Nociceptive Sensory Neurons Mediate Inflammation Induced by Bacillus Anthracis Edema Toxin.

Bacterial products are able to act on nociceptive neurons during pathogenic infection. Neurogenic inflammation is an active part of pain signaling and has recently been shown to impact host-pathogen defense. Bacillus anthracis Edema Toxin (ET) produces striking edema in peripheral tissues, but the cellular mechanisms involved in tissue swelling are not completely understood. Here, we find that nociceptive neurons play a role in ET-induced edema and inflammation in mice. Subcutaneous footpad infection of B. anthracis Sterne caused ET-dependent local mechanical allodynia, paw swelling and body weight gain. Subcutaneous administration of ET induced paw swelling and vascular leakage, the early phases of which were attenuated in the absence of Trpv1+ or Nav1.8+ nociceptive neurons. Nociceptive neurons express the anthrax toxin receptor ANTXR2, but this did not mediate ET-induced edema. ET induced local cytokine expression and neutrophil recruitment, which were dependent in part on Trpv1+ nociceptive neurons. Ablation of Trpv1+ or Nav1.8+ nociceptive neurons also attenuated early increases in paw swelling and body weight gain during live B. anthracis infection. Our findings indicate that nociceptive neurons play an active role in inflammation caused by B. anthracis and Edema Toxin to potentially influence bacterial pathogenesis.

Anthrax Exposure, Belief in Exposure, and Postanthrax Symptoms Among Survivors of a Bioterrorist Attack on Capitol Hill.

BACKGROUND: Following chemical, biological, radiological, and nuclear disasters, medically unexplained symptoms have been observed among unexposed persons. OBJECTIVES: This study examined belief in exposure in relation to postdisaster symptoms in a volunteer sample of 137 congressional workers after the 2001 anthrax attacks on Capitol Hill. METHODS: Postdisaster symptoms, belief in exposure, and actual exposure status were obtained through structured diagnostic interviews and self-reported presence in offices officially designated as exposed through environmental sampling. Multivariate models were tested for associations of number of postdisaster symptoms with exposure and belief in exposure, controlling for sex and use of antibiotics. RESULTS: The sample was divided into 3 main subgroups: exposed, 41%; unexposed but believed they were exposed, 17%; and unexposed and did not believe that they were exposed, 42%. Nearly two-thirds (64%) of the volunteers reported experiencing symptoms after the anthrax attacks. Belief in anthrax exposure was significantly associated with the number of ear/nose/throat, musculoskeletal, and all physical symptoms. No significant associations were found between anthrax exposure and the number of postdisaster symptoms. CONCLUSIONS: Given the high incidence of these symptoms, these data suggest that even in the absence of physical injury or illness, there may be surges in health care utilization. (Disaster Med Public Health Preparedness. 2019;13:555-560).

Role of a Small Molecule in the Modulation of Cell Death Signal Transduction Pathways.

Inflammasomes activate caspase-1 in response to molecular signals from pathogens and other dangerous stimuli as a part of the innate immune response. A previous study discovered a small-molecule, 4-fluoro- N'-[1-(2-pyridinyl)ethylidene]benzohydrazide, which we named DN1, that reduces the cytotoxicity of anthrax lethal toxin (LT). We determined that DN1 protected cells irrespectively of LT concentration and reduced the pathogenicity of an additional bacterial exotoxin and several viruses. Using the LT cytotoxicity pathway, we show that DN1 does not prevent LT internalization and catalytic activity or caspase-1 activation. Moreover, DN1 does not affect the proteolytic activity of host cathepsin B, which facilitates the cytoplasmic entry of toxins. PubChem Bioactivities lists two G protein-coupled receptors (GPCR), type-1 angiotensin II receptor and apelin receptor, as targets of DN1. The inhibition of phosphatidylinositol 3-kinase, phospholipase C, and protein kinase B, which are downstream of GPCR signaling, synergized with DN1 in protecting cells from LT. We hypothesize that DN1-mediated antagonism of GPCRs modulates signal transduction pathways to induce a cellular state that reduces LT-induced pyroptosis downstream of caspase-1 activation. DN1 also reduced the susceptibility of Drosophila melanogaster to toxin-associated bacterial infections. Future experiments will aim to further characterize how DN1 modulates signal transduction pathways to inhibit pyroptotic cell death in LT-sensitive macrophages. DN1 represents a novel chemical probe to investigate host cellular mechanisms that mediate cell death in response to pathogenic agents.

