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Lethal toxin (LT) is the critical virulence factor of Bacillus anthracis, the causative agent of anthrax. One common symptom observed in patients with anthrax is thrombocytopenia, which has also been observed in mice injected with LT. Our previous study demonstrated that LT induces thrombocytopenia by suppressing megakaryopoiesis, but the precise molecular mechanisms behind this phenomenon remain unknown. In this study, we utilized 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced megakaryocytic differentiation in human erythroleukemia (HEL) cells to identify genes involved in LT-induced megakaryocytic suppression. Through cDNA microarray analysis, we identified Dachshund homolog 1 (DACH1) as a gene that was upregulated upon TPA treatment but downregulated in the presence of TPA and LT, purified from the culture supernatants of B. anthracis. To investigate the function of DACH1 in megakaryocytic differentiation, we employed short hairpin RNA technology to knock down DACH1 expression in HEL cells and assessed its effect on differentiation. Our data revealed that the knockdown of DACH1 expression suppressed megakaryocytic differentiation, particularly in polyploidization. We demonstrated that one mechanism by which B. anthracis LT induces suppression of polyploidization in HEL cells is through the cleavage of MEK1/2. This cleavage results in the downregulation of the ERK signaling pathway, thereby suppressing DACH1 gene expression and inhibiting polyploidization. Additionally, we found that known megakaryopoiesis-related genes, such as FOSB, ZFP36L1, RUNX1, FLI1, AHR, and GFI1B genes may be positively regulated by DACH1. Furthermore, we observed an upregulation of DACH1 during in vitro differentiation of CD34–megakaryocytes and downregulation of DACH1 in patients with thrombocytopenia. In summary, our findings shed light on one of the molecular mechanisms behind LT-induced thrombocytopenia and unveil a previously unknown role for DACH1 in megakaryopoiesis. [ABSTRACT FROM AUTHOR]

Bacillus anthracis lethal toxin (LT) is a determinant of lethal anthrax. Its function in myeloid cells is required for bacterial dissemination, and LT itself can directly trigger dysfunction of the cardiovascular system. The interplay between LT and the host responses is important in the pathogenesis, but our knowledge on this interplay remains limited. Tumor necrosis factor-α (TNF-α) is a pleiotropic pro-inflammatory cytokine induced by bacterial infections. Since LT accumulates and cytokines, predominantly TNF, amass during B. anthracis infection, co-treatment of TNF + LT in mice was used to mimic in vivo conditions for LT to function in inflamed hosts. Bone marrow transplantation and genetically engineered mice showed unexpectedly that the death of intestinal epithelial cells (IECs) rather than that of hematopoietic cells led to LT + TNF-induced lethality. Inhibition of p38α mitogen-activated protein kinase (MAPK) signaling by LT in IECs promoted TNF-induced apoptosis and necroptosis of IECs, leading to intestinal damage and mouse death. Consistently, p38α inhibition by LT enhanced TNF-mediated cell death in human colon epithelial HT-29 cells. As intestinal damage is one of the leading causes of lethality in anthrax patients, the IEC damage caused by LT + TNF would most likely be a mechanism underneath this clinical manifestation and could be a target for interventions. [ABSTRACT FROM AUTHOR]

The present study was undertaken to evaluate the protective efficacy of Taxifolin against anthrax toxins, viz., lethal toxin (LT) and edema toxin (ET) in in-vitro model. The MTT [3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide] assay and cAMP ELISA was deployed to estimate the LT induced cytoxicity and ET induced intracellular increase in cyclic AMP, respectively, wherein 10 μg/mL PA + 1 μg/mL LF/EF was deduced as the cytotoxic dose. The assays revealed an anticipated dose dependent decrease in cytotoxicity at varying concentrations (100 μM to 0.1 μM) of taxifolin when challenged against optimized cytotoxic dose of LT (lethal toxin). Similar dose dependent decrease in intracellular cAMP levels was observed at all the evaluated concentrations of taxifolin (100 μM to 0.1 μM) against cytotoxic dose of ET (edema toxin). The neutralization potential of taxifolin against both the anthrax toxins pave a way for future prospects of experimentation using combination of taxifolin with other such inhibitors and/or antibiotics in in vitro and in vivo model, which could therefore be helpful in formulating therapeutics against anthrax. [ABSTRACT FROM AUTHOR]

