood for some time, the receptor that allows animal cells to bind the toxin escaped identification until recently. The receptor was identified by its ability to confer toxin-sensitivity to mouse cells, which are normally toxin-resistant. Although mice are also naturally resistant, we now demonstrate that transgenic mice expressing the diphtheria toxin receptor are as sensitive to the toxin as are humans and other toxin-sensitive animals. These transgenic mice provide a suitable model for studying modern antidotes for diphtheria.     

Platelet-activating Factor Contributes to Bacillus anthracis Lethal Toxin-associated Damage

The lethal toxin (LeTx) of Bacillus anthracis plays a central role in the pathogenesis of anthrax-associated shock. Platelet-activating factor (PAF) is a potent lipid mediator that has been implicated in endotoxin-associated shock. In this study, we examined the contribution of PAF to the manifestations of lethal toxin challenge in WT mice. LeTx challenge resulted in transient increase in serum PAF levels and a concurrent decrease in PAF acetylhydrolase activity. Inhibition of PAF activity using PAF antagonists or toxin challenge of PAF receptor negative mice reversed or ameliorated many of the pathologic features of LeTx-induced damage, including changes in vascular permeability, hepatic necrosis, and cellular apoptosis. In contrast, PAF inhibition had minimal effects on cytokine levels. Findings from these studies support the continued study of PAF antagonists as potential adjunctive agents in the treatment of anthrax-associated shock.     

In vitro formation of 3'-hydroxy T-2 and 3'-hydroxy HT-2 toxins from T-2 toxin by liver homogenates from mice and monkeys.

In vitro metabolism of T-2 toxin was studied in homogenates of mouse and monkey livers. In addition to several hydrolyzed products, including HT-2 toxin, neosolaniol, 4-deacetylneosolaniol, 15-deacetylneosolaniol, and T-2 tetraol, two metabolic products were isolated from the incubation mixture. Their structures were confirmed as 3'-hydroxy T-2 toxin and 3'-hydroxy HT-2 toxin on the basis of mass and nuclear magnetic resonance spectroscopy. The formation of these hydroxylated metabolites was found in the microsomes in the presence of NADPH, and the hydroxylation reaction was enhanced by treating mice with phenobarbital. The results suggest that a cytochrome P-450 is catalyzing the hydroxylation at the C-3' position of T-2 and HT-2 toxins. An in vitro metabolic pathway of T-2 toxin in the hepatic homogenates containing the NADPH-generating system is proposed.     

Staphylococcal enterotoxin B toxicity in BALB/c mice: Effect on T-cells, plasma cytokine levels and biochemical markers

Abstract Groups of BALB/c mice were treated with a sub-lethal dose (60 μg) of staphylococcal enterotoxin B (SEB) intraperitoneally and were sacrificed at 2, 5, 8, or 10 h post-injection. Organ, blood plasma and lymph node samples from these mice were analyzed. Plasma levels of urea, creatinine and alanine aminotransferase were significantly raised above normal by 5 h post-injection. However, alkaline phosphatase levels showed an erratic increase after toxin administration and, after administration of 10–40 μg SEB per mouse, were consistently at least 30% below normal levels at 24 h post-injection. Weight change was also monitored but found to be inconsistent. Lung, spleen and kidney samples appeared normal on histopathological examination, but liver samples showed minor polymorph infiltration and congestion. TNF-α, and IL-1 α levels in the plasma were raised by 8 h to picogram levels per ml of plasma, whereas IFN-γ and IL-2 were raised by 2 h to nanogram levels per ml of plasma. Lymph node cells taken from mice treated with toxin were given a secondary stimulation with toxin in vitro. Although the response of the cells was lower than normal on assay at four days, a time response curve showed a peak in cell responsiveness to secondary stimulation with toxin at three days. These data indicate that biochemical markers and cytokine levels are affected by the administration of SEB to mice and may be used as indicators of toxicity.     

Ultrastructural changes in β-cells of pancreatic islets in α-amanitin-poisoned Mice

In mice poisoned by alpha-amanitin nuclear changes typical of this toxin were observed in beta-cells of pancreatic islets. The lesions became progressively more severe and at 48 h after toxin injection some cells were necrotic. The damage to these cells could have implications in the changes in glycogen metabolism which occur after alpha-aminitin poisoning.     