The Potential Pathogenic Contributions of Endothelial Barrier and Arterial Contractile Dysfunction to Shock Due to B. anthracis Lethal and Edema Toxins.

Shock with B. anthracis infection is particularly resistant to conventional cardiovascular support and its mortality rate appears higher than with more common bacterial pathogens. As opposed to many bacteria that lack exotoxins directly depressing hemodynamic function, lethal and edema toxin (LT and ET respectively) both cause shock and likely contribute to the high lethality rate with B. anthracis. Selective inhibition of the toxins is protective in infection models, and administration of either toxin alone in animals produces hypotension with accompanying organ injury and lethality. Shock during infection is typically due to one of two mechanisms: (i) intravascular volume depletion related to disruption of endothelial barrier function; and (ii) extravasation of fluid and/or maladaptive dilation of peripheral resistance arteries. Although some data suggests that LT can produce myocardial dysfunction, growing evidence demonstrates that it may also interfere with endothelial integrity thereby contributing to the extravasation of fluid that helps characterize severe B. anthracis infection. Edema toxin, on the other hand, while known to produce localized tissue edema when injected subcutaneously, has potent vascular relaxant effects that could lead to pathologic arterial dilation. This review will examine recent data supporting a role for these two pathophysiologic mechanisms underlying the shock LT and ET produce. Further research and a better understanding of these mechanisms may lead to improved management of B. anthracis in patients.

Using Telemetry Data to Refine Endpoints for New Zealand White Rabbits Challenged with Bacillus anthracis.

We used a continuous-monitoring digital telemetry system to investigate temperature response in New Zealand White rabbits after inhalation or subcutaneous challenge with Bacillus anthracis. Two spore preparations of B. anthracis Ames A2084 were evaluated by using a nose-only inhalation model, and 2 strains, B. anthracis Ames A2084 and B. anthracis UT500, were evaluated in a subcutaneous model. Animal body temperature greater than 3 SD above the mean baseline temperature was considered a significant increase in body temperature (SIBT). All rabbits that exhibited SIBT after challenge by either route of infection or bacterial strain eventually died or were euthanized due to infection, and all rabbits that died or were euthanized due to infection exhibited SIBT during the course of disease. The time at onset of SIBT preceded clinical signs of disease in 94% of the rabbits tested by as long as 2 days. In addition, continuous temperature monitoring facilitated discrimination between the 2 B. anthracis strains with regard to the time interval between SIBT and death. These data suggest that for the New Zealand White rabbit anthrax model, SIBT is a reliable indicator of infection, is predictive of experimental outcome in the absence of treatment, and is measurable prior to the appearance of more severe signs of disease. The use of digital telemetry to monitor infectious disease course in animal models of anthrax can potentially be used in conjunction with other clinical score metrics to refine endpoint euthanasia criteria.

Model Systems for Pulmonary Infectious Diseases: Paradigms of Anthrax and Tuberculosis.

Robert Koch utilized animal model systems to put forward his postulates while discovering the etiological agents of anthrax and tuberculosis, Bacillus anthracis and Mycobacterium tuberculosis, respectively. After more than 130 years, we have achieved limited success towards understanding these two pestilences, which have propagated as scourge against humans. B. anthracis and M. tuberculosis are diverse organisms, which share a common evolutionary path in tropics. They adapt unique strategies to overcome unfavorable conditions and surpass the host defense mechanisms. B. anthracis is an endospore forming bacteria that primarily acts by releasing toxins in the host cells.. M. tuberculosis is an intracellular bacteria that resides within the host macrophages by blocking phagosome-lysosome fusion events and ensuring its own survival. The bacterium can remain dormant for long periods, and when activated, it spreads in lungs and other extrapulmonary sites leading to formation of necrotic granulomas. The two diseases are immunologically distinct examples of inducing primarily either humoral or cell mediated immunity. Natural immune response to the two diseases probably explains early success achieved with the anthrax vaccine, while the hunt for successful tuberculosis prevention is still on. For comprehensive understanding of these diseases, model systems are of utmost importance that can alleviate detailed assessment of disease etiology and introductory treatment regimes. In this review, we discuss the various in vitro and in vivo model systems used to study these two diseases, discussing their contributions and recent themes.