Anthrax toxin is a critical virulence factor of Bacillus anthracis. The toxin comprises protective antigen (PA) and two enzymatic moieties, edema factor (EF) and lethal factor (LF), forming bipartite lethal toxin (LT) and edema toxin (ET). PA binds cellular surface receptors and is required for intracellular translocation of the enzymatic moieties. For this reason, anti-PA antibodies have been developed as therapeutics for prophylaxis and treatment of human anthrax infection. Assays described publicly for the control of anti-PA antibody potency quantify inhibition of LT-mediated cell death or the ET-induced increase in c-AMP levels. These assays do not fully reflect and/or capture the pathological functions of anthrax toxin in humans. Herein, we report the development of a cell-based gene reporter potency assay for anti-PA antibodies based on the rapid LT-induced degradation of c-Jun protein, a pathogenic effect that occurs in human cells. This new assay was developed by transducing Hepa1c1c7 cells with an AP-1 reporter lentiviral construct and has been qualified for specificity, accuracy, repeatability, intermediate precision, and linearity. This assay not only serves as a bioassay for LT activity, but has applications for characterization and quality control of anti-PA therapeutic antibodies or other products that target the AP-1 signaling pathway. [ABSTRACT FROM AUTHOR]

Anthrax toxin is a critical virulence factor of Bacillus anthracis. The toxin comprises protective antigen (PA) and two enzymatic moieties, edema factor (EF) and lethal factor (LF), forming bipartite lethal toxin (LT) and edema toxin (ET). PA binds cellular surface receptors and is required for intracellular translocation of the enzymatic moieties. For this reason, anti-PA antibodies have been developed as therapeutics for prophylaxis and treatment of human anthrax infection. Assays described publicly for the control of anti-PA antibody potency quantify inhibition of LT-mediated cell death or the ET-induced increase in c-AMP levels. These assays do not fully reflect and/or capture the pathological functions of anthrax toxin in humans. Herein, we report the development of a cell-based gene reporter potency assay for anti-PA antibodies based on the rapid LT-induced degradation of c-Jun protein, a pathogenic effect that occurs in human cells. This new assay was developed by transducing Hepa1c1c7 cells with an AP-1 reporter lentiviral construct and has been qualified for specificity, accuracy, repeatability, intermediate precision, and linearity. This assay not only serves as a bioassay for LT activity, but has applications for characterization and quality control of anti-PA therapeutic antibodies or other products that target the AP-1 signaling pathway.

Abstract Background Bacillus anthracis causes a highly lethal infectious disease primarily due to toxin-mediated injury. Antibiotics are no longer effective to treat the accumulation of anthrax toxin, thereby new strategies of antibody treatment are essential. Two anti- anthrax protective antigen (PA) antibodies, hmPA6 and PA21, have been reported by our lab previously. Methods The mechanisms of the two antibodies were elucidated by Electrophoresis, Competitive Enzyme-linked immune sorbent assay, Western blot analysis and immunoprecipitation test, and in vitro, in vivo (F344 rats) treatment test. The epitopes of the two antibodies were proved by Western blot and Enzyme-linked immune sorbent assay with different domains of PA. Results In this study, we compared affinity and neutralization of these two antibodies. PA21 was better in protecting cells and rats, whereas hmPA6 had higher affinity. Furthermore, the neutralization mechanisms of the two antibodies and their recognition domains of PA were studied. The results showed that hmPA6 recognized domain IV, thus PA could not bind to cell receptors. Conversely, PA21 recognized domain II, thereby limiting heptamer oligomerization of PA63 in cells. Conclusions Our studies elucidated the mechanisms and epitopes of hmPA6 and PA21. The present investigation can advance future use of the two antibodies in anthrax treatment or prophylaxis, and potentially as a combination treatment as the antibodies target different epitopes.