Binding and cytotoxic effects of Clostridium botulinum type A, C1 and E toxins in primary neuron cultures from foetal mouse brains

Binding of purified Clostridium botulinum type A, C1 and E toxins to cultured cells was studied by an immunocytochemical method. Type A and C1 toxins bound strongly to neuron cultures prepared from brains of foetal mice, but binding of type E toxin was weak. None of the toxin types bound to the feeder layer, composed of non-neuronal cells. The heavy-chain component of the type C1 toxin bound to neurons, but the light chain component did not. Type C1 toxin also bound only to cell lines of neuronal origin. When type C1 toxin [final concentration 4 x 10(2) LD50 (10 ng) per well] was added to primary neuron cultures in 96-well plates, degeneration of neuronal processes and rounding of neuronal somas were observed, but type A and E toxins did not produce such changes. The binding and cytotoxic activities of type C1 toxin were blocked by heat treatment (80 degrees C for 30 min) or by preincubation of the toxin with polyclonal anti-C1 IgG and some of the monoclonal antibodies which neutralized the toxin activity in mice. In the neuronal processes treated with C1 toxin, many degenerated mitochondria, membranous dense bodies and vesicles were observed by electron microscopy; these ultrastructural changes were similar to those of Wallerian degeneration in vivo.     

Evaluation of hypoxic state in experimental animals induced by Yersinia pestis antigens

Some characteristics of purine metabolism in experimental animals (white mice, clawed jirds and guinea pigs), injected intraperitoneally with Y. pestis "murine" toxin and capsular antigen (Fraction 1), were studied. Under the influence of sublethal doses of these antigens increased levels of guanine and xanthine in blood were noted. Changes in the content of xanthine oxidase in cells were insignificant. In white mice and clawed jirds the activity of succinate dehydrogenase decreased under the action of "murine" toxin and increased after the injection of Fraction 1.     

Bacterial and plant enterotoxin B subunit-autoantigen fusion proteins suppress diabetes insulitis

Several bacterial and plant enterotoxin B subunit-islet autoantigen fusion proteins were compared for their ability to serve as islet autoantigen carriers and adjuvants for reduction of pancreatic islet inflammation associated with type 1 diabetes. The cholera toxin B subunit (CTB), the heat-labile toxin B subunit from enterotoxigenic Escherichia coli (LTB), the Shigella toxin B subunit (STB), and the plant toxin ricin B subunit (RTB) were genetically linked to the islet autoantigens proinsulin (INS) and glutamic acid decarboxylase (GAD). The adjuvant-autoantigen gene fusions were transferred to a bacterial expression vector and the corresponding fusion proteins synthesized in E. coli. The purified adjuvant-autoantigen proteins were fed to 5-wk-old nonobese diabetic (NOD) mice once a week for 4 wk. Histological examination of pancreatic islets isolated from inoculated mice showed significant levels of insulitis reduction in comparison with uninoculated mice. The ratio of serum anti-INS and anti-GAD IgG_2c to IgG₁ antibody isotype titers increased in all ligand-autoantigen inoculated animal groups, suggesting an increase in effector Th2 lymphocytes in B subunit-mediated insulitis suppression. The results of these experiments indicate that bacterial and plant enterotoxin B subunit ligand-autoantigens enhance insulitis reduction in NOD mice. This research prompts further exploration of a multiadjuvant/autoantigen co-delivery strategy that may facilitate type 1 diabetes prevention and suppression in animals and humans.     