Soluble P-selectin rescues mice from anthrax lethal toxin-induced mortality through PSGL-1 pathway-mediated correction of hemostasis.

As one of the virulence factors of Bacillus anthracis, lethal toxin (LT) induces various pathogenic responses including the suppression of the coagulation system. In this study, we observed that LT markedly increased the circulating soluble P-selectin (sP-sel) levels and microparticle (MP) count in wild-type but not P-selectin (P-sel, Selp-/-) or P-sel ligand-1 (PSGL-1, Selplg-/-) knockout mice. Because sP-sel induces a hypercoagulable state through PSGL-1 pathway to generate tissue factor-positive MPs, we hypothesized that the increase in plasma sP-sel levels can be a self-rescue response in hosts against the LT-mediated suppression of the coagulation system. In agreement with our hypothesis, our results indicated that compared with wild-type mice, Selp-/- and Selplg-/- mice were more sensitive to LT. In addition, the recombinant sP-sel treatment markedly ameliorated LT-mediated pathogenesis and reduced mortality. As a result, elicitation of circulating sP-sel is potentially a self-rescue response, which is beneficial to host recovery from an LT-induced hypocoagulation state. These results suggest that the administration of sP-sel is likely to be useful in the development of a new strategy to treat anthrax.

Identifying Meningitis During an Anthrax Mass Casualty Incident: Systematic Review of Systemic Anthrax Since 1880.

BACKGROUND: Bacillus anthracis, the causative agent of anthrax, is a potential bioterrorism agent. Anthrax meningitis is a common manifestation of B. anthracis infection, has high mortality, and requires more aggressive treatment than anthrax without meningitis. Its rapid identification and treatment are essential for successful management of an anthrax mass casualty incident. METHODS: Three hundred six published reports from 1880 through 2013 met predefined inclusion criteria. We calculated descriptive statistics for abstracted cases and conducted multivariable regression on separate derivation and validation cohorts to identify clinical diagnostic and prognostic factors for anthrax meningitis. RESULTS: One hundred thirty-two of 363 (36%) cases with systemic anthrax met anthrax meningitis criteria. Severe headache, altered mental status, meningeal signs, and other neurological signs at presentation independently predicted meningitis in the derivation cohort and were tested as a 4-item assessment tool for use during anthrax mass casualty incidents. Presence of any 1 factor on admission had a sensitivity for finding anthrax meningitis of 89% (83%) in the adult (pediatric) validation cohorts. Anthrax meningitis was unlikely in the absence of any of these signs or symptoms (likelihood ratio [LR]- = 0.12 [0.19] for adult [pediatric] cohorts), while presence of 2 or more made meningitis very likely (LR+ = 26.5 [30.0]). Survival of anthrax meningitis was predicted by treatment with a bactericidal agent (P = .005) and use of multiple antimicrobials (P = .01). CONCLUSIONS: We developed an evidence-based assessment tool for screening patients for meningitis during an anthrax mass casualty incident. Its use could improve both patient outcomes and resource allocation in such an event.

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.

Molecular determinants for a cardiovascular collapse in anthrax.