Currently, live anthrax vaccine has been used for vaccine prophylaxis in Russia and neighbor countries for seve ral decades, but precise mechanism of post-vaccination protection mechanism remains unclear. Here, we provide data on examining serum antibody level against protective antigen (PA) and lethal factor (LF) in repeatedly vaccinated volun teers at early stage (5–8 days) and 1 month after the performing pre-scheduled annual revaccination. Amount of peripheral blood antigen-specific memory T cells after previous vaccinations was analyzed. It was showed that frequency of CD3+CD45RO+CD62L– memory effector T cells was increased in the majority of volunteers on day 5-8 day after performing pre-scheduled annual revaccination that peaked at day 7 by elevating it by 2-fold compared with the control group. Percentage of anthrax-specific central memory T cells did not increase at early stage after vaccination, whereas amount of activated CD3+CD45RO+CD62L+HLA-DR+ subset within this memory T cell population was increased. Likewise, percentage of activated CD3+CD45RO+CD62L–HLA-DR+ effector memory T cell subset was also increased. Moreover, serum anti-PA IgG were detected on day 5–8 day after pre-scheduled annual revaccination in half of volunteers, whereas anti-LF IgG were found only in a single volunteer. Rapidly elevated amount of serum anthrax-specific IgG antibodies evidences about sustained memory B cell response in peripheral blood samples in volunteers after pre-scheduled annual revaccination. However, percentage of CD19+CD27+ memory B cells was not significantly elevated at early stage after revaccination that tended to increase. Both helper and cytotoxic T cell subsets were activated on day 5–8 after revaccination revealed by upregulated expression of CD69 and/or CD25 markers, with the latter predominantly found on helper T cells, thereby accounting for their high proliferative activity, whereas the former — on cytotoxic T cell subsets. Detection of anti-PA IgG antibodies correlates with protection against anthrax, which was confirmed in animal models. Unfortunately, the level of serum anti-PA IgG antibodies rapidly declines after vaccination. Ability of memory B cells to rapidly trigger production of anthrax-specific antibodies in response to revaccination suggests that anti-anthrax immunity may be evaluated by measuring frequency of peripheral blood anthrax-specific memory B and T cells.

Abstract Background Disease caused by Bacillus anthracis is often accompanied by high mortality primarily due to toxin-mediated injury. In the early disease course, anthrax toxins are secreted; thus, antibiotic use is limited to the early stage. In this regard, antibodies against the toxin component, protective antigen (PA), play an important role in protecting against anthrax. Therefore, we developed PA21, a fully human anti-PA immunoglobulin G monoclonal antibody. Methods Combining human Fab was screened from a phage library with human heavy constant regions. Enzyme-linked immune sorbent assay, Western blot analysis and immunoprecipitation test evaluated the binding ability of PA21. Moreover, the affinity and neutralizing activity of the antibody was detected in vitro while the protective effectiveness in 60 rats was also examined in vivo. Results The Fischer 344 rats challenged with the lethal toxin can be protected by PA21 at a concentration of 0.067 mg/kg. All six rats remained alive although PA21 was injected 24 h before the toxin challenge. PA21 did not influence the binding of PA to cell receptors and that of a lethal factor to PA. Conclusion The PA21 monoclonal antibody against PA can be used for emergency prophylaxis and anthrax treatment.

Inflammasomes act as sensors of infection or damage to initiate immune responses. While extensively studied in rodents, understanding of livestock inflammasomes is limited. The NLRP1 inflammasome sensor in rodents is activated by Toxoplasma gondii, Bacillus anthracis lethal toxin (LT), and potentially other zoonotic pathogens. LT activates NLRP1 by N-terminal proteolysis, inducing macrophage pyroptosis and a pro-inflammatory cytokine response. In contrast, NLRP1 in macrophages from humans and certain rodent strains is resistant to LT cleavage, and pyroptosis is not induced. Evolution of NLRP1 sequences towards those leading to pyroptosis is of interest in understanding innate immune responses in different hosts. We characterized NLRP1 in cattle (Bos taurus) and American bison (Bison bison). Bovine NLRP1 is not cleaved by LT, and cattle and bison macrophages do not undergo toxin-induced pyroptosis. Additionally, we found a predicted Nlrp1 splicing isoform in cattle macrophages lacking the N-terminal domain. Resistance to LT in bovine and human NLRP1 correlates with evolutionary sequence similarity to rodents. Consistent with LT-resistant rodents, bovine macrophages undergo a slower non-pyroptotic death in the presence of LPS and LT. Overall, our findings support the model that NLRP1 activation by LT requires N-terminal cleavage, and provide novel information on mechanisms underlying immune response diversity. [ABSTRACT FROM AUTHOR]