Neutrophil elastase mediates pathogenic effects of anthrax lethal toxin in the murine intestinal tract

Neutrophils isolated from BALB/c or C57BL/6 mice and treated in vitro with anthrax lethal toxin release bioactive neutrophil elastase, a proinflammatory mediator of tissue destruction. Similarly, neutrophils isolated from mice treated with anthrax lethal toxin in vivo and cultured ex vivo release greater amounts of elastase than neutrophils from vehicle-treated controls. Direct measurements from murine intestinal tissue samples demonstrate an anthrax lethal toxin-dependent increase in neutrophil elastase activity in vivo as well. These findings correlate with marked lethal toxin-induced intestinal ulceration and bleeding in neutrophil elastase(+/+) animals, but not in neutrophil elastase(-/-) animals. Moreover, neutrophil elastase(-/-) mice have a significant survival advantage over neutrophil elastase(+/+) animals following exposure to anthrax lethal toxin, thereby establishing a key role for neutrophil elastase in mediating the deleterious effects of anthrax lethal toxin.     

Clostridium spiroforme toxin is a binary toxin which ADP-ribosylates cellular actin

We have purified from Clostridium spiroforme strain 246 an heterogeneous population of proteins (Sa) ranging from 43 to 47 kilodaltons exhibiting ADP-ribosyl transferase activity as do C. botulinum C2 toxin component I or the ia chain of C. perfringens E iota toxin. C. spiriforme Sa had alone no activity upon injection in mice or inoculated to Vero cells. When spiroforme ADP ribosyl transferase were mixed with a trypsin activated protein (Sb) separated from C. spiroforme bacterial supernatant, a lethal effect in mice and cytotoxicity on Vero cells were recorded. The Sa cross-reacted immunologically with either the light chain of C. perfringens E iota toxin or the ADP-ribosyl transferase from C. difficile 196 strain. No immunological relatedness was observed between Sa and C2 toxin component I. C. spiroforme toxin is thus another binary toxin close to iota.     

Neutralising antibodies against ricin toxin

The Centers for Disease Control and Prevention have listed the potential bioweapon ricin as a Category B Agent. Ricin is a so-called A/B toxin produced by plants and is one of the deadliest molecules known. It is easy to prepare and no curative treatment is available. An immunotherapeutic approach could be of interest to attenuate or neutralise the effects of the toxin. We sought to characterise neutralising monoclonal antibodies against ricin and to develop an effective therapy. For this purpose, mouse monoclonal antibodies (mAbs) were produced against the two chains of ricin toxin (RTA and RTB). Seven mAbs were selected for their capacity to neutralise the cytotoxic effects of ricin in vitro. Three of these, two anti-RTB (RB34 and RB37) and one anti-RTA (RA36), when used in combination improved neutralising capacity in vitro with an IC(50) of 31 ng/ml. Passive administration of association of these three mixed mAbs (4.7 μg) protected mice from intranasal challenges with ricin (5 LD(50)). Among those three antibodies, anti-RTB antibodies protected mice more efficiently than the anti-RTA antibody. The combination of the three antibodies protected mice up to 7.5 hours after ricin challenge. The strong in vivo neutralising capacity of this three mAbs combination makes it potentially useful for immunotherapeutic purposes in the case of ricin poisoning or possibly for prevention.     

Specificity and detection limit of a dermal temperature histamine sensitization test for absence of residual pertussis toxin in vaccines

Currently, an assay based on fatal sensitization of mice to histamine challenge is widely used for testing absence of residual pertussis toxin in acellular pertussis containing vaccines. For replacement of this lethal end-point assay, an alternative method based on body temperature measurement in mice has been presented, and in this study the specificity and detection limit of a dermal temperature-based assay were assessed. Test preparations containing pertussis toxin were prepared in aluminum-adjuvanted pertussis toxoid vaccine and injected intraperitoneally in histamine sensitive mice. Later the mice were challenged with histamine and the pertussis toxin-induced decrease in dermal temperature recorded. By comparison of mice treated with pertussis toxoid vaccine spiked with pertussis toxin with mice treated with pertussis toxoid vaccine alone, the assay gave a response that specifically could detect presence of pertussis toxin. The acellular pertussis containing vaccine did not interfere with the pertussis toxin-induced temperature response recorded. In tests for presence of pertussis toxin in the pertussis vaccine preparation, the detection limit of the assay was estimated to approximately 5 ng pertussis toxin per human dose of pertussis toxoid. The dermal temperature-based assay was found to be a valid method to be applied in routine quality control of vaccines.