Bacillus anthracis releases two bipartite proteins, lethal toxin and edema factor, that contribute significantly to the progression of anthrax-associated shock. As blocking the anthrax toxins prevents disease, the toxins are considered the main virulence factors of the bacterium. The anthrax bacterium and the anthrax toxins trigger multi-organ failure associated with enhanced vascular permeability, hemorrhage and cardiac dysfunction in animal challenge models. A recent study using mice that either lacked the anthrax toxin receptor in specific cells and corresponding mice expressing the receptor in specific cell types demonstrated that cardiovascular cells are critical for disease mediated by anthrax lethal toxin. These studies are consistent with involvement of the cardiovascular system, and with an increase of cardiac failure markers observed in human anthrax and in animal models using B. anthracis and anthrax toxins. This review discusses the current state of knowledge regarding the pathophysiology of anthrax and tries to provide a mechanistic model and molecular determinants for the circulatory shock in anthrax.

B. anthracis associated cardiovascular dysfunction and shock: the potential contribution of both non-toxin and toxin components.

The development of cardiovascular dysfunction and shock in patients with invasive Bacillus anthracis infection has a particularly poor prognosis. Growing evidence indicates that several bacterial components likely play important pathogenic roles in this injury. As with other pathogenic Gram-positive bacteria, the B. anthracis cell wall and its peptidoglycan constituent produce a robust inflammatory response with its attendant tissue injury, disseminated intravascular coagulation and shock. However, B. anthracis also produces lethal and edema toxins that both contribute to shock. Growing evidence suggests that lethal toxin, a metalloprotease, can interfere with endothelial barrier function as well as produce myocardial dysfunction. Edema toxin has potent adenyl cyclase activity and may alter endothelial function, as well as produce direct arterial and venous relaxation. Furthermore, both toxins can weaken host defense and promote infection. Finally, B. anthracis produces non-toxin metalloproteases which new studies show can contribute to tissue injury, coagulopathy and shock. In the future, an understanding of the individual pathogenic effects of these different components and their interactions will be important for improving the management of B. anthracis infection and shock.

A New Murine Model for Gastrointestinal Anthrax Infection.

The scientific community has been restricted by the lack of a practical and informative animal model of gastrointestinal infection with vegetative Bacillus anthracis. We herein report the development of a murine model of gastrointestinal anthrax infection by gavage of vegetative Sterne strain of Bacillus anthracis into the complement-deficient A/J mouse strain. Mice infected in this manner developed lethal infections in a dose-dependent manner and died 30 h-5 d following gavage. Histological findings were consistent with penetration and growth of the bacilli within the intestinal villi, with subsequent dissemination into major organs including the spleen, liver, kidney and lung. Blood cultures confirmed anthrax bacteremia in all moribund animals, with approximately 1/3 showing co-infection with commensal enteric organisms. However, no evidence of immune activation was observed during infection. Time-course experiments revealed early compromise of the intestinal epithelium, characterized by villus blunting and ulceration in the ileum and jejunum. A decrease in body temperature was most predictive of near-term lethality. Antibiotic treatment of infected animals 24 h following high-dose bacterial gavage protected all animals, demonstrating the utility of this animal model in evaluating potential therapeutics.

Infections of the developing world.

Access to critical care is rapidly growing in areas of the world where it was previously nonexistent and where infectious diseases often comprise the largest disease burden. Additionally, with crowding, mass migrations, and air travel, infectious diseases previously geographically confined are quickly spread across the planet, often in shorter time frames than disease incubation periods. Hence, critical care practitioners must be familiar with infectious diseases previously confined to the developing world. This article reviews selected tropical diseases that are seen in diverse locales and often require critical care services.

The sepsis model: an emerging hypothesis for the lethality of inhalation anthrax.

Inhalation anthrax is often described as a toxin-mediated disease. However, the toxaemia model does not account for the high mortality of inhalation anthrax relative to other forms of the disease or for the pathology present in inhalation anthrax. Patients with inhalation anthrax consistently show extreme bacteraemia and, in contrast to animals challenged with toxin, signs of sepsis. Rather than toxaemia, we propose that death in inhalation anthrax results from an overwhelming bacteraemia that leads to severe sepsis. According to our model, the central role of anthrax toxin is to permit the vegetative bacteria to escape immune detection. Other forms of B. anthracis infection have lower mortality because their overt symptoms early in the course of disease cause patients to seek medical care at a time when the infection and its sequelae can still be reversed by antibiotics. Thus, the sepsis model explains key features of inhalation anthrax and may offer a more complete understanding of disease pathology for researchers as well as those involved in the care of patients.