Abstract Background Although anthrax immune globulin (AIG) improved survival in antibiotic-treated Bacillus anthracis-challenged animal models, whether it adds to the benefit of conventional hemodynamic support for B. anthracis toxin-associated shock is unknown. Methods We therefore tested AIG in sedated, mechanically ventilated canines challenged with 24-h B. anthracis lethal and edema toxin infusions and supported for 96 h with a previously demonstrated protective regimen of titrated normal saline and norepinephrine. Results Compared to controls, proportional survival (%) was increased with AIG treatment started 4 h before (33 vs. 100%, n = 6 each) or 2 h (17 vs. 86%, n = 6 and 7 respectively) or 5 h (0 vs. 67%, n = 3 each) after the start of toxin (p ≤ 0.05) and overall [3 survivors of 15 controls (20%) vs. 14 of 16 AIG animals (88%); p = 0.006]. Averaged across treatment times, AIG increased blood pressure at 48 h and decreased norepinephrine requirements at 72 h (p ≤ 0.02), increased left ventricular ejection fraction at 48 and 72 h (p ≤ 0.02), and increased urine output and decreased net fluid balance at 72 and 96 h (p ≤ 0.04). AIG also reduced acidosis and renal and hepatic injury markers between 24 and 96 h. Conclusions These findings further support AIG’s potential benefit for patients with B. anthracis infection and developing toxin-associated shock.

Inhibition of the RAF-MEK1/2-ERK signaling pathway is an ideal strategy for treating cancers with NRAS or BRAF mutations. However, the development of resistance due to incomplete inhibition of the pathway and activation of compensatory cell proliferation pathways is a major impediment of the targeted therapy. The anthrax lethal toxin (LT), which cleaves and inactivates MEKs, is a modifiable biomolecule that can be delivered selectively to tumor cells and potently kills various tumor cells. However, resistance to LT and the mechanism involved are yet to be explored. Here, we show that LT, through inhibiting MEK1/2-ERK activation, inhibits the proliferation of cancer cells with NRAS/BRAF mutations. Among them, the human colorectal tumor HT-29 and murine melanoma B16-BL6 cells developed resistance to LT in 2 to 3 days of treatment. These resistant cells activated AKT through a histone deacetylase (HDAC) 8-dependent pathway. Using an Affymetrix microarray, followed by qPCR validation, we identified that the differential expression of the phospholipase C-β1 (PLCB1) and squamous cell carcinoma-1 (DESC1) played an important role in HDAC8-mediated AKT activation and resistance to MEK1/2-ERK inhibition. By using inhibitors, small interference RNAs and/or expression vectors, we found that the inhibition of HDAC8 suppressed PLCB1 expression and induced DESC1 expression in the resistant cells, which led to the inhibition of AKT and re-sensitization to LT and MEK1/2 inhibition. These results suggest that targeting PLCB1 and DESC1 is a novel strategy for inhibiting the resistance to MEK1/2 inhibition.

Although lethal toxin (LT) and edema toxin (ET) contribute to lethality during Bacillus anthracis infection, whether they increase vascular permeability and the extravascular fluid accumulation characterizing this infection is unclear. We employed an isolated perfused Sprague-Dawley rat lung model to investigate LT and ET effects on pulmonary vascular permeability. Lungs (n ≥ 6 per experimental group) were isolated, ventilated, suspended from a force transducer, and perfused. Lung weight and pulmonary artery (Ppa) and left atrial pressures were measured over 4 h, after which pulmonary capillary filtration coefficients (Kf.c) and lung wet-to-dry weight ratios (W/D) were determined. When compared with controls, LT increased Ppa over 4 h and Kf.c and W/D at 4 h (P < 0.0001). ET decreased Ppa in a significant trend (P = 0.09) but did not significantly alter Kf.c or W/D (P ≥ 0.29). Edema toxin actually blocked LT increases in Ppa but not LT increases in Kf.c and W/D. When Ppa was maintained at control levels, LT still increased Kf.c and W/D (P ≤ 0.004). Increasing the dose of each toxin five times significantly increased and a toxin-directed monoclonal antibody decreased the effects of each toxin (P ≤ 0.05). Two rho-kinase inhibitors (GSK269962 and Y27632) decreased LT increases in P pa (P ≤ 0.02) but actually increased Kf.c and W/D in LT and control lungs (P ≤ 0.05). A vascular endothelial growth factor receptor inhibitor (ZM323881) had no significant effect (P ≥ 0.63) with LT. Thus, LT but not ET can increase pulmonary vascular permeability independent of increased Ppa and could contribute to pulmonary fluid accumulation during anthrax infection. However, pulmonary vascular dilation with ET could disrupt protective hypoxic vasoconstriction. NEW & NOTEWORTHY The most important findings from the present study are that Bacillus anthracis lethal toxin increases pulmonary artery pressure and pulmonary permeability independently in the isolated rat lung, whereas edema toxin decreases the former and does not increase permeability. Each effect could be a basis for organ dysfunction in patients with this lethal infection. These findings further support the need for adjunctive therapies that limit the effects of both toxins during infection. [ABSTRACT FROM AUTHOR]