Pathology and pathophysiology of inhalational anthrax in a guinea pig model.

Nonhuman primates (NHPs) and rabbits are the animal models most commonly used to evaluate the efficacy of medical countermeasures against anthrax in support of licensure under the FDA's "Animal Rule." However, a need for an alternative animal model may arise in certain cases. The development of such an alternative model requires a thorough understanding of the course and manifestation of experimental anthrax disease induced under controlled conditions in the proposed animal species. The guinea pig, which has been used extensively for anthrax pathogenesis studies and anthrax vaccine potency testing, is a good candidate for such an alternative model. This study was aimed at determining the median lethal dose (LD50) of the Bacillus anthracis Ames strain in guinea pigs and investigating the natural history, pathophysiology, and pathology of inhalational anthrax in this animal model following nose-only aerosol exposure. The inhaled LD50 of aerosolized Ames strain spores in guinea pigs was determined to be 5.0 × 10(4) spores. Aerosol challenge of guinea pigs resulted in inhalational anthrax with death occurring between 46 and 71 h postchallenge. The first clinical signs appeared as early as 36 h postchallenge. Cardiovascular function declined starting at 20 h postexposure. Hematogenous dissemination of bacteria was observed microscopically in multiple organs and tissues as early as 24 h postchallenge. Other histopathologic findings typical of disseminated anthrax included suppurative (heterophilic) inflammation, edema, fibrin, necrosis, and/or hemorrhage in the spleen, lungs, and regional lymph nodes and lymphocyte depletion and/or lymphocytolysis in the spleen and lymph nodes. This study demonstrated that the course of inhalational anthrax disease and the resulting pathology in guinea pigs are similar to those seen in rabbits and NHPs, as well as in humans.

Activated protein C ameliorates Bacillus anthracis lethal toxin-induced lethal pathogenesis in rats.

BACKGROUND: Lethal toxin (LT) is a major virulence factor of Bacillus anthracis. Sprague Dawley rats manifest pronounced lung edema and shock after LT treatments, resulting in high mortality. The heart failure that is induced by LT has been suggested to be a principal mechanism of lung edema and mortality in rodents. Since LT-induced death occurs more rapidly in rats than in mice, suggesting that other mechanisms in addition to the heart dysfunction may be contributed to the fast progression of LT-induced pathogenesis in rats. Coagulopathy may contribute to circulatory failure and lung injury. However, the effect of LT on coagulation-induced lung dysfunction is unclear. METHODS: To investigate the involvement of coagulopathy in LT-mediated pathogenesis, the mortality, lung histology and coagulant levels of LT-treated rats were examined. The effects of activated protein C (aPC) on LT-mediated pathogenesis were also evaluated. RESULTS: Fibrin depositions were detected in the lungs of LT-treated rats, indicating that coagulation was activated. Increased levels of plasma D-dimer and thrombomodulin, and the ameliorative effect of aPC further suggested that the activation of coagulation-fibrinolysis pathways plays a role in LT-mediated pathogenesis in rats. Reduced mortality was associated with decreased plasma levels of D-dimer and thrombomodulin following aPC treatments in rats with LT-mediated pathogenesis. CONCLUSIONS: These findings suggest that the activation of coagulation in lung tissue contributes to mortality in LT-mediated pathogenesis in rats. In addition, anticoagulant aPC may help to develop a feasible therapeutic strategy.

Impaired function of the Tie-2 receptor contributes to vascular leakage and lethality in anthrax.