Bacillus anthracis, the causative agent of inhalation anthrax, is a serious concern as a bioterrorism weapon. The vegetative form produces two exotoxins: Lethal toxin (LT) and edema toxin (ET). We recently characterized and compared six human airway and alveolar-resident phagocyte (AARP) subsets at the transcriptional and functional levels. In this study, we examined the effects of LT and ET on these subsets and human leukocytes. AARPs and leukocytes do not express high levels of the toxin receptors, tumor endothelium marker-8 (TEM8) and capillary morphogenesis protein-2 (CMG2). Less than 20% expressed surface TEM8, while less than 15% expressed CMG2. All cell types bound or internalized protective antigen, the common component of the two toxins, in a dose-dependent manner. Most protective antigen was likely internalized via macropinocytosis. Cells were not sensitive to LT-induced apoptosis or necrosis at concentrations up to 1000 ng/mL. However, toxin exposure inhibited B. anthracis spore internalization. This inhibition was driven primarily by ET in AARPs and LT in leukocytes. These results support a model of inhalation anthrax in which spores germinate and produce toxins. ET inhibits pathogen phagocytosis by AARPs, allowing alveolar escape. In late-stage disease, LT inhibits phagocytosis by leukocytes, allowing bacterial replication in the bloodstream.

Protective immunity against anthrax is inferred from measurement of vaccine antigen-specific neutralizing antibody titers in serum samples. In animal models, in vivo challenges with toxin and/or spores can also be performed. However, neither of these approaches considers toxin-induced damage to specific organ systems. It is therefore important to determine to what extent anthrax vaccines and existing or candidate adjuvants can provide organ-specific protection against intoxication. We therefore compared the ability of Alum, CpG DNA and the CD1d ligand α-galactosylceramide (αGC) to enhance protective antigen-specific antibody titers, to protect mice against challenge with lethal toxin, and to block cardiotoxicity and hepatotoxicity. By measurement of serum cardiac Troponin I (cTnI), and hepatic alanine aminotransferase (ALT), and aspartate aminotransferase (AST), it was apparent that neither vaccine modality prevented hepatic intoxication, despite high Ab titers and ultimate survival of the subject. In contrast, cardiotoxicity was greatly diminished by prior immunization. This shows that a vaccine that confers survival following toxin exposure may still have an associated morbidity. We propose that organ-specific intoxication should be monitored routinely during research into new vaccine modalities.

Bacillus anthracis, the causative agent of anthrax, has been a focus of study in host-pathogen dynamics since the nineteenth century. While the interaction between anthrax and host macrophages has been extensively modeled, comparatively little is known about the effect of anthrax on the immune function of neutrophils, a key frontline effector of innate immune defense. Here we showed that depletion of neutrophils significantly enhanced mortality in a systemic model of anthrax infection in mice. Ex vivo, we found that freshly isolated human neutrophils can rapidly kill anthrax, with specific inhibitor studies showing that phagocytosis and reactive oxygen species (ROS) generation contribute to this efficient bacterial clearance. Anthrax toxins, comprising lethal toxin (LT) and edema toxin (ET), are known to have major roles in B. anthracis macrophage resistance and systemic toxicity. Employing isogenic wild-type and mutant toxin-deficient B. anthracis strains, we show that despite previous studies that reported inhibition of neutrophil function by purified LT or ET, endogenous production of these toxins by live vegetative B. anthracis failed to alter key neutrophil functions. The lack of alteration in neutrophil function is accompanied by rapid killing of B. anthracis by neutrophils, regardless of the bacteria's expression of anthrax toxins. Lastly, our study demonstrates for the first time that anthrax induced neutrophil extracellular trap (NET) formation.

Bacillus anthracis, the causative agent of anthrax, has been a focus of study in host-pathogen dynamics since the nineteenth century. While the interaction between anthrax and host macrophages has been extensively modeled, comparatively little is known about the effect of anthrax on the immune function of neutrophils, a key frontline effector of innate immune defense. Here we showed that depletion of neutrophils significantly enhanced mortality in a systemic model of anthrax infection in mice. Ex vivo, we found that freshly isolated human neutrophils can rapidly kill anthrax, with specific inhibitor studies showing that phagocytosis and reactive oxygen species (ROS) generation contribute to this efficient bacterial clearance. Anthrax toxins, comprising lethal toxin (LT) and edema toxin (ET), are known to have major roles in B. anthracis macrophage resistance and systemic toxicity. Employing isogenic wild-type and mutant toxin-deficient B. anthracis strains, we show that despite previous studies that reported inhibition of neutrophil function by purified LT or ET, endogenous production of these toxins by live vegetative B. anthracis failed to alter key neutrophil functions. The lack of alteration in neutrophil function is accompanied by rapid killing of B. anthracis by neutrophils, regardless of the bacteria's expression of anthrax toxins. Lastly, our study demonstrates for the first time that anthrax induced neutrophil extracellular trap (NET) formation. [ABSTRACT FROM AUTHOR]

Although edema toxin (ETx) and lethal toxin (LTx) contribute to Bacillus anthracis shock and lethality, the mechanisms underlying their cardiovascular effects are unclear. We have previously shown that ETx but not LTx inhibited phenylephrine-stimulated contraction of aortic rings prepared from healthy rats and that adefovir, a selective inhibitor of ETx cAMP production, blocked this effect. Here, we examined arterial function in rats that received 24-h ETx or LTx infusions. Compared with control rats, ETx reduced mean arterial pressure (MAP) and survival over 48 h (P ≤ 0.0003) and increased plasma cAMP at 4, 24, and 48 h (P < 0.0001) and nitric oxide (NO) at 24 and 48 h (P ≤ 0.01). Compared with control animals, at 24- and 48-h phenylephrine stimulation of aortic rings from ETx animals produced decreased maximal contractile force (MCF; P = 0.05 and 0.006) and in vivo phenylephrine infusion in ETx animals produced decreased proportional increases in MAP (P < 0.0001 and P = 0.05). In ETx-treated animals, compared with placebo-treated animals, adefovir treatment prevented all lethality (P = 0.01), increased MAP (P ≤ 0.0001), decreased plasma and aortic tissue cAMP at 24 and 48 h, respectively (P ≤ 0.03), and plasma NO at both times (P ≤ 0.004), and increased phenylephrine-stimulated increases in MCF in aortic rings and MAP in vivo at 48 h (P = 0.02). LTx decreased MAP and survival also, but it did not alter the response to phenylephrine of MCF in aortic rings prepared from LTx animals or of MAP in vivo. In conclusion, in rats, hypotension and lethality are associated with reduced arterial contractile function with ETx but not LTx and adefovir improves ETx-induced hypotension and lethality. [ABSTRACT FROM AUTHOR]

Background: Although anthrax immune globulin (AIG) improved survival in antibiotic-treated Bacillus anthracis-challenged animal models, whether it adds to the benefit of conventional hemodynamic support for B. anthracis toxin-associated shock is unknown. Methods: We therefore tested AIG in sedated, mechanically ventilated canines challenged with 24-h B. anthracis lethal and edema toxin infusions and supported for 96 h with a previously demonstrated protective regimen of titrated normal saline and norepinephrine. Results: Compared to controls, proportional survival (%) was increased with AIG treatment started 4 h before (33 vs. 100%, n = 6 each) or 2 h (17 vs. 86%, n = 6 and 7 respectively) or 5 h (0 vs. 67%, n = 3 each) after the start of toxin ( p ≤ 0.05) and overall [3 survivors of 15 controls (20%) vs. 14 of 16 AIG animals (88%); p = 0.006]. Averaged across treatment times, AIG increased blood pressure at 48 h and decreased norepinephrine requirements at 72 h ( p ≤ 0.02), increased left ventricular ejection fraction at 48 and 72 h ( p ≤ 0.02), and increased urine output and decreased net fluid balance at 72 and 96 h ( p ≤ 0.04). AIG also reduced acidosis and renal and hepatic injury markers between 24 and 96 h. Conclusions: These findings further support AIG's potential benefit for patients with B. anthracis infection and developing toxin-associated shock. [ABSTRACT FROM AUTHOR]

Clostridium perfringens epsilon toxin (ETX) is considered as one of the most dangerous potential biological weapons. The goal of this work was to identify inhibitors of ETX using a novel approach for the inactivation of pore-forming toxins. The approach is based on the blocking of the target pore with molecules having the same symmetry as the pore itself. About 200 various β-cyclodextrin derivatives were screened for inhibitors of ETX activity using a colorimetric cell viability assay. Several compounds with dose-dependent activities at low micromolar concentrations have been identified. The same compounds were also able to inhibit lethal toxin of Bacillus anthracis . [ABSTRACT FROM AUTHOR]