The anthrax lethal toxin (LT) enters host cells and enzymatically cleaves MAPKKs or MEKs. How these molecular events lead to death from anthrax remains poorly understood, but published reports suggest a direct effect of LT on vascular permeability. We have found that LT challenge in mice disrupts signaling through Tie-2, a tonically activated receptor tyrosine kinase in the endothelium. Genetic manipulations favoring Tie-2 activation enhanced interendothelial junctional contacts, prevented vascular leakage, and promoted survival following a lethal dose of LT. Cleavage of MEK1/2 was necessary for LT to induce endothelial barrier dysfunction, and activated Tie-2 signaled through the uncleaved fraction of MEKs to prevent LT's effects on the endothelium. Finally, primates infected with toxin-secreting Bacillus anthracis bacilli developed a rapid and marked imbalance in the endogenous ligands that signal Tie-2, similar to that seen in LT-challenged mice. Our results show that B. anthracis LT blunts signaling through Tie-2, thereby weakening the vascular barrier and contributing to lethality of the disease. Measurement of circulating Tie-2 ligands and manipulation of Tie-2 activity may represent future prognostic and therapeutic avenues for humans exposed to B. anthracis.

New insights into the biological effects of anthrax toxins: linking cellular to organismal responses.

The anthrax toxins lethal toxin (LT) and edema toxin (ET) are essential virulence factors produced by Bacillus anthracis. These toxins act during two distinct phases of anthrax infection. During the first, prodromal phase, which is often asymptomatic, anthrax toxins act on cells of the immune system to help the pathogen establish infection. Then, during the rapidly progressing (or fulminant) stage of the disease bacteria disseminate via a hematological route to various target tissues and organs, which are typically highly vascularized. As bacteria proliferate in the bloodstream, LT and ET begin to accumulate rapidly reaching a critical threshold level that will cause death even when the bacterial proliferation is curtailed by antibiotics. During this final phase of infection the toxins cause an increase in vascular permeability and a decrease in function of target organs including the heart, spleen, kidney, adrenal gland, and brain. In this review, we examine the various biological effects of anthrax toxins, focusing on the fulminant stage of the disease and on mechanisms by which the two toxins may collaborate to cause cardiovascular collapse. We discuss normal mechanisms involved in maintaining vascular integrity and based on recent studies indicating that LT and ET cooperatively inhibit membrane trafficking to cell-cell junctions we explore several potential mechanisms by which the toxins may achieve their lethal effects. We also summarize the effects of other potential virulence factors secreted by B. anthracis and consider the role of toxic factors in the evolutionarily recent emergence of this devastating disease.

Bacillus anthracis lethal toxin alters regulation of visceral sympathetic nerve discharge.

Bacillus anthracis infection is a pathophysiological condition that is complicated by progressive decreases in mean arterial pressure (MAP). Lethal toxin (LeTx) is central to the pathogenesis of B. anthracis infection, and the sympathetic nervous system plays a critical role in physiological regulation of acute stressors. However, the effect of LeTx on sympathetic nerve discharge (SND), a critical link between central sympathetic neural circuits and MAP regulation, remains unknown. We determined visceral (renal, splenic, and adrenal) SND responses to continuous infusion of LeTx [lethal factor (100 µg/kg) + protective antigen (200 µg/kg) infused at 0.5 ml/h for ≤6 h] and vehicle (infused at 0.5 ml/h) in anesthetized, baroreceptor-intact and baroreceptor (sinoaortic)-denervated (SAD) Sprague-Dawley rats. LeTx infusions produced an initial state of cardiovascular and sympathetic nervous system activation in intact and SAD rats. Subsequent to peak LeTx-induced increases in arterial blood pressure, intact rats demonstrated a marked hypotension that was accompanied by significant reductions in SND (renal and splenic) and heart rate (HR) from peak levels. After peak LeTx-induced pressor and sympathoexcitatory responses in SAD rats, MAP, SND (renal, splenic, and adrenal), and HR were progressively and significantly reduced, supporting the hypothesis that LeTx alters the central regulation of sympathetic nerve outflow. These findings demonstrate that the regulation of visceral SND is altered in a complex manner during continuous anthrax LeTx infusions and suggest that sympathetic nervous system dysregulation may contribute to the marked hypotension accompanying B. anthracis infection.