A major difference between two currently licensed anthrax vaccines is presence (United Kingdom Anthrax Vaccine Precipitated, AVP) or absence (United States Anthrax Vaccine Adsorbed, AVA) of quantifiable amounts of the Lethal Toxin (LT) component Lethal Factor (LF). The primary immunogen in both vaccine formulations is Protective Antigen (PA), and LT-neutralizing antibodies directed to PA are an accepted correlate of vaccine efficacy; however, vaccination studies in animal models have demonstrated that LF antibodies can be protective. In this report we compared humoral immune responses in cohorts of AVP (n = 39) and AVA recipients (n = 78) matched 1:2 for number of vaccinations and time post-vaccination, and evaluated whether the LF response contributes to LT neutralization in human recipients of AVP. PA response rates (≥95%) and PA IgG concentrations were similar in both groups; however, AVP recipients exhibited higher LT neutralization ED 50 values (AVP: 1464.0 ± 214.7, AVA: 544.9 ± 83.2, p < 0.0001) and had higher rates of LF IgG positivity (95%) compared to matched AVA vaccinees (1%). Multiple regression analysis revealed that LF IgG makes an independent and additive contribution to the LT neutralization response in the AVP group. Affinity purified LF antibodies from two independent AVP recipients neutralized LT and bound to LF Domain 1, confirming contribution of LF antibodies to LT neutralization. This study documents the benefit of including an LF component to PA-based anthrax vaccines. [ABSTRACT FROM AUTHOR]

Lethal toxin (LT) of Bacillus anthracis reduces the production of a number of inflammatory mediators, including transcription factors, chemokines and cytokines in various human cell lines, leading to down-regulation of the host inflammatory response. Previously we showed that the reduction of interleukin-8 (IL-8) is a sensitive marker of LT-mediated intoxication in human neutrophil-like NB-4 cells and that IL-8 levels are restored to normality when therapeutic monoclonal antibodies (mAb) with toxin-neutralising (TN) activity are added. We used this information to develop cell-based assays that examine the effects of TN therapeutic mAbs designed to treat LT intoxication and here we extend these findings. We present an in vitro assay based on human endothelial cell line HUVEC jr2, which measures the TN activity of therapeutic anti-LT mAbs using IL-8 as a marker for intoxication. HUVEC jr2 cells have the advantage over NB-4 cells that they are adherent, do not require a differentiation step and can be used in a microtitre plate format and therefore can facilitate high throughput analysis. This human cell-based assay provides a valid alternative to the mouse macrophage assay as it is a more biologically relevant model of the effects of toxin-neutralising antibodies in human infection.

Outbreaks of Bacillus anthracis in the US and Europe over the past 10 years have emphasized the health threat this lethal bacteria poses even for developed parts of the world. In contrast to cutaneous anthrax, inhalational disease in the US during the 2001 outbreaks and the newly identified injectional drug use form of disease in the UK and Germany have been associated with relatively high mortality rates. One notable aspect of these cases has been the difficulty in supporting patients once shock has developed. Anthrax bacilli produce several different components which likely contribute to this shock. Growing evidence indicates that both major anthrax toxins may produce substantial cardiovascular dysfunction. Lethal toxin (LT) can alter peripheral vascular function; it also has direct myocardial depressant effects. Edema toxin (ET) may have even more pronounced peripheral vascular effects than LT, including the ability to interfere with the actions of conventional vasopressors. Additionally, ET also appears capable of interfering with renal sodium and water retention. Importantly, the two toxins exert their actions via quite different mechanisms and therefore have the potential to worsen shock and outcome in an additive fashion. Finally, both toxins have the ability to inhibit host defense and microbial clearance, possibly contributing to the very high bacterial loads noted in patients dying with anthrax. This last point is clinically relevant since emerging data has begun to implicate other bacterial components such as anthrax cell wall in the shock and organ injury observed with infection. Taken together, accumulating evidence regarding the potential contribution of LT and ET to anthrax-associated shock supports efforts to develop adjunctive therapies that target both toxins in patients with progressive shock.

A major virulence factor of Bacillus anthracis is the anthrax Lethal Toxin (LeTx), a bipartite toxin composed of Protective Antigen and Lethal Factor. Systemic administration of LeTx to laboratory animals leads to death associated with vascular leakage and pulmonary edema. In this study, we investigated whether systemic exposure of mice to LeTx would induce gene expression changes associated with vascular/capillary leakage in lung tissue. We observed enhanced susceptibility of A/J mice to death by systemic LeTx administration compared to the C57BL/6 strain. LeTx-induced groups of both up- and down-regulated genes were observed in mouse lungs 6 h after systemic administration of wild type toxin compared to lungs of mice exposed to an inactive mutant form of the toxin. Lungs of the less susceptible C57BL/6 strain showed 80% fewer differentially expressed genes compared to lungs of the more sensitive A/J strain. Expression of genes known to regulate vascular permeability was modulated by LeTx in the lungs of the more susceptible A/J strain. Unexpectedly, the largest set of genes with altered expression was immune specific, characterized by the up-regulation of lymphoid genes and the down-regulation of myeloid genes. Transcripts encoding neutrophil chemoattractants, modulators of tumor regulation and angiogenesis were also differentially expressed in both mouse strains. These studies provide new directions for the investigation of vascular leakage and pulmonary edema induced by anthrax LeTx.

Bacillus anthracis, similar to other bacterial pathogens, has evolved effective immune evasion strategies to prolong its survival in the host, thus ensuring the unchecked spread of the infection. This function is subserved by lethal (LT) and edema (ET) toxins, two exotoxins produced by vegetative anthrax bacilli following germination of the spores. The structure of these toxins and the mechanism of cell intoxication are topics covered by other reviews in this issue. Here we shall discuss how B. anthracis uses LT and ET to suppress the immune defenses of the host, focusing on T lymphocytes, the key players in adaptive immunity. We shall also summarize recent findings showing that, depending on its concentration, ET has the ability not only to suppress T cell activation but also to promote the polarization of CD4+ T cells to the Th2 and Th17 subsets, highlighting the potential use of this toxin as an immunomodulator.

Anthrax lethal toxin (LeTx) and edema toxin (EdTx) have been shown to alter hemodynamics in the rodent model, while LeTx primarily is reported to induce extensive tissue pathology. However, the rodent model has limitations when used for comparison to higher organisms such as humans. The rabbit model, on the other hand, has gained recognition as a useful model for studying anthrax infection and its pathophysiological effects. In this study, we assessed the hemodynamic effects of lethal toxin (LeTx) and edema toxin (EdTx) in the rabbit model using physiologically relevant amounts of the toxins. Moreover, we further examine the pathological effects of LeTx on cardiac tissue. We intravenously injected Dutch-belted rabbits with either low-dose and high-dose recombinant LeTx or a single dose of EdTx. The animals’ heart rate and mean arterial pressure were continuously monitored via telemetry until either 48 or 72 h post-challenge. Additional animals challenged with LeTx were used for cardiac troponin I (cTnI) quantitation, cardiac histopathology, and echocardiography. LeTx depressed heart rate at the lower dose and mean arterial pressure (MAP) at the higher dose. EdTx, on the other hand, temporarily intensified heart rate while lowering MAP. Both doses of LeTx caused cardiac pathology with the higher dose having a more profound effect. Lastly, left-ventricular dilation due to LeTx was not apparent at the given time-points. Our study demonstrates the hemodynamic effects of anthrax toxins, as well as the pathological effects of LeTx on the heart in the rabbit model, and it provides further evidence for the toxins’ direct impact on the heart.

Anthrax caused by Bacillus anthracis is a lethal infectious disease, especially when inhaled, and the mortality rate approaches 100% without treatment. The anthrax antitoxin monoclonal antibody (MAb) 5E11 is a humanized antibody that targets the anthrax protective antigen (PA). The efficacy of 5E11 needs proper animal models. However, anthrax spores are extremely dangerous, so experiments must be conducted under Biosafety Level 3 conditions. Considering the critical effects of lethal toxin (LT) on hosts during infection, we report the establishment of a LT-challenged rabbit model, which caused 100% mortality with a dose of 2 mg PA + 1 mg LF, while a 4 mg PA + 2 mg LF challenge could limit death to within three days. Then, we evaluated 5E11 efficacy against LT. A prophylactic study showed that the i.v. administration of 40 mg/kg 5E11 four days before lethal dose LT challenge could lead to 100% survival. In therapeutic studies, the i.v. administration of 40 mg/kg 5E11 10 min after lethal dose LT challenge could provide complete protection. Overall, we developed a new LT-challenged rabbit model, and our results indicate that 5E11 shows potential for the clinical application in anthrax treatment.